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WO2005090593A1 - Marqueurs hotes pour le diagnostic de l'encephalomyelite equine a protozoaire et l'infection de sarcocystis neurona - Google Patents

Marqueurs hotes pour le diagnostic de l'encephalomyelite equine a protozoaire et l'infection de sarcocystis neurona Download PDF

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Publication number
WO2005090593A1
WO2005090593A1 PCT/AU2005/000401 AU2005000401W WO2005090593A1 WO 2005090593 A1 WO2005090593 A1 WO 2005090593A1 AU 2005000401 W AU2005000401 W AU 2005000401W WO 2005090593 A1 WO2005090593 A1 WO 2005090593A1
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sequence
epm
polynucleotide
polypeptide
level
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PCT/AU2005/000401
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English (en)
Inventor
Richard Bruce Brandon
Mervyn Rees Thomas
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Genomics Research Partners Pty Ltd
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Priority claimed from AU2004901448A external-priority patent/AU2004901448A0/en
Application filed by Genomics Research Partners Pty Ltd filed Critical Genomics Research Partners Pty Ltd
Publication of WO2005090593A1 publication Critical patent/WO2005090593A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56905—Protozoa
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • This invention relates generally to protozoal diseases. More particularly, the 5 present invention relates to disease-associated molecules and assays, which are useful for diagnosing Equine Protozoal Myeloencephalitis (EPM).
  • EPM Equine Protozoal Myeloencephalitis
  • the invention has practical use in the early diagnosis of disease, in monitoring an animal's immune response to the disease, and in enabling better treatment and management decisions to be made in clinically and sub-clinically affected animals.
  • Equine Protozoal Myeloencephalitis is the leading infectious neurologic or abortigenic equine disease in the Western Hemisphere and is caused at least in part by the Apicomplexan parasite Sarcocystis neurona.
  • S. neurona has a host life cycle stage consisting of natural prey species, an intermediate host and a definitive host.
  • the opossum has 15 been determined to be the definitive host species.
  • the feral opossum ⁇ Didelphis virginiana) and the South American opossum ⁇ D. albiventris consume the intermediate host's muscle tissue infected with protozoal sarcocysts.
  • the sporozoites Following excystation, the sporozoites penetrate the intestinal mucosa ofthe horse, and undergo a series of 25 replicative cycles in the vascular endothelial cells, and possibly in the white blood cells. The merozoites then migrate to the central nervous system where they continually divide without encysting (i.e., they do not form cysts). The merozoites divide by polygeny and often leave a residual body that gradually destroys the nervous tissue ofthe infected horse causing spasticity, hypermetria, ataxia, paralysis, recumbency and death. The life-cycle stage ofthe protozoa that is 30 found in horses cannot be transmitted to other horses nor can the tissue of horses, even if eaten by opossums, infect the opossum.
  • a horse of any age, breed, or sex may be affected by EPM.
  • the disease has been reported in a horse as young as two months of age, as well as one in its thirties (Gray et al, 2001, VetRec. 149(9):269-273).
  • Clinical signs of a horse with EPM do not develop (and may not develop at all) until the organism has crossed the blood brain barrier and is within the central nervous system. These signs include weakness, muscle atrophy, spinal ataxia, or "wobbling" and/or head tilt with asymmetry ofthe face (e.g., eyelid, ear, or lip).
  • a severely EPM-affected horse may go down and be unable to rise. Lameness not traceable to orthopedic disease or any combination ofthe above signs may occur in early or less severe infections. In most cases, an affected horse is bright and alert with a normal appetite, haematological and biochemical blood values are usually in the normal range.
  • EPM West Nile Virus
  • WNV West Nile Virus
  • rabies hind limb lameness
  • cervical stenotic myelopathy Wibbler syndrome
  • botulism Equine Herpes Virus (EHV-1) infection (neurological strain)
  • EEE Eastern Equine Encephalitis Virus
  • WEE Western Equine Encephalitis Virus
  • VEE Venezuelan Equine Encephalitis Virus
  • protozoan parasites including Neospora caninum and Toxoplasma species, have been implicated in the aaetiology and pathogenesis of EPM.
  • EPM cerebrospinal fluid
  • EPM protozoan parasites
  • antiprotozoal agents including sulphonamides, pyrimethamine (see, U.S. Pat. No. 5,747,476) and coccidiostats (e.g., diclazuril and toltrazuril) are available for treatment but are expensive. These drugs do not cure the disease and a typical treatment course lasts for 90 days and costs typically in the range of $US800- $US1200 per horse. Response to treatment is often the most effective method of diagnosis.
  • Susceptibility to any disease is dependent upon exposure to conditions that are conducive to developing disease and the ability ofthe host animal to respond appropriately to these conditions.
  • the host animal response is generally orchestrated by the immune system.
  • the immune response can be influenced by factors (amongst others) such as gene expression, gene alleles or haplotype.
  • factors such as gene expression, gene alleles or haplotype.
  • United States Patent No. 6,376,176 discloses that susceptibility to Crohn's disease, type I diabetes mellitus, and rheumatoid arthritis can be determined through the presence or absence of a particular haplotype (set of alleles) ofthe genes for Notch4, hsp40-HOM and MHC Class III.
  • This supposition may be based on an over-representation of these breeds in necropsy studies. Necropsy numbers could be influenced by a number of other factors including owner interest in post-mortem results. Only one pony with EPM has been reported in the literature suggesting resistance in this breed. The ability to determine susceptibility to EPM would assist in the diagnosis of disease, allow horse owners to implement appropriate management routines, and veterinarians to advise owners on appropriate therapies. [0012] As such, there currently exists a need for more effective modalities for identifying equines susceptible to developing active disease, for diagnosing EPM, and for identifying equines amenable to treatment with antiprotozoal agents.
  • the present invention addresses the problem of diagnosing EPM by detecting a response to EPM that may be measured in host cells.
  • Advantageous embodiments involve monitoring the expression of certain genes in peripheral leukocytes ofthe immune system, which may be reflected in changing patterns of RNA levels or protein production that correlate with the presence of EPM.
  • the present invention provides methods for diagnosing the presence of EPM or S. neurona infection in a test subject, especially in an equine test subject.
  • These methods generally comprise detecting in the test subject aberrant expression of at least one gene (also referred to herein as an "EPM marker gene") selected from the group consisting of: (a) a gene having a polynucleotide expression product comprising a nucleotide sequence that shares at least 50% (and at least 51%> to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in any one of SEQ ID NO: 1, 3, 5, 7, 8, 10, 12, 14, 16, 17, 18, 20, 22, 24, 26, 27, 28, 30, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 54, 55, 56 or 57, or a complement thereof; (b) a gene having a polynucleotide expression product comprising a nucleotide
  • EPM marker polynucleotides Polypeptide expression products of the EPM marker genes are referred to herein as “EPM marker polypeptides.”
  • EPM marker polypeptides Polypeptide expression products ofthe EPM marker genes are referred to herein as “EPM marker polypeptides.”
  • the method broadly described above is used to diagnose acute EPM or S. neurona acute infection.
  • such aberrant expression is detected by: (1) measuring in a biological sample obtained from the test subject the level or functional activity of an expression product of at least one EPM marker gene and (2) comparing the measured level or functional activity of each expression product to the level or functional activity of a corresponding expression product in a reference sample obtained from one or more normal subjects or from one or more subjects lacking EPM, wherein a difference in the level or functional activity ofthe expression product in the biological sample, as compared to the level or functional activity of the corresponding expression product in the reference sample, is indicative ofthe presence of EPM or of or S. neurona infection in the test subject.
  • the methods comprise detecting aberrant expression of an EPM marker polynucleotide selected from the group consisting of (a) a polynucleotide comprising a nucleotide sequence that shares at least 50% (and at least 51% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in any one of SEQ ID NO: 1, 3, 5, 7, 8, 10, 12, 14, 16, 17, 18, 20, 22, 24, 26, 27, 28, 30, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 54, 55, 56, 57 or 422 or 422, or a complement thereof; (b) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NO: 2, 4, 6, 9, 11, 13, 15, 19, 21, 23, 25, 29, 31, 33, 51, 53 or 58; (c) a polynucleotide
  • the methods comprise detecting aberrant expression of an EPM marker polypeptide selected from the group consisting of: (i) a polypeptide comprising an amino acid sequence that shares at least 50%> (and at least 51% to at least 99% and all integer percentages in between) sequence similarity with the sequence set forth in any one of SEQ ID NO: 2, 4, 6, 9, 11, 13, 15, 19, 21, 23, 25, 29, 31, 33, 51, 53 or 58; (ii) a polypeptide comprising a portion ofthe sequence set forth in any one of SEQ ID NO: 2, 4, 6, 9, 11, 13, 15, 19, 21, 23, 25, 29, 31, 33, 51, 53 or 58, wherein the portion comprises at least 5 contiguous amino acid residues of that sequence; (iii) a polypeptide comprising an amino acid sequence that shares at least 30% similarity with at least 15 contiguous amino acid residues of the sequence set forth in any one of SEQ ID NO: 2, 4, 6, 9, 11, 13, 15, 19, 21, 23, 25, 29, 31, 33, 51, 53 or 58; and (iv)
  • the methods further comprise diagnosing the presence, stage, degree of EPM or the presence, stage, degree of S. neurona infection in the test subject when the measured level or functional activity ofthe or each expression product is different than the measured level or functional activity ofthe or each corresponding expression product.
  • the difference typically represents an at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, or even an at least about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% increase, or an at least about 10%, 20%, 30% 40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 97%, 98% or 99%, or even an at least about 99.5%, 99.9%, 99.95%, 99.99%, 99.995% or 99.999% decrease in the level or functional activity of an individual expression product as compared to the level or functional activity of an individual corresponding expression product, which is hereafter referred to as "aberrant expression.”
  • the presence of EPM or S In illustrative examples of this type, the presence of EPM or S.
  • EPM marker polynucleotide selected from (a) a polynucleotide comprising a nucleotide sequence that shares at least 50% (and at least 51% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in any one of SEQ ID NO: 3, 5, 7, 8, 18, 20, 22, 24, 26, 27, 38, 39, 44, 45, 50, 52, 54, 55, 56 or 57, or a complement thereof; (b) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NO: 4, 6, 9, 19, 21, 23, 25, 51, 53, or 58; (c) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide that shares at least 50% (and at least 51% to at least 99% and all integer percentages in between) sequence similarity with at least
  • the presence of EPM or S. neurona infection is determined by detecting a decrease in the level or functional activity of at least one EPM marker polynucleotide selected from (a) a polynucleotide comprising a nucleotide sequence that shares at least 50% (and at least 51% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in any one of SEQ ID NO: 1, 10, 12, 14, 16, 17, 28, 30, 32, 34, 35, 36, 37, 40, 41, 42, 43, 46, 47, 48, 49 or 422, or a complement thereof; (b) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NO: 2, 11, 13, 15, 29, 31 or 33; (c) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide that shares at least 50% (and at least 5 EPM marker polynucleotide selected from
  • the method further comprises diagnosing the absence of EPM or the absence of S- neurona infection or the absence of S. neurona infection in the test subject when the measured level or functional activity ofthe or each expression product is the same as or similar to the measured level or functional activity ofthe or each corresponding expression product.
  • the measured level or functional activity of an individual expression product varies from the measured level or functional activity of an individual corresponding expression product by no more than about 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.1%, which is hereafter referred to as "normal expression.”
  • the methods comprise measuring the level or functional activity of individual expression products of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 EPM marker genes.
  • the methods may comprise measuring the level or functional activity of an EPM marker polynucleotide either alone or in combination with as much as 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 other EPM marker polynucleotide(s).
  • the methods may comprise measuring the level or functional activity of an EPM marker polypeptide either alone or in combination with as much as 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 other EPM marker polypeptides(s).
  • the methods comprise measuring the level or functional activity of individual expression products of at least 1, 2, 3, 4, 5 or 6 EPM marker genes that have a very high correlation with the presence or risk of EPM (hereafter referred to as "level one correlation EPM marker genes"), representative examples of which include, but are not limited to, (a) a polynucleotide comprising a nucleotide sequence that shares at least 50% (and at least 51% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in any one of SEQ ID NO: 18, 22, 26, 27, 34, 35, 36, 37, 38, 39, 44 or 45, or a complement thereof; (b) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NO: 19 or 23; (c) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide that shares at
  • the methods comprise measuring the level or functional activity of individual expression products of at least 1, 2, 3, 4, 5, 6, 7 or 8 EPM marker genes that have a high correlation with the presence or risk of EPM (hereafter referred to as "level two correlation EPM marker genes"), representative examples of which include, but are not limited to, (a) a polynucleotide comprising a nucleotide sequence that shares at least 50% (and at least 51% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in any one of SEQ ID NO: 10, 16, 17, 20, 30, 40, 41, 48, 49 or 57, or a complement thereof; (b) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NO: 11, 21, 31 or 58; (c) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide that
  • the methods comprise measuring the level or functional activity of individual expression products of at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 EPM marker genes that have a medium correlation with the presence or risk of EPM (hereafter referred to as "level three correlation EPM marker genes"), representative examples of which include, but are not limited to, (a) a polynucleotide comprising a nucleotide sequence that shares at least 50% (and at least 51% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in any one of SEQ ID NO: 1, 3, 12, 14, 24, 28, 42, 43, 46, 47, 52 or 56, or a complement thereof; (b) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NO: 2, 4, 13, 15, 25, 29 or 53; (c) a polynucleotide comprising a nucleotide sequence that encode
  • the methods comprise measuring the level or functional activity of individual expression products of at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 EPM marker genes that have a lower correlation with the presence or risk of EPM (hereafter referred to as "level four correlation EPM marker genes"), representative examples of which include, but are not limited to, (a) a polynucleotide comprising a nucleotide sequence that shares at least 50% (and at least 51% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in any one of SEQ ID NO: 5, 7, 8, 32, 50, 54, 55 or 422, or a complement thereof; (b) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NO: 6, 9, 33 or 51; (c) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide that shares
  • the methods comprise measuring the level or functional activity of an expression product of at least 1 level one correlation EPM marker gene. In other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 2 level one correlation EPM marker genes. In still other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 1 level one correlation EPM marker gene and the level or functional activity of an expression product of at least 1 level two EPM marker gene. In still other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 2 level one correlation EPM marker genes and the level or functional activity of an expression product of at least 1 level two correlation EPM marker gene.
  • the methods comprise measuring the level or functional activity of an expression product of at least 1 level one correlation EPM marker gene and the level or functional activity of an expression product of at least 2 level two correlation EPM marker genes. [0028] In some embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 1 level one correlation EPM marker gene and the level or functional activity of an expression product of at least 1 level three correlation EPM marker gene. In other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 2 level one correlation EPM marker genes and the level or functional activity of an expression product of at least 1 level three correlation EPM marker gene.
  • the methods comprise measuring the level or functional activity of an expression product of at least 1 level one correlation EPM marker gene and the level or functional activity of an expression product of at least 2 level three correlation EPM marker genes. In still other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 1 level one correlation EPM marker gene and the level or functional activity of an expression product of at least 3 level three correlation EPM marker genes. [0029] In some embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 1 level one correlation EPM marker gene and the level or functional activity of an expression product of at least 1 level four correlation EPM marker gene.
  • the methods comprise measuring the level or functional activity of an expression product of at least 2 level one correlation EPM marker genes and the level or functional activity of an expression product of at least 1 level four correlation EPM marker gene. In still other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 1 level one correlation EPM marker gene and the level or functional activity of an expression product of at least 2 level four correlation EPM marker gene. In still other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 1 level one correlation EPM marker gene and the level or functional activity of an expression product of at least 2 level four correlation EPM marker genes.
  • the methods comprise measuring the level or functional activity of an expression product of at least 1 level one correlation EPM marker gene and the level or functional activity of an expression product of at least 3 level four correlation EPM marker genes. In still other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 1 level one correlation EPM marker gene and the level or functional activity of an expression product of at least 4 level four correlation EPM marker genes. [0030] In some embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 1 level two correlation EPM marker gene. In other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 2 level two correlation EPM marker genes.
  • the methods comprise measuring the level or functional activity of an expression product of at least 1 level two correlation EPM marker gene and the level or functional activity of an expression product of at least 1 level three correlation EPM marker gene. In other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 2 level two correlation EPM marker genes and the level or functional activity of an expression product of at least 1 level three correlation EPM marker gene. In still other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 1 level two correlation EPM marker gene and the level or functional activity of an expression product of at least 2 level three correlation EPM marker genes.
  • the methods comprise measuring the level or functional activity of an expression product of at least 1 level two correlation EPM marker gene and the level or functional activity of an expression product of at least 2 level three correlation EPM marker genes. In still other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 1 level two correlation EPM marker gene and the level or functional activity of an expression product of at least 3 level three correlation EPM marker genes. In still other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 1 level two correlation EPM marker gene and the level or functional activity of an expression product of at least 4 level three correlation EPM marker genes.
  • the methods comprise measuring the level or functional activity of an expression product of at least 1 level two correlation EPM marker gene and the level or functional activity of an expression product of at least 1 level four correlation EPM marker gene. In other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 2 level two correlation EPM marker genes and the level or functional activity of an expression product of at least 1 level four correlation EPM marker gene. In still other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 1 level two correlation EPM marker gene and the level or functional activity of an expression product of at least 2 level four correlation EPM marker genes.
  • the methods comprise measuring the level or functional activity of an expression product of at least 1 level two correlation EPM marker gene and the level or functional activity of an expression product of at least 2 level four correlation EPM marker genes. In still other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 1 level two correlation EPM marker gene and the level or functional activity of an expression product of at least 3 level four correlation EPM marker genes. In still other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 1 level two correlation EPM marker gene and the level or functional activity of an expression product of at least 4 level four correlation EPM marker genes.
  • the methods comprise measuring the level or functional activity of an expression product of at least 1 level two correlation EPM marker gene and the level or functional activity of an expression product of at least 5 level four correlation EPM marker genes. [0032] In some embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 1 level three correlation EPM marker gene. In other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 2 level three correlation EPM marker genes. In still other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 1 level three correlation EPM marker gene and the level or functional activity of an expression product of at least 1 level four correlation EPM marker gene.
  • the methods comprise measuring the level or functional activity of an expression product of at least 2 level three correlation EPM marker genes and the level or functional activity of an expression product of at least 1 level four correlation EPM marker gene. In still other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 1 level three correlation EPM marker gene and the level or functional activity of an expression product of at least 2 level four correlation EPM marker genes. In still other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 1 level three correlation EPM marker gene and the level or functional activity of an expression product of at least 2 level four correlation EPM marker genes.
  • the methods comprise measuring the level or functional activity of an expression product of at least 1 level three correlation EPM marker gene and the level or functional activity of an expression product of at least 3 level four correlation EPM marker genes. In still other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 1 level three correlation EPM marker gene and the level or functional activity of an expression product of at least 4 level four correlation EPM marker genes. In still other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 1 level three correlation EPM marker gene and the level or functional activity of an expression product of at least 5 level four correlation EPM marker genes. [0033] In some embodiments, the methods comprise measuring the level or - functional activity of an expression product of at least 1 level four correlation EPM marker gene.
  • the methods comprise measuring the level or functional activity of an expression product of at least 2 level four correlation EPM marker genes. In other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 3 level four correlation EPM marker genes. In still other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 3 level four correlation EPM marker genes. In still other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 4 level four correlation EPM marker genes. In still other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 5 level four correlation EPM marker genes. In still other embodiments, the methods comprise measuring the level or functional activity of an expression product of at least 6 level four correlation EPM marker genes.
  • the biological sample comprises blood, especially peripheral blood, which suitably includes leukocytes.
  • the expression product is selected from a RNA molecule or a polypeptide.
  • the expression product is the same as the corresponding expression product.
  • the expression product is a variant (e.g., an allelic variant) ofthe corresponding expression product.
  • the expression product or corresponding expression product is a target RNA (e.g., mRNA) or a DNA copy ofthe target RNA whose level is measured using at least one nucleic acid probe that hybridizes under at least low, medium or high stringency conditions to the target RNA or to the DNA copy, wherein the nucleic acid probe comprises at least 15 contiguous nucleotides of an EPM marker gene.
  • the measured level or abundance ofthe target RNA or its DNA copy is normalized to the level or abundance of a reference RNA or a DNA copy ofthe reference RNA that is present in the same sample.
  • the nucleic acid probe is immobilized on a solid or semi-solid support.
  • the nucleic acid probe forms part of a spatial array of nucleic acid probes.
  • the level of nucleic acid probe that is bound to the target RNA or to the PNA copy is measured by hybridization (e.g., using a nucleic acid array).
  • the level of nucleic acid probe that is bound to the target RNA or to the DNA copy is measured by nucleic acid amplification (e.g., using a polymerase chain reaction (PCR)).
  • PCR polymerase chain reaction
  • the level of nucleic acid probe that is bound to the target RNA or to the DNA copy is measured by nuclease protection assay.
  • the expression product or corresponding expression product is a target polypeptide whose level is measured using at least one antigen-binding molecule that is immuno-interactive with the target polypeptide.
  • the measured level ofthe target polypeptide is normalized to the level of a reference polypeptide that is present in the same sample.
  • the antigen-binding molecule is immobilized on a solid or semi-solid support.
  • the antigen-binding molecule forms part of a spatial array of antigen-binding molecule.
  • the level of antigen-binding molecule that is bound to the target polypeptide is measured by immunoassay (e.g., using an ELISA).
  • the expression product or corresponding expression product is a target polypeptide whose level is measured using at least one substrate for the target polypeptide with which it reacts to produce a reaction product.
  • the measured functional activity ofthe target polypeptide is normalized to the functional activity of a reference polypeptide that is present in the same sample.
  • a system is used to perform the method, which suitably comprises at least one end station coupled to a base station.
  • the base station is suitably caused (a) to receive subject data from the end station via a communications network, wherein the subject data represents parameter values corresponding to the measured or normalized level or functional activity of at least one expression product in the biological sample, and (b) to compare the subject data with predetermined data representing the measured or normalized level or functional activity of at least one corresponding expression product in the reference sample to thereby determine any difference in the level or functional activity ofthe expression product in the biological sample as compared to the level or functional activity ofthe corresponding expression product in the reference sample.
  • the base station is further caused to provide a diagnosis for the presence, absence of EPM.
  • the base station may be further caused to transfer an indication ofthe diagnosis to the end station via the communications network.
  • the present invention provides methods for treating, preventing or inhibiting the development of EPM in a subject. These methods generally comprise detecting aberrant expression of at least one EPM diagnostic marker gene in the subject, and administering to the subject an effective amount of an agent that treats or ameliorates the symptoms or reverses or inhibits the development of EPM in the subject.
  • the present invention provides isolated EPM marker polynucleotides, which are generally selected from: (a) a polynucleotide comprising a nucleotide sequence that shares at least 50% (and at least 51% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in any one of SEQ ID NO: 16, 17, 26, 27, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 54, 55, 56 or 422, or a complement thereof; (b) a polynucleotide comprising a portion ofthe sequence set forth in any one of SEQ ID NO: 16, 17, 26, 27, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 54, 55, 56 or 422, or a complement thereof, wherein the portion comprises at least 15 contiguous nucleotides of that sequence or complement; (c) a polynucleo
  • the present invention provides a nucleic acid construct comprising a polynucleotide as broadly described above in operable connection with a regulatory element, which is operable in a host cell.
  • the construct is in the form of a vector, especially an expression vector.
  • the present invention provides isolated host cells containing a nucleic acid construct or vector as broadly described above.
  • the host cells are selected from bacterial cells, yeast cells and insect cells.
  • the present invention provides probes for detecting the presence of a polynucleotide as broadly described above. These probes generally comprise a nucleotide sequence that hybridizes under at least low, medium or high stringency conditions to a polynucleotide as broadly described above.
  • the probes consist essentially of a nucleic acid sequence which corresponds or is complementary to at least a portion of a nucleotide sequence encoding the amino acid sequence set forth in any one of SEQ ID NO: 2, 4, 6, 9, 11, 13, 15, 19, 21, 23, 25, 29, 31, 33, 51, 53 or 58, wherein the portion is at least 15 nucleotides in length.
  • the probes comprise a nucleotide sequence which is capable of hybridizing to at least a portion of a nucleotide sequence encoding the amino acid sequence set forth in any one of SEQ ID NO: 2, 4, 6, 9, 11, 13, 15, 19, 21, 23, 25, 29, 31, 33, 51, 53 or 58 under at least low, medium or high stringency conditions, wherein the portion is at least 15 nucleotides in length.
  • the probes comprise a nucleotide sequence that is capable of hybridizing to at least a portion of any one of SEQ ID NO: 1, 3, 5, 7, 8, 10, 12, 14, 16, 17, 18, 20, 22, 24, 26, 27, 28, 30, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 54, 55, 56, 57 or 422 under at least low, medium or high stringency conditions, wherein the portion is at least 15 nucleotides in length.
  • Illustrative probes for detecting the presence of a polynucleotide as broadly described above are set forth in SEQ ID NO: 59-421 (see Table 2).
  • the invention provides a solid or semi-solid support comprising at least one nucleic acid probe as broadly described above immobilized thereon.
  • the solid or semi-solid support comprises a spatial array of nucleic acid probes immobilized thereon.
  • the present invention provides isolated polypeptides, referred to herein as "EPM marker polypeptides," which are generally selected from: (i) a polypeptide comprising an amino acid sequence that shares at least 50% (and at least 51%> to at least 99% and all integer percentages in between) sequence similarity with the sequence set forth in any one ofSEQ ID NO: 2, 4, 6, 9, 11, 13, 15, 19, 21, 23, 25, 29, 31, 33, 51, 53 or 58;
  • polypeptide comprising an amino acid sequence that shares at least 50%. (and at least 51% to at least 99% and all integer percentages in between) sequence similarity with a polypeptide expression product of an EPM marker gene as broadly described above, for example, especially a gene that comprises a nucleotide sequence that shares at least 50% (and at least 51% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in any one of SEQ ID NO: 16, 17, 26, 27, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 54, 55, 56 or 422; (iii) a portion ofthe polypeptide according to (i) or (ii) wherein the portion comprises at least 5 contiguous amino acid residues of that polypeptide; (iv) a polypeptide comprising an amino acid sequence that shares at least 30% similarity (and at least 31% to at least 99% and all integer percentages in between) with at least 15 contig
  • Still a further aspect of the present invention provides an antigen-binding molecule that is immuno-interactive with an EPM marker polypeptide as broadly described above.
  • the antigen-binding molecule is immuno-interactive with an EPM diagnostic marker polypeptide as broadly described above.
  • the invention provides a solid or semi-solid support comprising at least one antigen-binding molecule as broadly described above immobilized thereon.
  • the solid or semi-solid support comprises a spatial array of antigen-binding molecules immobilized thereon.
  • Still another aspect of the invention provides the use of one or more EPM marker polynucleotides as broadly described above, or the use of one or more probes as broadly described above, or the use of one or more EPM marker polypeptides as broadly described above, or the use of one or more antigen-binding molecules as broadly described above, in the manufacture of a kit for diagnosing the presence of EPM in a subject.
  • BRIEF DESCRIPTION OF THE DRAWINGS [0049]
  • Figure 1 is a graphical representation of a ROC for Day 2 post-infection with S neurona. The sensitivity and selectivity are 0.917 and 0.857 respectively. The areas under the curve are 0.92 and 0.87 using raw and Lloyd's method respectively.
  • FIG. 1 is a graphical representation of a ROC for Day 4 post-infection with S neurona. The sensitivity and selectivity are 0.750 and 0.714 respectively. The areas under the curve are 0.86 and 0.81 using raw and Lloyd's method respectively.
  • the ROC was calculated as per the ROC described in Figure 1.
  • Figure 3 is a graphical representation of a ROC for Day 7 post-infection with
  • FIG. 1 S neurona.
  • the sensitivity and selectivity are 0-833 and 0.714 respectively.
  • the areas under the curve are 0.89 and 0.84 using raw and Lloyd's method respectively.
  • the ROC was calculated as per the ROC described in Figure 1.
  • Figure 4 is a graphical representation of a ROC for Day 9 post-infection with S neurona.
  • the sensitivity and selectivity are 0.833 and 0.857 respectively.
  • the areas under the curve are 0.86 and 0.82 using raw and Lloyd's method respectively.
  • the ROC was calculated as per the ROC described in Figure 1.
  • Figure 5 is a graphical representation of a ROC for Day 11 post-infection with S neurona.
  • the sensitivity and selectivity are 0.833 and 0.857 respectively.
  • FIG. 1 The areas under the curve are 0.93 and 0.86 using raw and Lloyd's method respectively.
  • the ROC was calculated as per the ROC described in Figure 1.
  • Figure 6 is a graphical representation of a ROC for Day 14 post-infection with S neurona. The sensitivity and selectivity are 0.917 and 1.000 respectively. The areas under the curve are 0.94 and 0.91 using raw and Lloyd's method respectively. The ROC was calculated as per the ROC described in Figure 1.
  • Figure 7 is a graphical representation of a ROC for Day 17 post-infection with S neurona. The sensitivity and selectivity are 1.000 and 0.857 respectively. The areas under the curve are 0.98and 0.92 using raw and Lloyd's method respectively. The ROC was calculated as per the ROC described in Figure 1.
  • Figure ⁇ is a graphical representation of a ROC for Day 21 post-infection with S neurona.
  • the sensitivity and selectivity are 0.833 and 0.857 respectively.
  • the areas under the curve are 0.94 and 0.86 using raw and Lloyd's method respectively.
  • the ROC was calculated as per the ROC described in Figure 1.
  • Figure 9 is a graphical representation of a ROC for Day 24 post-infection with S neurona.
  • the sensitivity and selectivity are 1.000 and 0.857 respectively.
  • the areas under the curve are 0.94 and 0.91 using raw and Lloyd's method respectively.
  • the ROC was calculated as per the ROC described in Figure 1.
  • Figure 10 is a graphical representation of a ROC for Day 28 post-infection with S neurona.
  • the sensitivity and selectivity are 1 and 0.714 respectively.
  • the areas under the curve are 0.94 and 0.93 using raw and Lloyd's method respectively.
  • the ROC was calculated as per the ROC described in Figure 1.
  • an EPM marker gene refers to the overexpression or underexpression of an EPM marker gene relative to the level of expression ofthe EPM marker gene or variant thereof in cells obtained from a healthy subject or from a subject lacking EPM, and/or to a higher or lower level of an EPM marker gene product (e.g., transcript or polypeptide) in a tissue sample or body fluid obtained from a healthy subject or from a subject lacking EPM.
  • an EPM marker gene product e.g., transcript or polypeptide
  • an EPM marker gene is aberrantly expressed if the level of expression ofthe EPM marker gene is higher by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, or even an at least about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000%, or lower by at least about 10%, 20%, 30% 40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 97%, 98% or 99%, or even an at least about 99.5%, 99.9%, 99.95%, 99.99%, 99.995% or 99.999% than the level of expression ofthe EPM marker gene by cells obtained from a healthy subject or from a subject without EPM, and/or relative to the level of expression ofthe EPM marker gene in a tissue sample or body fluid obtained from a healthy subject or from a subject without EPM.
  • amplicon refers to a target sequence for amplification, and/or the amplification products of a target sequence for amplification. In certain other embodiments an "amplicon” may include the sequence of probes or primers used in amplification.
  • antigen-binding molecule is meant a molecule that has binding affinity for a target antigen.
  • the term "binds specifically," “specifically immuno- interactive” and the like when referring to an antigen-binding molecule refers to a binding reaction which is determinative of the presence of an antigen in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antigen-binding molecules bind to a particular antigen and do not bind in a significant amount to other proteins or antigens present in the sample.
  • antigen-binding molecules that is selected for its specificity for a particular antigen.
  • antigen-binding molecules can be raised to a selected protein antigen, which bind to that antigen but not to other proteins present in a sample.
  • immunoassay formats may be used to select antigen-binding molecules specifically immuno- interactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immuno-interactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor
  • biologically active portion is meant a portion of a full-length parent peptide or polypeptide which portion retains an activity ofthe parent molecule.
  • biologically active portion includes deletion mutants and peptides, for example of at least about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 300, 400, 500, 600, 700, 800, 900, 1000 contiguous amino acids, which comprise an activity of a parent molecule.
  • Portions of this type may be obtained through the application of standard recombinant nucleic acid techniques or synthesized using conventional liquid or solid phase synthesis techniques.
  • reference may be made to solution synthesis or solid phase synthesis as described, for example, in Chapter 9 entitled “Peptide Synthesis” by Atherton and Shephard which is included in a publication entitled “Synthetic Vaccines” edited by Nicholson and published by Blackwell Scientific Publications.
  • peptides can be produced by digestion of a peptide or polypeptide ofthe invention with proteinases such as endoLys-C, endoArg-C, endoGlu-C and staphylococcus V8- protease.
  • the digested fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques. Recombinant nucleic acid techniques can also be used to produce such portions.
  • the term "biological sample” as used herein refers to a sample that may be extracted, untreated, treated, diluted or concentrated from an animal.
  • the biological sample may include a biological fluid such as whole blood, serum, plasma,- saliva, urine, sweat, ascitic fluid, peritoneal fluid, synovial fluid, amniotic fluid, cerebrospinal fluid, tissue biopsy, and the like.
  • the biological sample is blood, especially peripheral blood.
  • cis-acting sequence As used herein, the term "cis-acting sequence,” “cis-acting element” or “cis- regulatory region” or “regulatory region” or similar term shall be taken to mean any sequence of nucleotides, which when positioned appropriately relative to an expressible genetic sequence, is capable of regulating, at least in part, the expression ofthe genetic sequence.
  • a cis-regulatory region may be capable of activating, silencing, enhancing, repressing or otherwise altering the level of expression and/or cell-type-specif ⁇ city and/or developmental specificity of a gene sequence at the transcriptional or post-transcriptional level.
  • the cis-acting sequence is an activator sequence that enhances or stimulates the expression of an expressible genetic sequence.
  • a polynucleotide (a) having a nucleotide sequence that is substantially identical or complementary to all or a portion of a reference polynucleotide sequence or (b) encoding an amino acid sequence identical to an amino acid sequence in a peptide or protein.
  • This phrase also includes within its scope a peptide or polypeptide having an amino acid sequence that is substantially identical to a sequence of amino acids in a reference peptide or protein.
  • an effective amount in the context of treating or preventing a condition is meant the administration of that amount of active to an individual in need of such treatment or prophylaxis, either in a single dose or as part of a series, that is effective for the prevention of incurring a symptom, holding in check such symptoms, and/or treating existing symptoms, of that condition.
  • the effective amount will vary depending upon the health and physical condition ofthe individual to be treated, the taxonomic group of individual to be treated, the formulation ofthe composition, the assessment ofthe medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • expression vector any autonomous genetic element capable of directing the transcription of a polynucleotide contained within the vector and suitably the synthesis of a peptide or polypeptide encoded by the polynucleotide. Such expression vectors are known to practitioners in the art.
  • the term “functional activity” generally refers to the ability of a molecule (e.g., a transcript or polypeptide) to perform its designated function including a biological, enzymatic, or therapeutic function. In certain embodiments, the functional activity of a molecule corresponds to its specific activity as determined by any suitable assay known in the art.
  • the term "gene” as used herein refers to any and all discrete coding regions ofthe cell's genome, as well as associated non-coding and regulatory regions. The gene is also intended to mean the open reading frame encoding specific polypeptides, introns, and adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression.
  • the gene may further comprise control signals such as promoters, enhancers, termination and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals.
  • the DNA sequences may be cDNA or genomic DNA or a fragment thereof.
  • the gene may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into the host.
  • high density polynucleotide arrays and the like is meant those arrays that contain at least 400 different features per cm 2 .
  • the phrase “high discrimination hybridization conditions” refers to hybridization conditions in which single base mismatch may be determined.
  • hybridization is used herein to denote the pairing of complementary nucleotide sequences to produce a DNA-DNA hybrid or a DNA-RNA hybrid.
  • Complementary base sequences are those sequences that are related by the base-pairing rules.
  • a pairs with T and C pairs with G In RNA, U pairs with A and C pairs with G.
  • match and mismatch as used herein refer to the hybridization potential of paired nucleotides in complementary nucleic acid strands.
  • hybridize efficiently such as the classical A-T and G-C base pair mentioned above. Mismatches are other combinations of nucleotides that do not hybridize efficiently.
  • hybridizing specifically to and the like refer to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • Reference herein to "immuno-interactive" includes reference to any interaction, reaction, or other form of association between molecules and in particular where one ofthe molecules is, or mimics, a component ofthe immune system.
  • isolated is meant material that is substantially or essentially free from components that normally accompany it in its native state.
  • an “isolated polynucleotide”, as used herein, refers to a polynucleotide, which has been purified from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment.
  • marker gene is meant a gene that imparts a distinct phenotype to cells expressing the marker gene and thus allows such transformed cells to be distinguished from cells that do not have the marker.
  • a selectable marker gene confers a trait for which one can 'select' based on resistance to a selective agent (e.g., a herbicide, antibiotic, radiation, heat, or other treatment damaging to untransformed cells).
  • a screenable marker gene confers a trait that one can identify through observation or testing, i.e., by 'screening' (e.g. ⁇ - glucuronidase, luciferase, or other enzyme activity not present in untransformed cells).
  • a "naturally-occurring" nucleic acid molecule refers to a
  • RNA or DNA molecule having a nucleotide sequence that occurs in nature For example a naturally-occurring nucleic acid molecule can encode a protein that occurs in nature.
  • a sample such as, for example, a nucleic acid extract or polypeptide extract is isolated from, or derived from, a particular source.
  • the extract may be isolated directly from fungal pathogens and fungal related plant pathogens including oomycete plant pathogens (e.g., Phytophthora and Pythium).
  • the polypeptide extract is isolated directly from zoospores, especially zoospore ventral vesicles.
  • oligonucleotide refers to a polymer composed of a multiplicity of nucleotide residues (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogues thereof, including nucleotides with modified or substituted sugar groups and the like) linked via phosphodiester bonds (or related structural variants or synthetic analogues thereof).
  • oligonucleotide typically refers to a nucleotide polymer in which the nucleotide residues and linkages between them are naturally-occurring
  • the term also includes within its scope various analogues including, but not restricted to, peptide nucleic acids (PNAs), phosphorothioate, phosphorodithioate, phophoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoroamidate, methyl phosphonates, 2-O-methyl ribonucleic acids, and the like.
  • PNAs peptide nucleic acids
  • phosphorothioate phosphorodithioate
  • phophoroselenoate phosphorodiselenoate
  • phosphoroanilothioate phosphoraniladate
  • phosphoroamidate methyl phosphonates
  • 2-O-methyl ribonucleic acids 2-O-methyl rib
  • Oligonucleotides are a polynucleotide subset with 200 bases or fewer in length. Preferably, oligonucleotides are 10 to 60 bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are usually single stranded, e.g., for probes; although oligonucleotides may be double stranded, e.g., for use in the construction of a variant nucleic acid sequence. Oligonucleotides ofthe invention can be either sense or antisense oligonucleotides.
  • oligonucleotide array refers to a substrate having oligonucleotide probes with different known sequences deposited at discrete known locations associated with its surface.
  • the substrate can be in the form of a two dimensional substrate as described in U.S. Patent No. 5,424,186. Such substrate may be used to synthesize two- dimensional spatially addressed oligonucleotide (matrix) arrays.
  • the substrate may be characterized in that it forms a tubular array in which a two dimensional planar sheet is rolled into a three-dimensional tubular configuration.
  • the substrate may also be in the form of a microsphere or bead connected to the surface of an optic fiber as, for example, disclosed by Chee et al. in WO 00/39587.
  • Oligonucleotide arrays have at least two different features and a density of at least 400 features per cm 2 .
  • the arrays can have a density of about 500, at least one thousand, at least 10 thousand, at least 100 thousand, at least one million or at least 10 million features per cm 2 .
  • the substrate may be silicon or glass and can have the thickness of a glass microscope slide or a glass cover slip, or may be composed of other synthetic polymers. Substrates that are transparent to light are useful when the method of performing an assay on the substrate involves optical detection.
  • operably connected means placing a structural gene under the regulatory control of a promoter, which then controls the transcription and optionally translation ofthe gene.
  • a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning ofthe element in its natural setting; i.e., the genes from which it is derived.
  • pathogen is used herein in its broadest sense to refer to an organism or an infectious agent whose infection of cells of viable animal tissue elicits a disease response.
  • polynucleotide or “nucleic acid” as used herein designates mRNA, RNA, cRNA, cDNA or DNA.
  • polynucleotide variant and “variant” refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter. These terms also encompass polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides.
  • polynucleotide variant and “variant” also include naturally-occurring allelic variants.
  • Polypeptide and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues ofthe same.
  • polypeptide variant refers to polypeptides which are distinguished from a reference polypeptide by the addition, deletion or substitution of at least one amino acid residue.
  • one or more amino acid residues of a reference polypeptide are replaced by different amino acids. It is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature ofthe activity ofthe polypeptide (conservative substitutions) as described hereinafter.
  • primer an oligonucleotide which, when paired with a strand of DNA, is capable of initiating the synthesis of a primer extension product in the presence of a suitable polymerizing agent.
  • the primer is preferably single-stranded for maximum efficiency in amplification but can alternatively be double-stranded.
  • a primer must be sufficiently long to prime the synthesis of extension products in the presence ofthe polymerization agent. The length ofthe primer depends on many factors, including application, temperature to be employed, template reaction conditions, other reagents, and source of primers.
  • the primer may be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500, to one base shorter in length than the template sequence at the 3' end ofthe primer to allow extension of a nucleic acid chain, though the 5' end ofthe primer may extend in length beyond the 3' end ofthe template sequence.
  • primers can be large polynucleotides, such as from about 35 nucleotides to several kilobases or more.
  • Primers can be selected to be “substantially complementary” to the sequence on the template to which it is designed to hybridize and serve as a site for the initiation of synthesis.
  • substantially complementary it is meant that the primer is sufficiently complementary to hybridize with a target polynucleotide.
  • the primer contains no mismatches with the template to which it is designed to hybridize but this is not essential.
  • non- complementary nucleotide residues can be attached to the 5' end ofthe primer, with the remainder ofthe primer sequence being complementary to the template.
  • non- complementary nucleotide residues or a stretch of non-complementary nucleotide residues can be interspersed into a primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize therewith and thereby form a template for synthesis ofthe extension product ofthe primer.
  • Probe refers to a molecule that binds to a specific sequence or subsequence or other moiety of another molecule. Unless otherwise indicated, the term “probe” typically refers to a polynucleotide probe that binds to another polynucleotide, often called the "target polynucleotide", through complementary base pairing.
  • Probes can bind target polynucleotides lacking complete sequence complementarity with the probe, depending on the stringency ofthe hybridization conditions. Probes can be labeled directly or indirectly and include primers within their scope.
  • the term "recombinant polynucleotide" as used herein refers to a polynucleotide formed in vitro by the manipulation of nucleic acid into a form not normally found in nature.
  • the recombinant polynucleotide may be in the form of an expression vector.
  • expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleotide sequence.
  • recombinant polypeptide is meant a polypeptide made using recombinant techniques, i.e., through the expression of a recombinant or synthetic polynucleotide.
  • regulatory element or “regulatory sequence” is meant nucleic acid sequences (e.g., DNA) necessary for expression of an operably linked coding sequence in a particular host cell.
  • the regulatory sequences that are suitable for prokaryotic cells for example, include a promoter, and optionally a cis-acting sequence such as an operator sequence and a ribosome binding site.
  • Control sequences that are suitable for eukaryotic cells include promoters, polyadenylation signals, transcriptional enhancers, translational enhancers, leader or trailing sequences that modulate mRNA stability, as well as targeting sequences that target a product encoded by a transcribed polynucleotide to an intracellular compartment within a cell or to the extracellular environment.
  • sequence identity refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a "percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, I
  • the identical amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His
  • sequence identity will be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. [0100] “Similarity” refers to the percentage number of amino acids that are identical or constitute conservative substitutions as defined in Table A infra. Similarity may be determined using sequence comparison programs such as GAP (Deveraux et al. 1984, Nucleic Acids Research 12, 387-395).
  • sequences of a similar or substantially different length to those cited herein might be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.
  • Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence,” “comparison window,” “sequence identity,” “percentage of sequence identity” and “substantial identity”.
  • a “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length.
  • two polynucleotides may each comprise (1) a sequence (i.e., only a portion ofthe complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences ofthe two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence ofthe same number of contiguous positions after the two sequences are optimally aligned.
  • the comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment ofthe two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any ofthe various methods selected.
  • GAP Garnier et al.
  • BESTFIT Pearson FASTA
  • FASTA Pearson's Alignment of sequences
  • TFASTA Pearson's Alignin Altschul et al.
  • a detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al, "Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998, Chapter 15.
  • vertebrate subject refers to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired.
  • Suitable vertebrate animals include, but are not restricted to, primates, avians, livestock animals ⁇ e.g., sheep, cows, horses, donkeys, pigs), laboratory test animals ⁇ e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals ⁇ e.g., cats, dogs) and captive wild animals ⁇ e.g., foxes, deer, dingoes).
  • a preferred subject is an equine animal in need of treatment or prophylaxis of EPM However, it will be understood that the aforementioned terms do not imply that symptoms are present.
  • substantially similar affinities refers herein to target sequences having similar strengths of detectable hybridization to their complementary or substantially complementary oligonucleotide probes under a chosen set of stringent conditions.
  • template refers to a nucleic acid that is used in the creation of a complementary nucleic acid strand to the "template” strand.
  • the template may be either RNA and/or DNA, and the complementary strand may also be RNA and/or DNA.
  • the complementary strand may comprise all or part ofthe complementary sequence to the "template,” and/or may include mutations so that it is not an exact, complementary strand to the "template”. Strands that are not exactly complementary to the template strand may hybridize specifically to the template strand in detection assays described here, as well as other assays known in the art, and such complementary strands that can be used in detection assays are part ofthe invention.
  • transformation means alteration ofthe genotype of an organism, for example a bacterium, yeast, mammal, avian, reptile, fish or plant, by the introduction of a foreign or endogenous nucleic acid.
  • vector is meant a polynucleotide molecule, suitably a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast, virus, mammal, avian, reptile or fish into which a polynucleotide can be inserted or cloned.
  • a vector preferably contains one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome ofthe defined host such that the cloned sequence is reproducible.
  • the vector can be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
  • the vector can contain any means for assuring self-replication.
  • the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • a vector system can comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome ofthe host cell, or a transposon.
  • the choice ofthe vector will typically depend on the compatibility ofthe vector with the host cell into which the vector is to be introduced.
  • the vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are known to those of skill in the art.
  • the terms "wild-type” and "normal” are used interchangeably to refer to the phenotype that is characteristic of most ofthe members ofthe species occurring naturally and contrast for example with the phenotype of a mutant.
  • markers of EPM in the form of RNA molecules of specified sequences, or polypeptides expressed from these RNA molecules in cells, especially in blood cells, and more especially in peripheral blood cells, of subjects with or susceptible to EPM, are disclosed. These markers are indicators of EPM and, when differentially expressed as compared to their expression in normal subjects or in subjects lacking EPM, are diagnostic for the presence of EPM in tested subjects. Such markers provide considerable advantages over the prior art in this field. [0111] It will be apparent that the nucleic acid sequences disclosed herein will find utility in a variety of applications in EPM detection, diagnosis, prognosis and treatment.
  • Examples of such applications within the scope ofthe present disclosure comprise amplification of EPM markers using specific primers, detection of EPM markers by hybridization with oligonucleotide probes, incorporation of isolated nucleic acids into vectors, expression of vector-incorporated nucleic acids as RNA and protein, and development of immunological reagents corresponding to marker encoded products.
  • the identified EPM markers may in turn be used to design specific oligonucleotide probes and primers.
  • probes and primers may be of any length that would specifically hybridize to the identified marker gene sequences and may be at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500 nucleotides in length and in the case of probes, up to the full length ofthe sequences ofthe marker genes identified herein. Probes may also include additional sequence at their 5' and/or 3' ends so that they extent beyond the target sequence with which they hybridize. [0113] When used in combination with nucleic acid amplification procedures, these probes and primers enable the rapid analysis of biological samples (e.g., peripheral blood samples) for detecting marker genes or for detecting or quantifying marker gene transcripts.
  • biological samples e.g., peripheral blood samples
  • the identified markers may also be used to identify and isolate full-length gene sequences, including regulatory elements for gene expression, from genomic DNA libraries, which are suitably but not exclusively of equine origin.
  • the cDNA sequences identified in the present disclosure may be used as hybridization probes to screen genomic DNA libraries by conventional techniques. Once partial genomic clones have been identified, full- length genes may be isolated by "chromosomal walking" (also called “overlap hybridization") using, for example, the method disclosed by Chinault & Carbon (1979, Gene 5: 111-126).
  • a partial genomic clone Once a partial genomic clone has been isolated using a cDNA hybridization probe, non-repetitive segments at or near the ends of the partial genomic clone may be used as hybridization probes in further genomic library screening, ultimately allowing isolation of entire gene sequences for the EPM markers of interest.
  • full-length genes may be obtained using the full-length or partial cDNA sequences or short expressed sequence tags (ESTs) described in this disclosure using standard techniques as disclosed for example by Sambrook, et al. (MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbor Press, 1989) and Ausubel et al, (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. 1994).
  • sequences may be used to identify and isolate full- length cDNA sequences using standard techniques as disclosed, for example, in the above- referenced texts. Sequences identified and isolated by such means may be useful in the detection ofthe EPM marker genes using the detection methods described herein, and are part ofthe invention. [0115] One of ordinary skill in the art could select segments from the identified marker genes for use in the different detection, diagnostic, or prognostic methods, vector constructs, antigen-binding molecule production, kit, and/or any ofthe embodiments described herein as part ofthe present invention.
  • Marker gene sequences that are desirable for use in the invention are those set fort in SEQ ID NO: 1, 3, 5, 7, 8, 10, 12, 14, 16, 17, 18, 20, 22, 24, 26, 27, 28, 30, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 54, 55, 56, 57 or 422 (see Table 1).
  • Nucleic acid molecules ofthe invention [0116] As described in the Examples and in Tables 1 , the present disclosure provides 31 markers of EPM, identified by GeneChip® analysis of blood obtained from normal horses and from horses with clinical evidence of EPM. Ofthe 31 markers identified, 18 comprise coding regions sequences (see the markers relating to SEQ ID NO: 1, 3, 5, 7, 8, 10, 12, 14, 18, 20, 22, 24, 28, 30, 32, 50, 52 and 57) and 13 comprise 5' and/or 3' untranslated sequences only (see the markers relating to SEQ ID NO: 7, 16, 17; 26, 27; 34, 35; 36, 37; 38, 39; 40, 41; 42, 43; 44, 45; 46, 47; 48, 49; 54, 55, 56 and 422).
  • EPM marker polynucleotides are diagnostic for the presence, stage or degree of EPM (also referred to herein as "EPM marker polynucleotides”). Sequence analysis has revealed that the EPM marker genes can be classified into subgroups. For example, several EPM marker genes encode membrane associated polypeptides (e.g., SEQ ID NO: 2, 11, 13, 17, 21, 25, 33 and 58), whereas others encode cytoplasm associated polypeptides (e.g., SEQ ID NO: 4, 8, 15, 23, 51 and 53), while still others encode nucleus associated polypeptides (e.g., SEQ ID NO: 19 and 31), whereas still others encode immune-modulating molecules (e.g., 2, 11, 13, 15, 17, 21, 25, 33, 51 and 58) and still others encode house-keeping molecules (e.g., SEQ ID NO: 4, 6, 19, 23, 38, 39 and 42).
  • membrane associated polypeptides e.g., SEQ ID NO: 2, 11, 13, 17, 21, 25, 33 and 58
  • the sequences of isolated nucleic acids disclosed herein find utility inter alia as hybridization probes or amplification primers. These nucleic acids may be used, for example, in diagnostic evaluation of biological samples or employed to clone full-length cDNAs or genomic clones corresponding thereto.
  • these probes and primers represent oligonucleotides, which are of sufficient length to provide specific hybridization to a RNA or DNA sample extracted from the biological sample.
  • the sequences typically will be about 10-20 nucleotides, but may be longer. Longer sequences, e.g., of about 30, 40, 50, 100, 500 and even up to full-length, are desirable for certain embodiments.
  • probes may be used that hybridize to multiple target sequences without compromising their ability to effectively diagnose EPM.
  • the hybridization probes described herein are useful both as reagents in solution hybridization, as in PCR, for detection of expression of corresponding genes, as well as in embodiments employing a solid phase.
  • Various probes and primers may be designed around the disclosed nucleotide sequences.
  • the sequences used to design probes and primers may include repetitive stretches of adenine nucleotides (poly-A tails) normally attached at the ends ofthe RNA for the identified marker genes.
  • probes and primers may be specifically designed to not include these or other segments from the identified marker genes, as one of ordinary skilled in the art may deem certain segments more suitable for use in the detection methods disclosed. In any event, the choice of primer or probe sequences for a selected application is within the realm ofthe ordinary skilled practitioner. Illustrative probe sequences for detection of EPM marker genes are presented in Tables 2. [0120] Primers may be provided in double-stranded or single-stranded form, although the single-stranded form is desirable. Probes, while perhaps capable of priming, are designed to bind to a target DNA or RNA and need not be used in an amplification process.
  • the probes or primers are labeled with radioactive species 32 P, 14 C, 35 S, 3 H, or other label), with a fluorophore (e.g., rhodamine, fluorescein) or with a chemillumiscent label (e.g., luciferase).
  • a fluorophore e.g., rhodamine, fluorescein
  • a chemillumiscent label e.g., luciferase.
  • the present invention provides substantially full-length cDNA sequences as well as EST and partial cDNA sequences that are useful as markers of EPM. It will be understood, however, that the present disclosure is not limited to these disclosed sequences and is intended particularly to encompass at least isolated nucleic acids that are hybridizable to nucleic acids comprising the disclosed sequences or that are variants of these nucleic acids.
  • a nucleic acid of partial sequence may be used to identify a structurally-related gene or the full-length genomic or cDNA clone from which it is derived.
  • Methods for generating cDNA and genomic libraries which may be used as a target for the above-described probes are known in the art (see, for example, Sambrook et al, 1989, supra and Ausubel et al, 1994, supra). All such nucleic acids as well as the specific nucleic acid molecules disclosed herein are collectively referred to as "EPM marker polynucleotides.”
  • the present invention includes within its scope isolated or purified expression products of EPM marker polynucleotides (i.e., RNA transcripts and polypeptides).
  • the present invention encompasses isolated or substantially purified nucleic acid or protein compositions.
  • An "isolated” or “purified” nucleic acid molecule or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the nucleic acid molecule or protein as found in its naturally occurring environment.
  • an isolated or purified polynucleotide or polypeptide is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • an "isolated" polynucleotide is free of sequences (especially protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends ofthe polynucleotide) in the genomic DNA ofthe organism from which the polynucleotide was derived.
  • an isolated EPM marker polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the polynucleotide in genomic DNA ofthe cell from which the polynucleotide was derived.
  • a polypeptide that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein.
  • culture medium suitably represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.
  • the present invention also encompasses portions of the full-length or substantially full-length nucleotide sequences ofthe EPM marker genes or their transcripts or DNA copies of these transcripts.
  • Portions of an EPM marker nucleotide sequence may encode polypeptide portions or segments that retain the biological activity ofthe native polypeptide.
  • portions of an EPM marker nucleotide sequence that are useful as hybridization probes generally do not encode amino acid sequences retaining such biological activity.
  • portions of an EPM marker nucleotide sequence may range from at least about 15, 16, 17, 18,
  • a portion of an EPM marker nucleotide sequence that encodes a biologically active portion of an EPM marker polypeptide ofthe invention may encode at least about 5, 6, 1, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 300, 400, 500, 600, 700, 800, 900 or 1000, or even at least about 2000, 3000, 4000 or 5000 contiguous amino acid residues, or almost up to the total number of amino acids present in a full-length EPM marker polypeptide.
  • Portions of an EPM marker nucleotide sequence that are useful as hybridization probes or PCR primers generally need not encode a biologically active portion of an EPM marker polypeptide.
  • a portion of an EPM marker nucleotide sequence may encode a biologically active portion of an EPM marker polypeptide, or it may be a fragment that can be used as a hybridization probe or PCR primer using standard methods known in the art.
  • a biologically active portion of an EPM marker polypeptide can be prepared by isolating a portion of one ofthe EPM marker nucleotide sequences ofthe invention, expressing the encoded portion ofthe EPM marker polypeptide (e.g., by recombinant expression in vitro), and assessing the activity ofthe encoded portion ofthe EPM marker polypeptide.
  • Nucleic acid molecules that are portions of an EPM marker nucleotide sequence comprise at least about 15, 16, 17, 18, 19,
  • EPM marker nucleotide sequences can be naturally-occurring, such as allelic variants (same locus), homologues (different locus), and orfhologues (different organism) or can be non naturally- occurring.
  • Naturally occurring variants such as these can be identified with the use of well- known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as known in the art.
  • Non-naturally occurring variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms.
  • the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions.
  • the variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product).
  • nucleotide sequences conservative variants include those sequences that, because ofthe degeneracy ofthe genetic code, encode the amino acid sequence of one ofthe EPM marker polypeptides ofthe invention.
  • Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis but which still encode an EPM marker polypeptide ofthe invention.
  • variants of a particular nucleotide sequence ofthe invention will have at least about 30%, 40% 50%, 55%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%, desirably about 90% to 95% or more, and more suitably about 98% or more sequence identity to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein using default parameters.
  • the EPM marker nucleotide sequences ofthe invention can be used to isolate corresponding sequences and alleles from other organisms, particularly other mammals, especially other equine species. Methods are readily available in the art for the hybridization of nucleic acid sequences.
  • Coding sequences from other organisms may be isolated according to well known techniques based on their sequence identity with the coding sequences set forth herein. In these techniques all or part ofthe known coding sequence is used as a probe which selectively hybridizes to other EPM marker coding sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism. Accordingly, the present invention also contemplates polynucleotides that hybridize to the EPM marker gene nucleotide sequences, or to their complements, under stringency conditions described below.
  • hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions describes conditions for hybridization and washing.
  • Guidance for performing hybridization reactions can be found in Ausubel et al, (1998, supra), Sections 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in that reference and either can be used.
  • Reference herein to low stringency conditions include and encompass from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization at 42° C, and at least about 1 M to at least about 2 M salt for washing at 42° C.
  • Low stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO (pH 7.2), 5% SDS for washing at room temperature.
  • BSA Bovine Serum Albumin
  • 1 mM EDTA 1 mM EDTA, 0.5 M NaHPO 4
  • pH 7.2 7% SDS for hybridization at 65° C
  • 2 x SSC 0.1% SDS
  • BSA Bovine Serum Albumin
  • BSA Bovine Serum Albumin
  • SSC sodium chloride/sodium citrate
  • Medium stringency conditions include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization at 42° C, and at least about 0.1 M to at least about 0.2 M salt for washing at 55° C.
  • Medium stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO 4 (pH 7.2), 5% SDS for washing at 60-65° C.
  • BSA Bovine Serum Albumin
  • medium stringency conditions includes hybridizing in 6 x SSC at about 45° C, followed by one or more washes in 0.2 x SSC, 0.1% SDS at 60° C.
  • High stringency conditions include and encompass from at least about 31% v/v to at least about 50% v/v formamide and from about 0.01 M to about 0.15 M salt for hybridization at 42° C, and about 0.01 M to about 0.02 M salt for washing at 55° C.
  • High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 0.2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO 4 (pH 7.2), 1% SDS for washing at a temperature in excess of 65° C.
  • One embodiment of high stringency conditions includes hybridizing in 6 x SSC at about 45° C, followed by one or more washes in 0.2 x SSC, 0.1% SDS at 65° C.
  • an antigen-binding molecule ofthe invention is encoded by a polynucleotide that hybridizes to a disclosed nucleotide sequence under very high stringency conditions.
  • very high stringency conditions includes hybridizing 0.5 M sodium phosphate, 7% SDS at 65° C, followed by one or more washes at 0.2 x SSC, 1% SDS at 65° C.
  • Other stringency conditions are well known in the art and a skilled addressee will recognize that various factors can be manipulated to optimize the specificity ofthe hybridization. Optimization ofthe stringency ofthe final washes can serve to ensure a high degree of hybridization. For detailed examples, see Ausubel et al, supra at pages 2.10.1 to 2.10.16 and Sambrook et al. (1989, supra) at sections 1.101 to 1.104. [0130] While stringent washes are typically carried out at temperatures from about
  • T m is the melting temperature, or temperature at which two complementary polynucleotide sequences dissociate. Methods for estimating T m are well known in the art (see Ausubel et al, supra at page 2.10.8) .
  • T m 81.5 + 16.6 (log I0 M) + 0.41 (%G+C) - 0.63 (% formamide) - (600/length) [0132] wherein: M is the concentration of Na + , preferably in the range of 0.01 molar to 0.4 molar; %G+C is the sum of guanosine and cytosine bases as a percentage ofthe total number of bases, within the range between 30% and 75% G+C; % formamide is the percent formamide concentration by volume; length is the number of base pairs in the DNA duplex.
  • T m of a duplex DNA decreases by approximately 1 ° C with every increase of 1 % in the number of randomly mismatched base pairs. Washing is generally carried out at T m - 15° C for high stringency, or T m - 30° C for moderate stringency.
  • a membrane e.g., a nitrocellulose membrane or a nylon membrane
  • immobilized DNA is hybridized overnight at 42° C in a hybridization buffer (50% deionised formamide, 5 x SSC, 5 x
  • Denhardt's solution (0.1% ficoll, 0.1% polyvinylpyrollidone and 0.1% bovine serum albumin), 0.1% SDS and 200 mg/mL denatured salmon sperm DNA containing labeled probe.
  • the membrane is then subjected to two sequential medium stringency washes (i.e., 2 x SSC, 0.1% SDS for 15 min at 45° C, followed by 2 x SSC, 0.1% SDS for 15 min at 50° C), followed by two sequential higher stringency washes (i.e., 0.2 x SSC, 0.1% SDS for 12 min at 55° C followed by 0.2 x SSC and 0.1%SDS solution for 12 min at 65-68° C.
  • polypeptides ofthe invention also contemplates full-length polypeptides encoded by the EPM marker genes ofthe invention as well as the biologically active portions of those polypeptides, which are referred to collectively herein as "EPM marker polypeptides.”
  • Biologically active portions of full-length EPM marker polypeptides include portions with immuno-interactive activity of at least about 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 60 amino acid residues in length.
  • immuno-interactive fragments contemplated by the present invention are at least 6 and desirably at least 8 amino acid residues in length, which can elicit an immune response in an animal for the production of antigen-binding molecules that are immuno-interactive with an EPM marker polypeptide ofthe invention.
  • antigen-binding molecules can be used to screen other mammals, especially equine mammals, for structurally and/or functionally related EPM marker polypeptides.
  • portions of a full-length EPM marker polypeptide may participate in an interaction, for example, an intramolecular or an inter- molecular interaction.
  • An inter-molecular interaction can be a specific binding interaction or an enzymatic interaction (e.g., the interaction can be transient and a covalent bond is formed or broken).
  • Biologically active portions of a full-length EPM marker polypeptide include peptides comprising amino acid sequences sufficiently similar to or derived from the amino acid sequences of a (putative) full-length EPM marker polypeptide, for example, the amino acid sequences shown in SEQ ED NO: 2, 4, 6, 9, 11, 13, 15, 19, 21, 23, 25, 29, 31, 33, 51, 53 or 58, which include less amino acids than a full-length EPM marker polypeptide, and exhibit at least one activity of that polypeptide.
  • biologically active portions comprise a domain or motif with at least one activity of a full-length EPM marker polypeptide.
  • a biologically active portion of a full-length EPM marker polypeptide can be a polypeptide which is, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 300, 400, 500, 600, 700, 800, 900 or 1000, or even at least about 2000 or 3000, or more amino acid residues in length.
  • the portion is a "biologically-active portion" having no less than about 1%, 10%>, 25% 50% ofthe activity ofthe full-length polypeptide from which it is derived.
  • the present invention also contemplates variant EPM marker polypeptides.
  • Variant polypeptides include proteins derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein.
  • Variant proteins encompassed by the present invention are biologically active, that is, they continue to possess the desired biological activity ofthe native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation.
  • Biologically active variants of a native EPM marker polyeptide ofthe invention will have at least 40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%, preferably about 90% to 95% or more, and more preferably about 98% or more sequence similarity with the amino acid sequence for the native protein as determined by sequence alignment programs described elsewhere herein using default parameters.
  • a biologically active variant of a protein ofthe invention may differ from that protein generally by as much 1000, 500, 400, 300, 200, 100, 50 or 20 amino acid residues or suitably by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
  • An EPM marker polypeptide of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of an EPM marker protein can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA 82:488-492), Kunkel et al. (1987, Methods in Enzymol. 154:367- 382), U.S. Pat. No. 4,873,192, Watson, J. D. et al.
  • REM Recursive ensemble mutagenesis
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as follows: [0138] Acidic: The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having an acidic side chain include glutamic acid and aspartic acid.
  • Basic The residue has a positive charge due to association with H ion at physiological pH or within one or two pH units thereof (e.g., histidine) and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH.
  • Amino acids having a basic side chain include arginine, lysine and histidine.
  • Charged The residues are charged at physiological pH and, therefore, include amino acids having acidic or basic side chains (i.e., glutamic acid, aspartic acid, arginine, lysine and histidine).
  • Hydrophobic The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium.
  • Amino acids having a hydrophobic side chain include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan.
  • Neutral/polar The residues are not charged at physiological pH, but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium.
  • Amino acids having a neutral/polar side chain include asparagine, glutamine, cysteine, histidine, serine and threonine. [0143] This description also characterizes certain amino acids as "small” since their side chains are not sufficiently large, even if polar groups are lacking, to confer hydrophobicity. With the exception of proline, "small" amino acids are those with four carbons or less when at least one polar group is on the side chain and three carbons or less when not. Amino acids having a small side chain include glycine, serine, alanine and threonine. The gene-encoded secondary amino acid proline is a special case due to its known effects on the secondary conformation of peptide chains.
  • proline differs from all the other naturally- occurring amino acids in that its side chain is bonded to the nitrogen ofthe cu-amino group, as well as the c-carbon.
  • amino acid similarity matrices e.g., PAM120 matrix and PAM250 matrix as disclosed for example by Dayhoff et al (1978) A model of evolutionary change in proteins. Matrices for determining distance relationships In M. O. Dayhoff, (ed.), Atlas of protein sequence and structure, Vol. 5, pp.
  • proline in the same group as glycine, serine, alanine and threonine. Accordingly, for the purposes ofthe present invention, proline is classified as a "small" amino acid. [0144] The degree of attraction or repulsion required for classification as polar or nonpolar is arbitrary and, therefore, amino acids specifically contemplated by the invention have been classified as one or the other. Most amino acids not specifically named can be classified on the basis of known behavior.
  • Amino acid residues can be further sub-classified as cyclic or noncyclic, and aromatic or nonaromatic, self-explanatory classifications with respect to the side-chain substituent groups ofthe residues, and as small or large.
  • the residue is considered small if it contains a total of four carbon atoms or less, inclusive ofthe carboxyl carbon, provided an additional polar substituent is present; three or less if not.
  • Small residues are, of course, always nonaromatic.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Amino acid substitutions falling within the scope ofthe invention are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure ofthe peptide backbone in the area ofthe substitution, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk ofthe side chain. After the substitutions are introduced, the variants are screened for biological activity. [0147] Alternatively, similar amino acids for making conservative substitutions can be grouped into three categories based on the identity ofthe side chains.
  • the first group includes glutamic acid, aspartic acid, arginine, lysine, histidine, which all have charged side chains;
  • the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, asparagine;
  • the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, methionine, as described in Zubay, G., Biochemistry, third edition, Wm.C. Brown Publishers (1993).
  • a predicted non-essential amino acid residue in an EPM marker polypeptide is typically replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of an EPM marker gene coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an activity ofthe parent polypeptide to identify mutants which retain that activity. Following mutagenesis ofthe coding sequences, the encoded peptide can be expressed recombinantly and the activity ofthe peptide can be determined.
  • the present invention also contemplates variants ofthe naturally-occurring EPM marker polypeptide sequences or their biologically-active fragments, wherein the variants are distinguished from the naturally-occurring sequence by the addition, deletion, or substitution of one or more amino acid residues.
  • variants will display at least about 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % similarity to a parent EPM marker polypeptide sequence as, for example, set forth in any one of SEQ ID NO: 2, 4, 6, 9, 11, 13, 15, 19, 21, 23, 25, 29, 31, 33, 51, 53 or 58.
  • variants will have at least 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % sequence identity to a parent EPM marker polypeptide sequence as, for example, set forth in any one of SEQ ID NO: 2, 4, 6, 9, 11, 13, 15, 19, 21, 23, 25, 29, 31, 33, 51, 53 or 58.
  • EPM marker polypeptides also include polypeptides that are encoded by polynucleotides that hybridize under stringency conditions as defined herein, especially high stringency conditions, to the EPM marker polynucleotide sequences ofthe invention, or the non-coding strand thereof, as described above.
  • variant polypeptides differ from an EPM marker sequence by at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s).
  • variant polypeptides differ from the corresponding sequence in any one of SEQ ID NO: 2, 4, 6, 9, 11, 13, 15, 19, 21, 23, 25, 29, 31, 33, 51, 53 or 58 by at least 1% but less than 20%, 15%, 10% or 5% ofthe residues. (If this comparison requires alignment the sequences should be aligned for maximum similarity. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, suitably, differences or changes at a non-essential residue or a conservative substitution.
  • a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of an embodiment polypeptide without abolishing or substantially altering one or more of its activities.
  • the alteration does not substantially alter one of these activities, for example, the activity is at least 20%, 40%, 60%>, 10% or 80% of wild-type.
  • An "essential” amino acid residue is a residue that, when altered from the wild-type sequence of an EPM marker polypeptide ofthe invention, results in abolition of an activity ofthe parent molecule such that less than 20% ofthe wild-type activity is present.
  • a variant polypeptide includes an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or more similarity to a corresponding sequence of an EPM marker polypeptide as, for example, set forth in any one of SEQ ID NO: 2, 4, 6, 9, 11, 13, 15, 19, 21, 23, 25, 29, 31, 33, 51, 53 or 58, and has the activity of that EPM marker polypeptide.
  • EPM marker polypeptides ofthe invention may be prepared by any suitable procedure known to those of skill in the art.
  • the polypeptides may be prepared by a procedure including the steps of: (a) preparing a chimeric construct comprising a nucleotide sequence that encodes at least a portion of an EPM marker polynucleotide and that is operably linked to a regulatory element; (b) introducing the chimeric construct into a host cell; (c) culturing the host cell to express the EPM marker polypeptide; and (d) isolating the EPM marker polypeptide from the host cell.
  • the nucleotide sequence encodes at least a portion ofthe sequence set forth in any one of SEQ ID NO: 2, 4, 6, 9, 11, 13, 15, 19, 21, 23, 25, 29, 31, 33, 51, 53 or 58 or a variant thereof.
  • the chimeric construct is typically in the form of an expression vector, which is suitably selected from self-replicating extra-chromosomal vectors (e.g., plasmids) and vectors that integrate into a host genome.
  • the regulatory element will generally be appropriate for the host cell employed for expression of the EPM marker polynucleotide. Numerous types of expression vectors and regulatory elements are known in the art for a variety of host cells.
  • Illustrative elements of this type include, but are not restricted to, promoter sequences (e.g., constitutive or inducible promoters which may be naturally occurring or combine elements of more than one promoter), leader or signal sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and termination sequences, and enhancer or activator sequences.
  • the expression vector comprises a selectable marker gene to permit the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell employed.
  • the expression vector may also include a fusion partner (typically provided by the expression vector) so that the EPM marker polypeptide is produced as a fusion polypeptide with the fusion partner.
  • fusion partners assist identification and/or purification ofthe fusion polypeptide.
  • it is necessary to ligate the EPM marker polynucleotide into an expression vector so that the translational reading frames ofthe fusion partner and the EPM marker polynucleotide coincide.
  • fusion partners include, but are not limited to, glutathione-S -transferase (GST), Fc potion of human IgG, maltose binding protein (MBP) and hexahistidine (HIS 6 ), which are particularly useful for isolation ofthe fusion polypeptide by affinity chromatography.
  • fusion polypeptides are purified by affinity chromatography using matrices to which the fusion partners bind such as but not limited to glutathione-, amylose-, and nickel- or cobalt-conjugated resins.
  • matrices to which the fusion partners bind such as but not limited to glutathione-, amylose-, and nickel- or cobalt-conjugated resins.
  • Many such matrices are available in "kit” form, such as the QIAexpressTM system (Qiagen) useful with (HIS 6 ) fusion partners and the Pharmacia GST purification system.
  • Other fusion partners known in the art are light-emitting proteins such as green fluorescent protein (GFP) and luciferase, which serve as fluorescent "tags" that permit the identification and/or isolation of fusion polypeptides by fluorescence microscopy or by flow cytometry.
  • GFP green fluorescent protein
  • luciferase serve as fluorescent "tags” that permit the identification and/
  • the fusion partners also possess protease cleavage sites, such as for Factor X a or Thrombin, which permit the relevant protease to partially digest the fusion polypeptide and thereby liberate the EPM marker polypeptide from the fusion construct. The liberated polypeptide can then be isolated from the fusion partner by subsequent chromatographic separation.
  • Fusion partners also include within their scope "epitope tags," which are usually short peptide sequences for which a specific antibody is available.
  • the chimeric constructs ofthe invention are introduced into a host by any suitable means including "transduction” and “transfection”, which are art recognized as meaning the introduction of a nucleic acid, for example, an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.
  • Transformation refers to a process in which a host's genotype is changed as a result ofthe cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell comprises the expression system ofthe invention.
  • transformation refers to a process in which a host's genotype is changed as a result ofthe cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell comprises the expression system ofthe invention.
  • the method employed will depend on the choice of host cell.
  • Technology for introduction of chimeric constructs into host cells is well known to those of skill in the art.
  • Four general classes of methods for delivering nucleic acid molecules into cells have been described: (1) chemical methods such as calcium phosphate precipitation, polyethylene glycol (PEG)-mediate precipitation and lipofection; (2) physical methods such as microinjection, electroporation, acceleration methods and vacuum infiltration; (3) vector based methods such as bacterial and viral vector-mediated transformation; and (4) receptor-mediated. Transformation techniques that fall within these and other classes are well known to workers in the art, and new techniques are continually becoming known.
  • EPM marker polypeptides may be produced by culturing a host cell transformed with a chimeric construct. The conditions appropriate for expression ofthe EPM marker polynucleotide will vary with the choice of expression vector and the host cell and are easily ascertained by one skilled in the art through routine experimentation.
  • Suitable host cells for expression may be prokaryotic or eukaryotic.
  • An illustrative host cell for expression of a polypeptide ofthe invention is a bacterium.
  • the bacterium used may be Escherichia coli.
  • the host cell may be a yeast cell or an insect cell such as, for example, SF9 cells that may be utilized with a baculovirus expression system.
  • Recombinant EPM marker polypeptides can be conveniently prepared using standard protocols as described for example in Sambrook, et al, (1989, supra), in particular
  • EPM marker polypeptides may be synthesized by chemical synthesis, e.g., using solution synthesis or solid phase synthesis as described, for example, in Chapter 9 of Atherton and Shephard ⁇ supra) and in Roberge et al (1995, Science 269: 202).
  • the invention also provides antigen-binding molecules that are specifically immuno-interactive with an EPM marker polypeptide ofthe invention.
  • the antigen-binding molecule comprise whole polyclonal antibodies. Such antibodies may be prepared, for example, by injecting an EPM marker polypeptide ofthe invention into a production species, which may include mice or rabbits, to obtain polyclonal antisera. Methods of producing polyclonal antibodies are well known to those skilled in the art.
  • monoclonal antibodies may be produced using the standard method as described, for example, by K ⁇ hler and Milstein (1975, Nature 256, 495-497), or by more recent modifications thereof as described, for example, in Coligan et al, (1991, supra) by immortalizing spleen or other antibody producing cells derived from a production species which has been inoculated with one or more ofthe EPM marker polypeptides ofthe invention.
  • the invention also contemplates as antigen-binding molecules Fv, Fab, Fab' and F(ab') 2 immunoglobulin fragments.
  • the antigen-binding molecule may comprise a synthetic stabilized Fv fragment.
  • Exemplary fragments of this type include single chain Fv fragments (sFv, frequently termed scFv) in which a peptide linker is used to bridge the N terminus or C terminus of a V # domain with the C terminus or N-terminus, respectively, of a Vi domain.
  • ScFv lack all constant parts of whole antibodies and are not able to activate complement.
  • ScFvs may be prepared, for example, in accordance with methods outlined in Kreber et al (Kreber et al. 1997, J. Immunol. Methods; 201(1): 35-55). Alternatively, they may be prepared by methods described in U.S.
  • the synthetic stabilized Fv fragment comprises a disulfide stabilized Fv (dsFv) in which cysteine residues are introduced into the Y H and Y L domains such that in the fully folded Fv molecule the two residues will form a disulfide bond between them.
  • dsFv disulfide stabilized Fv
  • Suitable methods of producing dsFv are described for example in (Glockscuther et al. Biochem. 29: 1363-1367; Reiter et al. 1994, J. Biol. Chem. 269: 18327-18331; Reiter et al. 1994, Biochem. 33: 5451-5459; Reiter et ⁇ /. 1994.
  • Phage display and combinatorial methods for generating anti-EPM marker polypeptide antigen-binding molecules are known in the art (as described in, e.g., Ladner et al. U.S. Patent No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et al.
  • the antigen-binding molecules can be used to screen expression libraries for variant EPM marker polypeptides. They can also be used to detect and/or isolate the EPM marker polypeptides ofthe invention.
  • the invention also contemplates the use of antigen-binding molecules to isolate EPM marker polypeptides using , for example, any suitable immunoaffinity based method including, but not limited to, immunochromatography and immunoprecipitation.
  • a suitable method utilises solid phase adsorption in which anti- EPM marker polypeptide antigen-binding molecules are attached to a suitable resin, the resin is contacted with a sample suspected of containing an EPM marker polypeptide, and the EPM marker polypeptide, if any, is subsequently eluted from the resin.
  • Illustrative resins include: Sepharose® (Pharmacia), Poros® resins (Roche Molecular Biochemicals, Indianapolis), Actigel SuperflowTM resins (Sterogene Bioseparations Inc., Carlsbad Calif.), and DynabeadsTM (Dynal Inc., Lake Success, N.Y.).
  • the antigen-binding molecule can be coupled to a compound, e.g., a label such as a radioactive nucleus, or imaging agent, e.g. a radioactive, enzymatic, or other, e.g., imaging agent, e.g., a NMR contrast agent. Labels which produce detectable radioactive emissions or fluorescence are preferred.
  • An anti- EPM marker polypeptide antigen-binding molecule e.g., monoclonal antibody
  • EPM marker polypeptides e.g., in a cellular lysate or cell supernatant
  • antigen-binding molecules can be used to monitor EPM marker polypeptides levels in biological samples (including whole cells and fluids) for diagnosing the presence, absence, degree, or stage of development of EPM. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance (i.e., antibody labeling).
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 1, 131 1, 35 S or 3 H.
  • the label may be selected from a group including a chromogen, a catalyst, an enzyme, a fluorophore, a chemiluminescent molecule, a lanthanide ion such as Europium (Eu 34 ), a radioisotope and a direct visual label.
  • a direct visual label use may be made of a colloidal metallic or non-metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like.
  • Enzyme labels useful in the present invention include alkaline phosphatase, horseradish peroxidase, luciferase, ⁇ - galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and the like.
  • the enzyme label may be used alone or in combination with a second enzyme in solution.
  • EPM marker genes genes whose transcripts include, but are not limited to, SEQ ID NQ: 1, 3, 5, 7, 8, 10, 12, 14, 16, 17, 18, 20, 22, 24, 26, 27, 28, 30, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 54, 55, 56, 57 or 422, as compared to normal horses or to horses lacking EPM;
  • the invention features a method for diagnosing the presence, absence, degree or stage of EPM in a subject, which is typically of equine origin, by detecting aberrant expression of an EPM diagnostic marker gene in a biological sample obtained from the subject.
  • the presence, degree, or stage of development of EPM is diagnosed when an EPM marker gene product is expressed at a detectably lower level in the biological sample as compared to the level at which that gene is expressed in a reference sample obtained from normal subjects or from subjects lacking EPM.
  • the presence, degree, or stage of development of EPM is diagnosed when an EPM marker gene product is expressed at a detectably higher level in the biological sample as compared to the level at which that gene is expressed in a reference sample obtained from normal subjects or from subjects lacking EPM.
  • diagnoses are made when the level or functional activity of an EPM marker gene product in the biological sample varies from the level or functional activity of a corresponding EPM marker gene product in the reference sample by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 97%, 98% or 99%, or even by at least about 99.5%, 99.9%, 99.95%, 99.99%, 99.995% or 99.999%, or even by at least about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000%.
  • the corresponding gene product is generally selected from the same gene product that is present in the biological sample, a gene product expressed from a variant gene (e.g., an homologous or orthologous gene) including an allelic variant, or a splice variant or protein product thereof.
  • the method comprises measuring the level or functional activity of individual expression products of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 29 or 30 EPM marker genes.
  • the biological sample contains blood, especially peripheral blood, or a fraction or extract thereof.
  • the biological sample comprises blood cells such as mature, immature and developing leukocytes, including lymphocytes, polymorphonuclear leukocytes, neutrophils, monocytes, reticulocytes, basophils, coelomocytes, hemocytes, eosinophils, megakaryocytes, macrophages, dendritic cells natural killer cells, or fraction of such cells (e.g., a nucleic acid or protein fraction).
  • the biological sample comprises leukocytes including peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • Nucleic acid used in polynucleotide-based assays can be isolated from cells contained in the biological sample, according to standard methodologies (Sambrook, et al, 1989, supra; and Ausubel et al, 1994, supra).
  • the nucleic acid is typically fractionated (e.g., poly A + RNA) or whole cell RNA. Where RNA is used as the subject of detection, it may be desired to convert the RNA to a complementary DNA.
  • the nucleic acid is amplified by a template-dependent nucleic acid amplification technique. A number of template dependent processes are available to amplify the EPM marker sequences present in a given template sample.
  • PCR polymerase chain reaction
  • the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides. By raising and lowering the temperature ofthe reaction mixture, the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated.
  • a reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al, 1989, supra. Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641.
  • the template-dependent amplification involves the quantification of transcripts in real-time.
  • RNA or DNA may be quantified using the Real-Time PCR technique (Higuchi, 1992, et al, Biotechnology 10: 413- 417).
  • the concentration ofthe amplified products ofthe target DNA in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations ofthe specific target sequence in the original DNA mixture. If the DNA mixtures are cDNAs synthesized from RNAs isolated from different tissues or cells, the relative abundance ofthe specific mRNA from which the target sequence was derived can be determined for the respective tissues or cells.
  • LCR ligase chain reaction
  • SDA Strand Displacement Amplification
  • RCR Repair Chain Reaction
  • SDA SDA
  • Target specific sequences can also be detected using a cyclic probe reaction (CPR).
  • CPR a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA that is present in a sample.
  • the reaction is treated with RNase H, and the products ofthe probe identified as distinctive products that are released after digestion.
  • the original template is annealed to another cycling probe and the reaction is repeated.
  • Still another amplification method described in GB Application No. 2 202 328, and in PCT Application No. PCT/US89/01025, may be used.
  • modified primers are used in a PCR-like, template- and enzyme-dependent synthesis.
  • the primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).
  • a capture moiety e.g., biotin
  • a detector moiety e.g., enzyme
  • an excess of labeled probes are added to a sample.
  • the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe.
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al, 1989, Proc. Natl. Acad. Sci. U.S.A., 86: 1173; Gingeras et al, PCT Application WO 88/10315).
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR Zaoh et al, 1989, Proc. Natl. Acad. Sci. U.S.A., 86: 1173; Gingeras et al, PCT Application WO 88/10315.
  • the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA.
  • RNA polymerase such as T7 or SP6.
  • T7 or SP6 an RNA polymerase
  • ssRNA single-stranded RNA
  • dsDNA double- stranded DNA
  • the ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse franscriptase (RNA-dependent DNA polymerase).
  • RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA).
  • RNase H ribonuclease H
  • the resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template.
  • This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA (“dsDNA”) molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence.
  • This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies ofthe DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because ofthe cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA. [0182] Miller et al. in PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA”) followed by transcription of many RNA copies ofthe sequence.
  • ssDNA target single-stranded DNA
  • Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence ofthe resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide may also be used for amplifying target nucleic acid sequences. Wu et al, (1989, Genomics 4: 560).
  • the EPM marker nucleic acid of interest is identified in the sample directly using a template-dependent amplification as described, for example, above, or with a second, known nucleic acid following amplification. Next, the identified product is detected.
  • the detection may be performed by visual means (e.g., ethidium bromide staining of a gel).
  • the detection may involve indirect identification ofthe product via chemiluminescence, radioactive scintigraphy of radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax Technology; Bellus, 1994, JMacromol Sci. Pure, Appl Chem., A31(l): 1355-1376).
  • amplification products or "amplicons” are visualized in order to confirm amplification ofthe EPM marker sequences.
  • One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light.
  • the amplification products are integrally labeled with radio- or fluorometrically- labelled nucleotides
  • the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation. In some embodiments, visualization is achieved indirectly.
  • a labeled nucleic acid probe is brought into contact with the amplified EPM marker sequence.
  • the probe is suitably conjugated to a chromophore but may be radiolabeled.
  • the probe is conjugated to a binding partner, such as an antigen-binding molecule, or biotin, and the other member ofthe binding pair carries a detectable moiety or reporter molecule.
  • target nucleic acids are quantified using blotting techniques, which are well l ⁇ iown to those of skill in the art. Southern blotting involves the use of DNA as a target, whereas Northern blotting involves the use of RNA as a target. Each provide different types of information, although cDNA blotting is analogous, in many aspects, to blotting or RNA species.
  • a probe is used to target a DNA or RNA species that has been immobilized on a suitable matrix, often a filter of nitrocellulose.
  • the different species should be spatially separated to facilitate analysis. This often is accomplished by gel electrophoresis of nucleic acid species followed by "blotting" on to the filter. Subsequently, the blotted target is incubated with a probe (usually labeled) under conditions that promote denaturation and rehybridization. Because the probe is designed to base pair with the target, the probe will bind a portion ofthe target sequence under renaturing conditions. Unbound probe is then removed, and detection is accomplished as described above.
  • biochip-based technologies such as those described by Hacia et al. (1996, Nature Genetics 14: 441-447) and Shoemaker et al. (1996, Nature Genetics 14: 450-456). Briefly, these techniques involve quantitative methods for analysing large numbers of genes rapidly and accurately. By tagging genes with oligonucleotides or using fixed probe arrays, one can employ biochip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization. See also Pease et al.
  • nucleic acid probes to EPM marker polynucleotides are made and attached to biochips to be used in screening and diagnostic methods, as outlined herein.
  • the nucleic acid probes attached to the biochip are designed to be substantially complementary to specific expressed EPM marker nucleic acids, i.e., the target sequence (either the target sequence ofthe sample or to other probe sequences, for example in sandwich assays), such that hybridization ofthe target sequence and the probes ofthe present invention occurs.
  • This complementarity need not be perfect; there may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the nucleic acid probes ofthe present invention. However, if the number of mismatches is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence.
  • more than one probe per sequence is used, with either overlapping probes or probes to different sections ofthe target being used. That is, two, three, four or more probes, with three being desirable, are used to build in a redundancy for a particular target.
  • the probes can be overlapping (i.e. have some sequence in common), or separate.
  • nucleic acids can be attached to or immobilized on a solid support in a wide variety of ways.
  • immobilized and grammatical equivalents herein is meant the association or binding between the nucleic acid probe and the solid support is sufficient to be stable under the conditions of binding, washing, analysis, and removal as outlined below.
  • the binding can be covalent or non-covalent.
  • non- covalent binding and grammatical equivalents herein is meant one or more of either electrostatic, hydrophilic, and hydrophobic interactions.
  • non-covalent binding is the covalent attachment of a molecule, such as, sfreptavidin to the support and the non-covalent binding ofthe biotinylated probe to the sfreptavidin.
  • covalent binding and grammatical equivalents herein is meant that the two moieties, the solid support and the probe, are attached by at least one bond, including sigma bonds, pi bonds and coordination bonds.
  • Covalent bonds can be formed directly between the probe and the solid support or can be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules. Immobilization may also involve a combination of covalent and non-covalent interactions.
  • the probes are attached to the biochip in a wide variety of ways, as will be appreciated by those in the art.
  • the nucleic acids can either be synthesized first, with subsequent attachment to the biochip, or can be directly synthesized on the biochip.
  • the biochip comprises a suitable solid or semi-solid substrate or solid support.
  • substrate or “solid support” is meant any material that can be modified to contain discrete individual sites appropriate for the attachment or association ofthe nucleic acid probes and is amenable to at least one detection method.
  • the number of possible substrates are very large, and include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes,
  • the substrates allow optical detection and do not appreciably fluorescese.
  • the substrate is planar, although as will be appreciated by those of skill in the art, other configurations of substrates may be used as well.
  • the probes may be placed on the inside surface of a tube, for flow-through sample analysis to minimize sample volume.
  • the substrate may be flexible, such as a flexible foam, including closed cell foams made of particular plastics.
  • oligonucleotides probes are synthesized on the substrate, as is l ⁇ iown in the art.
  • photoactivation techniques utilizing photopolymerisation compounds and techniques can be used.
  • the nucleic acids are synthesized in situ, using well known photolithographic techniques, such as those described in WO 95/25116; WO 95/35505; U.S. Pat. Nos. 5,700,637 and 5,445,934; and references cited within; these methods of attachment form the basis ofthe Affymetrix GeneChipTM technology.
  • oligonucleotide probes on the biochip are exposed to or contacted with a nucleic acid sample suspected of containing one or more EPM polynucleotides under conditions favoring specific hybridization.
  • RNA either single or double-stranded, may be prepared from fluid suspensions of biological materials, or by grinding biological materials, or following a cell lysis step which includes, but is not limited to, lysis effected by treatment with SDS (or other detergents), osmotic shock, guanidinium isothiocyanate and lysozyme.
  • Suitable DNA which may be used in the method of the invention, includes cDNA. Such DNA may be prepared by any one of a number of commonly used protocols as for example described in Ausubel, et al, 1994, supra, and Sambrook, et al, et al, 1989, supra.
  • RNA which may be used in the method of the invention, includes messenger RNA, complementary RNA transcribed from DNA (cRNA) or genomic or subgenomic RNA. Such RNA may be prepared using standard protocols as for example described in the relevant sections of Ausubel, et al. 1994, supra and Sambrook, et al. 1989, supra).
  • cDNA may be fragmented, for example, by sonication or by treatment with restriction endonucleases.
  • cDNA is fragmented such that resultant DNA fragments are of a length greater than the length ofthe immobilized oligonucleotide probe(s) but small enough to allow rapid access thereto under suitable hybridization conditions.
  • fragments of cDNA may be selected and amplified using a suitable nucleotide amplification technique, as described for example above, involving appropriate random or specific primers.
  • the target EPM marker polynucleotides are detectably labeled so that their hybridization to individual probes can be determined.
  • the target polynucleotides are typically detectably labeled with a reporter molecule illustrative examples of which include chromogens, catalysts, enzymes, fluorochromes, chemiluminescent molecules, bioluminescent molecules, lanthanide ions (e.g., Eu 34 ), a radioisotope and a direct visual label.
  • a direct visual label use may be made of a colloidal metallic or non-metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like.
  • Illustrative labels of this type include large colloids, for example, metal colloids such as those from gold, selenium, silver, tin and titanium oxide.
  • an enzyme is used as a direct visual label
  • biotinylated bases are incorporated into a target polynucleotide. Hybridization is detected by • incubation with streptavidin-reporter molecules.
  • Suitable fluorochromes include, but are not limited to, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), R-Phycoerythrin (RPE), and Texas Red.
  • FITC fluorescein isothiocyanate
  • TRITC tetramethylrhodamine isothiocyanate
  • RPE R-Phycoerythrin
  • Texas Red Texas Red
  • Radioactive reporter molecules include, for example, 32 P, which can be detected by an X-ray or phosphoimager techniques.
  • the hybrid-forming step can be performed under suitable conditions for hybridizing oligonucleotide probes to test nucleic acid including DNA or RNA.
  • hybridization is influenced by the length ofthe oligonucleotide probe and the polynucleotide sequence under test, the pH, the temperature, the concentration of mono- and divalent cations, the proportion of G and C nucleotides in the hybrid-forming region, the viscosity ofthe medium and the possible presence of denaturants. Such variables also influence the time required for hybridization.
  • the preferred conditions will therefore depend upon the particular application. Such empirical conditions, however, can be routinely determined without undue experimentation. [0202]
  • high discrimination hybridization conditions are used. For example, reference may be made to Wallace et al. (1979, Nucl. Acids Res.
  • a hybridization reaction can be performed in the presence of a hybridization buffer that optionally includes a hybridization-optimizing agent, such as an isostabilising agent, a denaturing agent and/or a renaturation accelerant.
  • a hybridization-optimizing agent such as an isostabilising agent, a denaturing agent and/or a renaturation accelerant.
  • isostabilising agents include, but are not restricted to, betaines and lower tetraalkyl ammonium salts.
  • Denaturing agents are compositions that lower the melting temperature of double stranded nucleic acid molecules by interfering with hydrogen bonding between bases in a double stranded nucleic acid or the hydration of nucleic acid molecules.
  • Denaturing agents include, but are not restricted to, formamide, formaldehyde, dimethylsulf oxide, tetraethyl acetate, urea, guanidium isothiocyanate, glycerol and chaotropic salts.
  • Hybridization accelerants include heterogeneous nuclear ribonucleoprotein (hnRP) Al and cationic detergents such as cetyltrimethylammonium bromide (CTAB) and dodecyl trimethylammonium bromide (DTAB), polylysine, spermine, spermidine, single stranded binding protein (SSB), phage T4 gene 32 protein and a mixture of ammonium acetate and ethanol.
  • Hybridization buffers may include target polynucleotides at a concentration between about 0.005 nM and about 50 nM, preferably between about 0.5 nM and 5 nM, more preferably between about 1 nM and 2 nM.
  • a hybridization mixture containing the target EPM marker polynucleotides is placed in contact with the array of probes and incubated at a temperature and for a time appropriate to permit hybridization between the target sequences in the target polynucleotides and any complementary probes.
  • Contact can take place in any suitable container, for example, a dish or a cell designed to hold the solid support on which the probes are bound.
  • incubation will be at temperatures normally used for hybridization of nucleic acids, for example, between about 20° C and about 75° C, example, about 25° C, about 30° C, about 35° C, about 40° C, about 45° C, about 50° C, about 55° C, about 60° C, or about 65° C.
  • a sample of target polynucleotides is incubated with the probes for a time sufficient to allow the desired level of hybridization between the target sequences in the target polynucleotides and any complementary probes.
  • the hybridization may be carried out at about 45° C +/-10° C in formamide for 1-2 days.
  • the probes are washed to remove any unbound nucleic acid with a hybridization buffer, which can typically comprise a hybridization optimizing agent in the same range of concentrations as for the hybridization step. This washing step leaves only bound target polynucleotides.
  • a signal may be instrumentally detected by irradiating a fluorescent label with light and detecting fluorescence in a fluorimeter; by providing for an enzyme system to produce a dye which could be detected using a specfrophotometer; or detection of a dye particle or a colored colloidal metallic or non metallic particle using a reflectometer; in the case of using a radioactive label or chemiluminescent molecule employing a radiation counter or autoradiography.
  • a detection means may be adapted to detect or scan light associated with the label which light may include fluorescent, luminescent, focussed beam or laser light.
  • a charge couple device (CCD) or a photocell can be used to scan for emission of light from a probe:target polynucleotide hybrid from each location in the micro-array and record the data directly in a digital computer.
  • electronic detection ofthe signal may not be necessary.
  • the detection means is suitably interfaced with pattern recognition software to convert the pattern of signals from the array into a plain language genetic profile.
  • oligonucleotide probes specific for different EPM marker gene products are in the form of a nucleic acid array and detection of a signal generated from a reporter molecule on the array is performed using a 'chip reader'.
  • a detection system that can be used by a 'chip reader' is described for example by Pirrung et al (U.S. Patent No. 5,143,854).
  • the chip reader will typically also incorporate some signal processing to determine whether the signal at a particular array position or feature is a true positive or maybe a spurious signal.
  • Exemplary chip readers are described for example by Fodor et al (U.S. Patent No., 5,925,525).
  • the reaction may be detected using flow cytometry.
  • the presence of an aberrant concentration of an EPM marker protein is indicative ofthe presence, degree, or stage of development of EPM.
  • EPM marker protein levels in biological samples can be assayed using any suitable method known in the art. For example, when an EPM marker protein is an enzyme, the protein can be quantified based upon its catalytic activity or based upon the number of molecules ofthe protein contained in a sample.
  • Antibody-based techniques may be employed, such as, for example, immunohistological and immunohistochemical methods for measuring the level of a protein of interest in a tissue sample.
  • specific recognition is provided by a primary antibody (polyclonal or monoclonal) and a secondary detection system is used to detect presence (or binding) ofthe primary antibody.
  • Detectable labels can be conjugated to the secondary antibody, such as a fluorescent label, a radiolabel, or an enzyme (e.g., alkaline phosphatase, horseradish peroxidase) which produces a quantifiable, e.g., coloured, product.
  • the primary antibody itself can be detectably labeled.
  • a protein extract is produced from a biological sample (e.g., tissue, cells) for analysis.
  • a biological sample e.g., tissue, cells
  • Such an extract e.g., a detergent extract
  • Other useful antibody-based methods include immunoassays, such as the enzyme-linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • a protein-specific monoclonal antibody can be used both as an immunoadsorbent and as an enzyme-labeled probe to detect and quantify an EPM marker protein of interest.
  • the amount of such protein present in a sample can be calculated by reference to the amount present in a standard preparation using a linear regression computer algorithm (see Lacobilli et al, 1988, Breast Cancer Research and Treatment 11: 19-30).
  • two different monoclonal antibodies to the protein of interest can be employed, one as the immunoadsorbent and the other as an enzyme-labeled probe.
  • recent developments in the field of protein capture arrays permit the simultaneous detection and/or quantification of a large number of proteins.
  • low-density protein arrays on filter membranes such as the universal protein array system (Ge, 2000 Nucleic Acids Res. 28(2):e3) allow imaging of arrayed antigens using standard ELISA techniques and a scanning charge-coupled device (CCD) detector.
  • Immuno- sensor arrays have also been developed that enable the simultaneous detection of clinical analytes. It is now possible using protein arrays, to profile protein expression in bodily fluids, such as in sera of healthy or diseased subjects, as well as in subjects pre- and post-drug treatment.
  • Protein capture arrays typically comprise a plurality of protein-capture agents each of which defines a spatially distinct feature ofthe array.
  • the protein-capture agent can be any molecule or complex of molecules which has the ability to bind a protein and immobilize it to the site ofthe protein-capture agent on the array.
  • the protein-capture agent may be a protein whose natural function in a cell is to specifically bind another protein, such as an antibody or a receptor.
  • the protein-capture agent may instead be a partially or wholly synthetic or recombinant protein which specifically binds a protein.
  • the protein-capture agent may be a protein which has been selected in vitro from a mutagenized, randomized, or completely random and synthetic library by its binding affinity to a specific protein or peptide target.
  • the selection method used may optionally have been a display method such as ribosome display or phage display, as l ⁇ iown in the art.
  • the protein- capture agent obtained via in vitro selection may be a DNA or RNA aptamer which specifically binds a protein target (see, e.g., Potyrailo et al, 1998 Anal. Chem. 70:3419-3425; Cohen et al, 1998, Proc. Natl. Acad. Sci. USA 95:14272-14277; Fukuda, et al, 1997 Nucleic Acids Symp. Ser. 37:237-238; available from SomaLogic).
  • aptamers are selected from libraries of oligonucleotides by the SelexTM process and their interaction with protein can be enhanced by covalent attachment, through incorporation of brominated deoxyuridine and UV-activated crosslinking (photoaptamers).
  • Aptamers have the advantages of ease of production by automated oligonucleotide synthesis and the stability and robustness of DNA; universal fluorescent protein stains can be used to detect binding.
  • the in vitro selected protein-capture agent may be a polypeptide (e.g., an antigen) (see, e.g., Roberts and Szostak,
  • Exemplary protein capture arrays include arrays comprising spatially addressed antigen-binding molecules, commonly referred to as antibody arrays, which can facilitate extensive parallel analysis of numerous proteins defining a proteome or subproteome.
  • Antibody arrays have been shown to have the required properties of specificity and acceptable background, and some are available commercially (e.g., BD Biosciences, Clontech, BioRad and Sigma). Various methods for the preparation of antibody arrays have been reported (see, e.g., Lopez et al, 2003 J. Chromatogr. B 787:19-27; Cahill, 2000 Trends in Biotechnology 7:47-51; U.S. Pat. App. Pub. 2002/0055186; U.S. Pat. App. Pub.
  • the antigen- binding molecules of such arrays may recognise at least a subset of proteins expressed by a cell or population of cells, illustrative examples of which include growth factor receptors, hormone receptors, neurotransmitter receptors, catecholamine receptors, amino acid derivative receptors, cytokine receptors, extracellular matrix receptors, antibodies, lectins, cytokines, serpins, proteases, kinases, phosphatases, ras-like GTPases, hydrolases, steroid hormone receptors, transcription factors, heat-shock transcription factors, DNA-binding proteins, zinc-finger proteins, leucine-zipper proteins, homeodomain proteins, intracellular signal transduction modulators and effectors, apoptosis-related factors, DNA synthesis factors, DNA repair factors, DNA recombination factors, cell-surface antigens, hepatitis C virus (HCV) proteases and HIV proteases.
  • HCV hepatitis C virus
  • Antigen-binding molecules for antibody arrays are made either by conventional immunization (e.g., polyclonal sera and hybridomas), or as recombinant fragments, usually expressed in E. coli, after selection from phage display or ribosome display libraries (e.g., available from Cambridge Antibody Technology, Biofcivent, Affitech and Biosite).
  • phage display or ribosome display libraries e.g., available from Cambridge Antibody Technology, Biofcivent, Affitech and Biosite.
  • 'combibodies' comprising non-covalent associations ofVH and VL domains, can be produced in a matrix format created from combinations of diabody-producing bacterial clones (e.g., available from Domantis).
  • Exemplary antigen-binding molecules for use as protein-capture agents include monoclonal antibodies, polyclonal antibodies, Fv, Fab, Fab' and F(ab') 2 immunoglobulin fragments, synthetic stabilized Fv fragments, e.g., single chain Fv fragments (scFv), disulfide stabilized Fv fragments (dsFv), single variable region domains (dAbs) minibodies, combibodies and multivalent antibodies such as diabodies and multi-scFv, single domains from camelids or engineered human equivalents.
  • Individual spatially distinct protein-capture agents are typically attached to a support surface, which is generally planar or contoured.
  • Common physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, and magnetic and other microbeads.
  • CD centrifugation devices based on developments in microfluidics (e.g., available from Gyros) and specialized chip designs, such as engineered microchannels in a plate (e.g., The Living ChipTM, available from Biofrove) and tiny 3D posts on a silicon surface (e.g., available from Zyomyx).
  • Particles in suspension can also be used as the basis of arrays, providing they are coded for identification; systems include color coding for microbeads (e.g., available from Luminex, Bio-Rad and Nanomics Biosystems) and semiconductor nanocrystals (e.g., QDotsTM, available from Quantum Dots), and barcoding for beads (UltraPlexTM, available from
  • NanobarcodesTM particles available from Surromed
  • Beads can also be assembled into planar arrays on semiconductor chips (e.g., available from LEAPS technology and BioArray Solutions).
  • individual protein- capture agents are typically attached to an individual particle to provide the spatial definition or separation ofthe array.
  • the particles may then be assayed separately, but in parallel, in a compartmentalized way, for example in the wells of a microtiter plate or in separate test tubes.
  • a protein sample which is optionally fragmented to form peptide fragments (see, e.g., U.S. Pat. App. Pub.
  • a protein- capture array under conditions suitable for protein or peptide binding, and the array is washed to remove unbound or non-specifically bound components ofthe sample from the array.
  • the presence or amount of protein or peptide bound to each feature ofthe array is detected using a suitable detection system.
  • the amount of protein bound to a feature ofthe array may be determined relative to the amount of a second protein bound to a second feature ofthe array. In certain embodiments, the amount ofthe second protein in the sample is already known or known to be invariant.
  • a protein sample of a first cell or population of cells is delivered to the array under conditions suitable for protein binding.
  • a protein sample of a second cell or population of cells to a second array is delivered to a second array which is identical to the first array. Both arrays are then washed to remove unbound or non-specifically bound components ofthe sample from the arrays.
  • the amounts of protein remaining bound to the features ofthe first array are compared to the amounts of protein remaining bound to the corresponding features ofthe second array.
  • the amount of protein bound to individual features ofthe first array is subtracted from the amount of protein bound to the corresponding features ofthe second array.
  • fluorescence labeling can be used for detecting protein bound to the array.
  • capture arrays e.g. antibody arrays
  • fluorescently labeled proteins from two different cell states, in which cell lysates are labeled with different fluorophores (e.g., Cy-3 and Cy-5) and mixed, such that the color acts as a readout for changes in target abundance.
  • Fluorescent readout sensitivity can be amplified 10-100 fold by tyramide signal amplification (TSA) (e.g., available from PerkinElmer Lifesciences).
  • TSA tyramide signal amplification
  • Planar waveguide technology e.g., available from Zeptosens
  • High sensitivity can also be achieved with suspension beads and particles, using phycoerythrin as label (e.g., available from Luminex) or the properties of semiconductor nanocrystals (e.g., available from Quantum Dot). Fluorescence resonance energy transfer has been adapted to detect binding of unlabelled ligands, which may be useful on arrays (e.g., available from Affibody).
  • label e.g., available from Luminex
  • semiconductor nanocrystals e.g., available from Quantum Dot
  • Fluorescence resonance energy transfer has been adapted to detect binding of unlabelled ligands, which may be useful on arrays (e.g., available from Affibody).
  • the techniques used for detection of EPM marker expression products will include internal or external standards to permit quantitative or semi- quantitative determination of those products, to thereby enable a valid comparison ofthe level or functional activity of these expression products in a biological sample with the corresponding expression products in a reference sample or samples.
  • standards can be determined by the skilled practitioner using standard protocols.
  • absolute values for the level or functional activity of individual expression products are determined.
  • the diagnostic method is implemented using a system as disclosed, for example, in International Publication No. WO 02/090579 and in copending PCT Application No. PCT/AU03/01517 filed November 14, 2003, comprising at least one end station coupled to a base station.
  • the base station is typically coupled to one or more databases comprising predetermined data from a number of individuals representing the level or functional activity of EPM marker expression products, together with indications ofthe actual status ofthe individuals (e.g., presence, absence, degree, or stage of development of EPM) when the predetermined data was collected.
  • the base station is adapted to receive from the end station, typically via a communications network, subject data representing a measured or normalized level or functional activity of at least one expression product in a biological sample obtained from a test subject and to compare the subject data to the predetermined data stored in the database(s). Comparing the subject and predetermined data allows the base station to determine the status ofthe subject in accordance with the results ofthe comparison.
  • kits All the essential materials and reagents required for detecting and quantifying EPM maker gene expression products may be assembled together in a kit.
  • the kits may also optionally include appropriate reagents for detection of labels, positive and negative controls, washing solutions, blotting membranes, microtiter plates dilution buffers and the like.
  • a nucleic acid-based detection kit may include (i) an EPM marker polynucleotide (which may be used as a positive control), (ii) a primer or probe that specifically hybridizes to an EPM marker polynucleotide. Also included may be enzymes suitable for amplifying nucleic acids including various polymerases (Reverse Transcriptase, Taq, SequenaseTM DNA ligase etc. depending on the nucleic acid amplification technique employed), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification. Such kits also generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each primer or probe.
  • a protein-based detection kit may include (i) an EPM marker polypeptide (which may be used as a positive control), (ii) an antigen-binding molecule that is immuno-interactive with an EPM marker polynucleotide.
  • the kit can also feature various devices and reagents for performing one ofthe assays described herein; and/or printed instructions for using the kit to quantify the expression of an EPM marker gene.
  • the present invention also extends to the treatment or prevention of EPM in subjects following positive diagnosis for the presence, or stage of development of EPM in the subjects.
  • the treatment will include administering to a positively diagnosed subject an effective amount of an agent, typically an antiprotozoal agent, that ameliorates the symptoms or reverses the development of EPM or that reduces or abrogates a pathogenic infection underlying EPM or that reduces potential ofthe animal to developing EPM or that inhibits the growth of an organism belonging to a protozoal organism (e.g., from the family Sarcocystidae, especially Sarcocystis neurona).
  • an agent typically an antiprotozoal agent
  • EPM EPM
  • DHFR diaminopyrimidine dihydrofolate reductase
  • sulphonamides 2, 4-diaminopyrimidine (pyrimethamine) and analogues of paraaminobenzoic acid or combinations thereof as disclosed, for example, in U.S. Pat. Nos.
  • anti-coccidial agents illustrative examples of which include triazine-based anticoccidials such as diclazuril, toltrazuril, ponazuril and sulphonotoltrazuril or combinations thereof as disclosed, for example, in U.S. Pat. No.
  • the present invention encompasses any agent that is useful for treating or preventing EPM and is not limited to the aforementioned illustrative compounds and formulations.
  • the agents will be administered in pharmaceutical (or veterinary) compositions together with a pharmaceutically acceptable carrier and in an effective amount to achieve their intended purpose.
  • the dose of active compounds administered to a subject should be sufficient to achieve a beneficial response in the subject over time such as a reduction in, or relief from, the symptoms of EPM.
  • the quantity ofthe pharmaceutically active compounds(s) to be administered may depend on the subject to be treated inclusive ofthe age, sex, weight and general health condition thereof. In this regard, precise amounts ofthe active compound(s) for administration will depend on the judgement ofthe practitioner.
  • the veterinarian may evaluate severity of any symptom associated with the presence of EPM including asymmetric neurological symptoms, lameness, muscle wasting, stumbling, incoordination, head tilt, inability to walk backwards or in a tight circle, nerve paralysis, soreness, sudden recumbency or sleep, seizures, spasticity, hypermefria, ataxia, paralysis and recumbency.
  • asymmetric neurological symptoms including asymmetric neurological symptoms, lameness, muscle wasting, stumbling, incoordination, head tilt, inability to walk backwards or in a tight circle, nerve paralysis, soreness, sudden recumbency or sleep, seizures, spasticity, hypermefria, ataxia, paralysis and recumbency.
  • the antiprotozoal agents may by administered in concert with adjunctive therapies to reduce inflammation ofthe nervous tissue in the affected subject.
  • adjunctive therapies include non steroidal-anti inflammatory drugs (NSAIDs) and dimethylsulfoxides (DMSO).
  • NSAIDs non steroidal-anti inflammatory drugs
  • DMSO dimethylsulfoxides
  • folic acid supplementation may also be administered to affected subjects to prevent haematological and fetal development side effects.
  • EXAMPLE 1 SPECIFIC DIAGNOSTIC GENES FOR EPM [0227] Animals were exposed to large numbers of S. neurona spores. A proportion of those animals developed clinical signs of EPM (including presence of antibodies to S. neurona in cerebro spinal fluid, and characteristic lesions in the central nervous system (revealed at autopsy)). Blood samples obtained from exposed animals were analyzed using GeneChipsTM (method of use is described below in detail in "Generation of Gene Expression Data") containing thousands of genes expressed in white blood cells of horses.
  • an assay that measures the RNA level in the sample from the expression of at least one and desirably at least two EPM diagnostic marker genes representative transcript sequences of which are set forth in SEQ ID NO: 1, 3, 5, 7, 8, 10, 12, 14, 16, 17, 18, 20, 22, 24, 26, 27, 28, 30, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 54, 55, 56, 57 and 422.
  • This provides a level of specificity and sensitivity both equal to 94%.
  • any combination of at least these two polynucleotides with any ofthe other 31 EPM diagnostic marker polynucleotides listed in Table 1 provides strong diagnostic capacity.
  • Blood is collected from a horse (in a non-agitated state) for the purpose of extraction of high quality RNA or protein.
  • Suitable blood collection tubes for the collection, preservation, transport and isolation of RNA include PAXgeneTM tubes (PreAnalytix Inc., Valencia, CA, USA).
  • blood can be collected into tubes containing solutions designed for the preservation of nucleic acids (available from Roche, Ambion, Invitrogen and ABI).
  • nucleic acids available from Roche, Ambion, Invitrogen and ABI.
  • 50 mL of blood is prevented from clotting by collection into a tube containing 4 mL of 4% sodium citrate.
  • White blood cells and plasma are isolated and stored frozen for later analysis and detection of specific proteins.
  • PAXgene tubes can be kept at room temperature prior to RNA extraction.
  • Total RNA Extraction A kit available from Qiagen Inc (Valencia, CA, USA) has the reagents and instructions for the isolation of total RNA from 2.5 mL blood collected in the PAXgene Blood RNA Tube. Isolation begins with a centrifugation step to pellet nucleic acids in the PAXgene blood RNA tube. The pellet is washed and resuspended and incubated in optimized buffers together with Proteinase K to bring about protein digestion. An additional centrifugation is carried out to remove residual cell debris and the supernatant is transferred to a fresh microcentrifuge tube.
  • RNA is selectively bound to the silica-gel membrane as contaminants pass through. Remaining contaminants are removed in three efficient wash steps and RNA is then eluted in Buffer BR5.
  • RNA levels in a tissue sample can be achieved using a variety of technologies. Two common and readily available technologies that are well known in the art are: [0232] • GeneChip® analysis using Affymetrix technology. [0233] • Real-Time Polymerase Chain Reaction (TaqManTM from Applied Biosystems for example). [0234] GeneChips® quantitate RNA by detection of labeled cRNA hybridized to short oligonucleotides built on a silicon substrate. Details on the technology and methodology can be found at www.affymetrix.com.
  • RT-PCR Real-Time Polymerase Chain Reaction quantitates RNA using two PCR primers, a labeled probe and a thermostable DNA polymerase. As PCR product is generated a dye is released into solution and detected. Internal controls such as 18S RNA probes are often used to determine starting levels of total RNA in the sample. Each gene and the internal confrol are run separately. Details on the technology and methods can be found at www.appliedbiosytems.com or www.qiagen.com or www.biorad..com. Applied Biosystems offer a service whereby the customer provides DNA sequence information and payment and is supplied in return all ofthe reagents required to perform RT-PCR analysis on individual genes.
  • GeneChip® analysis has the advantage of being able to analyze thousands of genes at a time. However it is expensive and takes over 3 days to perform a single assay. RT- PCR generally only analyses one gene at a time, but is inexpensive and can be completed within a single day. [0237] RT-PCR is the method of choice for gene expression analysis if the number of specific genes to be analyzed is less than 20. GeneChip® or other gene expression analysis technologies (such as Hlumina Bead Arrays) are the method of choice when many genes need to be analyzed simultaneously. [0238] The methodology for GeneChip® data generation and analysis and Real Time PCR is presented below in brief.
  • RNA quantity is determined on an Agilent "Lab-on-a-Chip" system (Agilent Technologies). Hybridization, Washing & Staining [0246] The steps are: [0247] • A hybridization cocktail is prepared containing 0.05 ⁇ g/ ⁇ L of labeled and fragmented cRNA, spike-in positive hybridization controls, and the Affymetrix oligonucleotides B2, bioB, bioC, bioD and ere. [0248] • The final volume (80 ⁇ L) ofthe hybridization cocktail is added to the GeneChip® cartridge. [0249] • The cartridge is placed in a hybridization oven at constant rotation for 16 hours.
  • GeneChip® called a .DAT file (see figure overleaf).
  • the .DAT file is then pre-processed prior to any statistical analysis.
  • Data pre-processing steps include: [0259] • . DAT File Quality Control (QC). [0260] • .CEL File Generation. [0261] • Scaling and Normalization.
  • the .DAT file is an image.
  • the image is inspected manually for artifacts (e.g. high low intensity spots, scratches, high regional or overall background).
  • artifacts e.g. high low intensity spots, scratches, high regional or overall background.
  • the B2 oligonucleotide hybridization performance is easily identified by an alternating pattern of intensities creating a border and array name.
  • the MAS 5 software used the B2 oligonucleotide border to align a grid over the image so that each square of oligonucleotides was centered and identified.
  • the other spiked hybridization controls bioB, bioC, bioD and ere
  • the .DAT file is of suitable quality it is converted to an intensity data file (.CEL file) by Affymetrix MAS 5 software).
  • the .CEL files generated by the MAS 5 software from .DAT files contain calculated raw intensities for the probe sets. Gene expression data is obtained by subfracting a calculated background from each cell value. To eliminate negative intensity values, a noise correction fraction based from a local noise value from the standard deviation ofthe lowest 2% ofthe background is applied. [0265] All .CEL files generated from the GeneChips® are subjected to specific quality mefrics parameters. [0266] Some mefrics are routinely recommended by Affymefrix and can be determined from Affymefrix internal confrols provided as part ofthe GeneChip®. Other mefrics are based on experience and the processing of many GeneChips®.
  • Affymetrix MAS 5 Algorithm [0271] .CEL files are used by Affymefrix MAS 5 software to normalize or scale the data. Scaled data from one chip are compared to similarly scaled data from other chips. [0272] Affymefrix MAS 5 normalization is achieved by applying the default "Global Scaling" option ofthe MAS 5 algorithm to the .CEL files. This procedure subtracts a robust estimate ofthe center ofthe distribution of probe values, and divides by a robust estimate ofthe probe variability. This produces a set of chips with common location and scale at the probe level. [0273] Gene expression indices are generated by a robust averaging procedure on all the probe pairs for a given gene. The results are constrained to be non-negative.
  • RMA Algorithm This algorithm quantifies the expression of a set of chips, rather than of a single chip. It estimates background intensities using a robust statistical model applied to perfect match probe data. It does not make use of mis-match probe data. Following implicit background correction, chips are processed using Quantile Quantile normalization (Rizarray et al, 2002, Biostatistics (in print)).
  • a kit available from Qiagen Inc has the reagents and instructions for the isolation of total DNA from 8.5 mL blood collected in the PAXgene Blood DNA Tube. Isolation begins with the addition of additional lysis solution followed by a centrifugation step. The pellet is washed and resuspended and incubated in optimized buffers together with Proteinase K to bring about protein digestion. DNA is precipitated using alcohol and an additional centrifugation is carried out to pellet the nucleic acid. Remaining contaminants are removed in a wash step and the DNA is then resuspended in Buffer BG4. [0278] Determination of DNA quantity and quality is necessary prior to proceeding and can be achieved using a specfrophotometer or agarose gel electrophoresis.
  • Genotvping Analysis [0279] Many methods are available to genotype DNA. A review of allelic discrimination methods can be found in Kristensen et al. (Biotechniques 30(2): 318-322 (2001). An illustrative method for genotyping using allele-specific PCR is described here.
  • Upstream and downstream PCR primers specific for particular alleles can be designed using freely available computer programs, such as Primer3 (http://frodo.wi.mit.edu/primer3/primer3 code.htm .
  • Primer3 http://frodo.wi.mit.edu/primer3/primer3 code.htm .
  • the DNA sequences ofthe various alleles can be aligned using a program such as ClustalW
  • PCR amplicon is designed to have a restriction enzyme site in one allele but not the other. Primers are generally 18-25 base pairs in length with similar melting temperatures.
  • PCR Applications of PCR, Dennis Lo (Editor), Blackwell Publishing, 1998). Briefly, a reaction contains primers, DNA, buffers and a thermostable polymerase enzyme. The reaction is cycled (up to 50 times) through temperature steps of denaturation, hybridization and DNA extension on a thermocycler such as the MJ Research Thermocycler model PTC-96V. DNA Analysis [0282] PCR products can be analyzed using a variety of methods including size differentiation using mass spectrometry, capillary gel electrophoresis and agarose gel electrophoresis.
  • the DNA in the PCR reaction is purified using DNA-binding columns or precipitation and re-suspended in water, and then restricted using the appropriate restriction enzyme.
  • the restricted DNA can then be run on an agarose gel where DNA is separated by size using electric current.
  • Various alleles of a gene will have different sizes depending on whether they contain restriction sites. Thus, homozygotes and heterozygotes can be determined.
  • Real-Time PCR Data Generation Background information for conducting Real-time PCR may be obtained, for example, at http://dorakmt.tripod.com genetics/realtime.html and in a review by Bustin SA (2000, J Mol Endocrinol 25:169-193).
  • TaqMan TM Primer and Probe Design Guidelines [0284] 1. The Primer ExpressTM (ABI) software designs primers with a melting temperature (Tm) of 58-60° C, and probes with a Tm value of 10° C higher. The Tm of both primers should be equal. [0285] 2. Primers should be 15-30 bases in length. [0286] 3. The G+C content should ideally be 30-80%. If a higher G+C content is unavoidable, the use of high annealing and melting temperatures, cosolvents such as glycerol, DMSO, or 7-deaza-dGTP may be necessary. [0287] 4. The run of an identical nucleotide should be avoided.
  • the probes should not have runs of identical nucleotides (especially four or more consecutive Gs), G+C content should be 30-80%, there should be more Cs than Gs, and not a G at the 5' end. The higher number of Cs produces a higher ⁇ Rn. The choice of probe should be made first.
  • TaqManTM Primers and Probes [0300] The TaqManTM probes ordered from ABI at midi-scale arrive already resuspended at 100 ⁇ M. If a 1/20 dilution is made, this gives a 5 ⁇ M solution. This stock solution should be aliquoted, frozen and kept in the dark. Using 1 ⁇ L of this in a 50 ⁇ L reaction gives the recommended 100 nM final concentration. [0301] The primers arrive lyophilized with the amount given on the tube in pmols
  • PDAR primers and probes are supplied as a mix in one tube. They have to be used 2.5 ⁇ L in a 50 ⁇ L reaction volume.
  • One-step real-time PCR uses RNA (as opposed to cDNA) as a template. This is the preferred method if the RNA solution has a low concentration but only if singleplex reactions are run. The disadvantage is that RNA carryover prevention enzyme AmpErase cannot be used in one-step reaction format. In this method, both reverse franscriptase and real-time PCR take place in the same tube. The downstream PCR primer also acts as the primer for reverse franscriptase (random hexamers or oligo-dT cannot be used for reverse transcription in one-step RT-PCR).
  • One-step reaction requires higher dNTP concentration (greater than or equal to 300 mM vs 200 mM) as it combines two reactions needing dNTPs in one.
  • a typical reaction mix for one-step PCR by Gold RT-PCR kit is as follows: * If a PDAR is used, 2.5 ⁇ L of primer + probe mix used. [0304] Ideally 10 pg - 100 ng RNA should be used in this reaction. Note that decreasing the amount of template from 100 ng to 50 ng will increase the C ⁇ value by 1. To decrease a C ⁇ value by 3, the initial amount of template should be increased 8-fold. ABI claims that 2 picograms of RNA can be detected by this system and the maximum amount of RNA that can be used is 1 microgram. For routine analysis, 10 pg - 100 ng RNA and 100 pg - 1 ⁇ g genomic DNA can be used.
  • PCR Reverse transcription (by MuLV) 48° C for 30 min.
  • PCR denaturation 95° C for 15 sec and annealing/extension 60° C for 1 min (repeated 40 times) (On ABI 7700, minimum holding time is 15 seconds.)
  • the recently introduced EZ one-stepTM RT-PCR kit allows the use of UNG as the incubation time for reverse transcription is 60° C thanks to the use of a thermostable reverse transcriptase. This temperature also a better option to avoid primer dimers and nonspecific bindings at 48° C.
  • Relative standard Known amounts ofthe target nucleic acid are included in the assay design in each run, [0324] 3.
  • Comparative C ⁇ method This method uses no l ⁇ iown amount of standard but compares the relative amount ofthe target sequence to any ofthe reference values chosen and the result is given as relative to the reference value (such as the expression level of resting lymphocytes or a standard cell line).
  • the standard curve method should be used for quantitation of gene expression.
  • the dynamic range should be determined for both (1) minimum and maximum concentrations ofthe targets for which the results are accurate and (2) minimum and maximum ratios of two gene quantities for which the results are accurate.
  • the dynamic range is limited to a target-to-competitor ratio of about 10: 1 to 1 : 10 (the best accuracy is obtained for 1 : 1 ratio).
  • the real-time PCR is able to achieve a much wider dynamic range.
  • the advantage of using the comparative C ⁇ method is that the need for a standard curve is eliminated (more wells are available for samples). It also eliminates the adverse effect of any dilution errors made in creating the standard curve samples. [0327] As long as the target and normalizer have similar dynamic ranges, the comparative C ⁇ method ( ⁇ C T method) is the most practical method. It is expected that the normalizer will have a higher expression level than the target (thus, a smaller C ⁇ value).
  • the optimal concentrations ofthe reagents are as follows: [0337] i. Magnesium chloride concentration should be between 4 and 7 mM. It is optimized as 5.5 mM for the primers/probes designed using the Primer Express software. [0338] ii. Concentrations of dNTPs should be balanced with the exception of dUTP (if used). Substitution of dUTP for dTTP for control of PCR product carryover requires twice dUTP that of other dNTPs.
  • dNTPs While the optimal range for dNTPs is 500 ⁇ M to 1 mM (for one-step RT-PCR), for a typical TaqMan reaction (PCR only), 200 ⁇ M of each dNTP (400 ⁇ M ofdUTP) is used. [0339] iii. Typically 0.25 ⁇ L (1.25 U) AmpliTaq DNA Polymerase (5.0 U/ ⁇ L) is added into each 50 ⁇ L reaction. This is the minimum requirement. If necessary, optimization can be done by increasing this amount by 0.25 U increments. [0340] iv. The optimal probe concentration is 50-200 nM, and the primer concenfration is 100-900 nM.
  • each primer pair should be optimized at three different temperatures (58, 60 and 62° C for TaqMan primers) and at each combination of three concentrations (50, 300, 900 nM). This means setting up three different sets (for three temperatures) with nine reactions in each (50/50 mM, 50/300 mM, 50/900, 300/50, 300/300, 300/900, 900/50, 900/300, 900/900 mM) using a fixed amount of target template. If necessary, a second round of optimization may improve the results. Optimal performance is achieved by selecting the primer concentrations that provide the lowest C ⁇ and highest ⁇ Rn. Similarly, the probe concentration should be optimized for 25-225 nM. [0341] 4.
  • AmpliTaq Gold DNA Polymerase there has to be a 9-12 min pre-PCR heat step at 92 - 95° C to activate it. If AmpliTaq Gold DNA Polymerase is used, there is no need to set up the reaction on ice.
  • a typical TaqMan reaction consists of 2 min at 50° C for UNG (see below) incubation, 10 min at 95° C for Polymerase activation, and 40 cycles of 15 sec at 95° C (denaturation) and 1 min at 60° C (annealing and extension).
  • a typical reverse transcription cycle (for cDNA synthesis), which should precede the TaqMan reaction if the starting material is total RNA, consists of 10 min at 25° C (primer incubation), 30 min at 48° C (reverse transcription with conventional reverse transcriptase) and 5 min at 95° C (reverse transcriptase inactivation). [0342] 5. AmpErase uracil-N-glycosylase (UNG) is added in the reaction to prevent the reamplification of carry-over PCR products by removing any uracil incorporated into amplicons. This is why dUTP is used rather than dTTP in PCR reaction. UNG does not function above 55 °C and does not cut single-stranded DNA with terminal dU nucleotides.
  • UNG- containing master mix should not be used with one-step RT-PCR unless xTth DNA polymerase is being used for reverse transcription and PCR (TaqMan EZ RT-PCR kit).
  • NAC No Amplification Controls
  • NTC No Template Controls
  • the dynamic range of a primer/probe system and its normalizer should be examined if the ⁇ C T method is going to be used for relative quantitation. This is done by running (in triplicate) reactions of five RNA concentrations (for example, 0, 80 pg/ ⁇ L, 400 pg/ ⁇ L, 2 ng/ ⁇ L and 50 ng/ ⁇ L).
  • the resulting plot of log ofthe initial amount vs C ⁇ values should be a (near) straight line for both the target and normalizer real-time RT- PCRs for the same range of total RNA concentrations.
  • the passive reference is a dye (ROX) included in the reaction (present in the TaqMan universal PCR master mix). It does not participate in the 5' nuclease reaction. It provides an internal reference for background fluorescence emission. This is used to normalize the reporter-dye signal. This normalization is for non-PCR-related fluorescence fluctuations occurring well-to-well (concentration or volume differences) or over time and different from the normalization for the amount of cDNA or efficiency of the PCR.
  • ROX dye
  • ABI 7700 can be used not only for quantitative RT-PCR but also end- point PCR. The latter includes presence/absence assays or allelic discrimination assays (such as SNP typing).
  • Shifting Rn values during the early cycles (cycle 0-5) of PCR means initial disequilibrium ofthe reaction components and does not affect the final results as long as the lower value of baseline range is reset.
  • the ABI 7700 should not be deactivated for extended periods of time. If it has ever been shutdown, it should be allowed to warm up for at least one hour before a run. Leaving the instrument on all times is recommended and is beneficial for the laser. If the machine has been switched on just before a run, an error box stating a firmware version conflict may appear. If this happens, choose the "Auto Download" option. [0362] 25.
  • the ABI 7700 is only one ofthe real-time PCR systems available, others include systems from BioRad, Cepheid, Corbett Research, Roche and Stratagene.
  • EXAMPLE 2 IDENTIFICATION OF DIAGNOSTIC MARKER GENES [0363] Differences in gene expression between animals with and without clinical evidence of EPM were analyzed using the empirical Bayes approach of Lonnstedt and Speed (Lonnstedt and Speed, 2002, Statistica Sinica 12: 31-46). A general linear model was fitted to each gene, with a term for clinical status (with or without clinical evidence of EPM). Genes were ranked according to their posterior odds of differential expression between clinical status groups. Only those genes with statistically significant changes (assessed using the t statistic based on the empirical Bayes shrunken standard deviations) were recorded.
  • ROC curve provides a useful summary of the diagnostic potential of an assay.
  • a perfect diagnostic assay has a ROC curve which is a horizontal line passing through the point with sensitivity and specificity both equal to one. The area under the ROC curve for such a perfect diagnostic is 1.
  • a useless diagnostic assay has a ROC curve which is given by a 45 degree line through the origin. The area for such an uninformative diagnostic is 0.5.
  • Sensitivity, and selectivity and the areas under the ROC curve are shown in Table 5, for samples taken 2, 4, 7, 9, 11, 14, 17, 21, 24 and 28 days after infection.
  • the EPM marker genes have little diagnostic ability at 2 and 4 days post post-infection, it is clearly apparent that they have some diagnostic ability from day 9 onwards. Clinical signs first appeared in the experimental animals in the study on Day 9 and persisted through to Day 28. Most importantly, there is a correlation between the area under the ROC curve and neurological clinical signs.
  • One ofthe principal objectives ofthe present invention is to disclose the method for a practical diagnostic test for EPM that distinguishes between exposure to the causative organism and active disease or aberrant host response. Active disease, or an aberrant host response to parasitic infection, is required for clinical signs to be evident in EPM. These results demonstrate that specific and sensitive changes can be measured in active disease in animals suffering from pathogenic protozoal infection.
  • Receiver Operator curves calculated in this way based on shrinkage estimates over the entire set of genes on the chip are conservative - that is, they tend to underestimate the diagnostic potential. Better diagnostic performance should be obtained in operational diagnostics, based on a selected subset ofthe genes.
  • Table 6 shows the sensitivity, selectivity and area under the ROC curve for an analysis based on 31 genes, selected because of their large differential expression following infection.
  • the sensitivities, specificities and ROC areas are greater than those for the shrinkage based analysis.
  • the ROC curves for the analysis based on 31 genes for days 2, 4, 7, 9, 11, 14, 17, 21, 24 and 28 are shown in Figures 1-10, respectively. The diagnostic capability is very high.
  • Table 9 shows the cross-validated classification success obtained from a linear discriminant analysis based on three genes selected from the diagnostic set. Only twenty sets of three genes are presented. It will be readily apparent to those of skill in the art that other suitable diagnostic selections based on three EPM marker genes can be made.
  • Table 10 shows the cross-validated classification success obtained from a linear discriminant analysis based on four genes selected from the diagnostic set. Only twenty sets of four genes are presented. It will be readily apparent to practitioners in the art that other suitable diagnostic selections based on four EPM marker genes can be made.
  • Table 11 shows the cross-validated classification success obtained from a linear discriminant analysis based on five genes selected from the diagnostic set. Only twenty sets of five genes are presented. It will be readily apparent to practitioners in the art that other suitable diagnostic selections based on five EPM marker genes can be made.
  • Table 12 shows the cross-validated classification success obtained from a linear discriminant analysis based on six genes selected from the diagnostic set. Only twenty sets of six genes are presented. It will be readily apparent to practitioners in the art that other suitable diagnostic selections based on six EPM marker genes can be made.
  • Table 13 shows the cross-validated classification success obtained from a linear discriminant analysis based on seven genes selected from the diagnostic set. Only twenty sets of seven genes are presented.
  • Table 14 shows the cross-validated classification success obtained from a linear discriminant analysis based on eight genes selected from the diagnostic set. Only twenty sets of eight genes are presented. It will be readily apparent to practitioners in the art that other suitable diagnostic selections based on eight EPM marker genes can be made.
  • Table 15 shows the cross-validated classification success obtained from a linear discriminant analysis based on nine genes selected from the diagnostic set. Only twenty sets of nine genes are presented. It will be readily apparent to practitioners in the art that other suitable diagnostic selections based on nine EPM marker genes can be made.
  • Table 16 shows the cross-validated classification success obtained from a linear discriminant analysis based on ten genes selected from the diagnostic set. Only twenty sets often genes are presented. It will be readily apparent to practitioners in the art that other suitable diagnostic selections based on ten EPM marker genes can be made.
  • Table 17 shows the cross-validated classification success obtained from a linear discriminant analysis based on 20 genes selected from the diagnostic set. Only 20 sets of twenty genes are presented. It will be readily apparent to practitioners in the art that other suitable diagnostic selections based on twenty EPM marker genes can be made.
  • the 30 EPM diagnostic genes were used as a training set against a gene expression database of over 850 GeneChips®. Gene expression results in the database were obtained from samples from horses with various diseases and conditions including; induced EPM in the acute stage of disease, induced EPM in the chronic stage of disease, clinical cases of EPM, herpes virus infection, degenerative osteoarthritis, stress, Rhodococcus infection, endotoxaemia, laminitis, gastric ulcer syndrome, animals in athletic training and clinically normal animals. [0389] An EPM index score was calculated for each GeneChip®, using the 30 genes in the training set.
  • the score was calculated from a regularized discriminant function, so that large values would be associated with high probability of EPM, and the variance ofthe score should be approximately 1.
  • GeneChips® were ranked on this score, from the largest to the smallest.
  • Specificity was investigated by varying a threshold value for a positive diagnosis. At each value ofthe threshold, specificity was defined as the proportion of positive results (i.e. GeneChip® index score greater than the threshold) which were true positives. A threshold value of two (i.e. two standard deviations) was adopted.
  • Only nine animals from the database had conditions that were not related to acute, chronic or clinical EPM and were two standard deviations above zero on discriminant function. A further 120 animals with acute EPM were two standard deviations above zero on discriminant function. No animals with chronic or clinical EPM were two standard deviations above zero on discriminant function. Using this method and a gene signature of 30 genes, a specificity of 93% for acute EPM was obtained from a population sample size of over 850.
  • BM735363 (592) Galectin 3 binding protein / 1 CGCTTCGTGGCCCACGTCGCTGATTTCAAGGGCTCGAAGGCCGTGATCCCCAGCGCCCTG SEQ ID NO: 7
  • Membrane 61 GGCACCAACAGTTCCAGGAGCGCCTCTCTCTTTCCCTGCCAGGCAGGGTCCTTCAGTGGC extracellular 121 TTCCAGGTGGTCATCCGCCTTCTACCTGACCAACCCCTCGGCGGAGGACTAGACGGGA space and 181 GGCTGGGTGAGCCGAGGGGGCGAGGGACAGGAGCACAGAGAAGCGAGGCGCCTCCCAGGA membrane, signal 241 TGCCCCCCGCCCCCAGCTGAGCCTCTCGCATCCTTCCTTCCTCTGCATGCACCTCCAGCA transduction, 301 GCTGCCACCAGATGTCCCCCCTGCTTCCACTGAGTGCTCTGAGCTTGGAGAAATTACTGG cellular defence 361 AAGGTTTCACCTAGTGCTCACCAGGGTGGTGAGAATTCCTGT

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Abstract

La présente invention a trait à des molécules et des dosages associés à la maladie, utiles pour le diagnostic de l'encéphalomyélite équine à protozoaire (EPM). L'invention présente une utilité pratique dans le diagnostic précoce de maladie, dans le suivi de la réponse immunitaire d'un animal à la maladie, et permet la prise de meilleures décisions de traitement et de contrôle pour des animaux cliniquement et sous-cliniquement affectés.
PCT/AU2005/000401 2004-03-19 2005-03-21 Marqueurs hotes pour le diagnostic de l'encephalomyelite equine a protozoaire et l'infection de sarcocystis neurona WO2005090593A1 (fr)

Applications Claiming Priority (6)

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AU2004901448A AU2004901448A0 (en) 2004-03-19 Screening and/or diagnostic agents and uses therefor
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US58184004P 2004-06-23 2004-06-23
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109068998A (zh) * 2016-04-25 2018-12-21 外分泌腺系统公司 用于检测汗液分析物的eab生物传感器

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BLADER ET AL: "Microarray Analysis Reveals Previously Unknown Changes in Toxoplasma gondii-infected Human Cells.", THE JOURNAL OF BIOLOGICAL CHEMISTRY., vol. 276, no. 26, 2001, pages 24223 - 24231 *
CHAUSSABEL ET AL: "Unique gene expression profiles of human macrophages and dendritic cells to phylogenetically distinct parasites.", BLOOD., vol. 15, no. 2, 2003, pages 672 - 681 *
DUBEY ET AL: "A review of Sarcocystis neurona and equine protozoal myeloencephalitis (EPM).", VETERINARY PARASITOLOGY., vol. 95, 2001, pages 89 - 131 *
FURR ET AL: "Clinical Diagnosis of equine Protozoal Myeloencephalitis (EPM).", J.VET INTERN MED., vol. 16, 2002, pages 618 - 621 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109068998A (zh) * 2016-04-25 2018-12-21 外分泌腺系统公司 用于检测汗液分析物的eab生物传感器

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