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WO1994021662A1 - Diagnosis of human inflammatory bowel diseases and nucleic acid reagents therefor - Google Patents

Diagnosis of human inflammatory bowel diseases and nucleic acid reagents therefor Download PDF

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Publication number
WO1994021662A1
WO1994021662A1 PCT/US1994/002806 US9402806W WO9421662A1 WO 1994021662 A1 WO1994021662 A1 WO 1994021662A1 US 9402806 W US9402806 W US 9402806W WO 9421662 A1 WO9421662 A1 WO 9421662A1
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Prior art keywords
nucleic acid
inflammatory bowel
human
disease
small rna
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PCT/US1994/002806
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French (fr)
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Sidney Altman
Peter U.H. Lundberg
Cecilia Guerrier-Takada
Shaji T. George
Hugh D. Robertson
Alan R. Goldberg
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Yale University
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Priority to AU64090/94A priority Critical patent/AU6409094A/en
Publication of WO1994021662A1 publication Critical patent/WO1994021662A1/en

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    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • 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
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • INTRODUCTION Detection methods are presented which may be used for the diagnosis of the human inflammatory bowel diseases, Crohn's disease and ulcerative colitis.
  • nucleic acids and nucleic acid detection methods are presented herein which may be utilized to detect small RNA molecules found in ulcerative colitis- and/or Crohn's-diseased tissues, but which are absent from, or are present in greatly reduced levels in, the corresponding non-diseased tissues.
  • Second, reagents and schemes are presented whereby peptides encoded by such RNA molecules may be identified.
  • diagnoses may be made very early in the course of the conditions, using tissue which does not yet even manifest visual aspects of disease.
  • the diagnostic procedures presented here therefore, represent a great improvement over the current diagnostic techniques which rely on imprecise visual observations and which may only be utilized once disease has become overtly established.
  • Ulcerative colitis is primarily an inflammatory disease which affects the superficial epithelial layer of the rectum and distal colon, although it may also extend to involve the entire colonic mucosa. The cause of the disease is unknown and exacerbations and remissions are common. Symptoms of ulcerative colitis usually develop in individuals between 25 and 45 years of age. Although the illness occurs less frequently in the elderly, attacks in patients older than 60 years of age may be severe, and mortality rates are very high for this group.
  • ulcerative colitis ulcerative colitis
  • diseases which are confined to the abdomen and perianal region usually indicate a mild or moderate manifestation of the disease, while prominent, generalized symptoms such as fatigue and/or weight loss often indicate a more severe disease manifestation.
  • the majority of patients with ulcerative colitis manifest a mild form of the disease, which is confined to the distal colon and rectum.
  • Such patients have intermittent diarrhea with no extracolonic or systemic manifestations.
  • Physical examinations are usually negative, although a localized tenderness over the distal colon may be detected.
  • ulcerative colitis cases are classified as moderately severe, with patients passing watery or pasty stools containing mucus and gross quantities of blood.
  • Systemic manifestations such as intermittent fatigue and an increased need for sleep, are often noted in these moderately severe cases, and, during exacerbations, low-grade fevers and mild weight loss may also occur.
  • a small percentage of individuals experience severe ulcerative colitis with recurrent exacerbations which may lead to hospitalization.
  • Patients with severe colitis typically exhibit temperatures of 100°F (38°C) or higher, and profuse, constant, bloody stools. If symptoms are allowed to persist, extensive weight loss may occur. In these cases, physical examination may be normal, but a distended abdomen with tympany and absent bowel sounds may indicate a full-blown flare up of the disease.
  • Crohn's disease or granulomatous ileocolitis, is a chronic inflammatory disease of unknown etiology which slowly destroys the alimentary tract.
  • the disease usually involves the terminal ileum and/or colon, but may manifest itself as a widespread illness involving any portion of the hollow gut from mouth to anus.
  • the chronic inflammation of the disease occurs in all layers of the bowel.
  • Crohn's disease is chiefly a disease of younger people. Nearly 60% of all cases begin between ages of ten to twenty-five, with peak incidence of onset occurring in teenagers and the majority of affected individuals being between 20 and 40 years of age. In the United States, the annual incidence is about 2 per 100,000 individuals, the world-wide incidence is approximately 6.1 per 100,000 individuals, and the world-wide prevalence is 60 per 100,000 individuals.
  • ulcerative colitis which often initially presents itself as an acute, severe disease
  • Crohn's disease is an indolent, or so- called smoldering, disease.
  • the disease often becomes chronic before a patient sees a physician, and the average individual has symptoms for approximately five years before the diagnosis of Crohn's disease is made.
  • Sigmoidoscopy may reveal lower colon abnormalities such as ulceration, granularity, and nodule formation, but because Crohn's most often affects only the terminal ileum, cecu , and ascending colon, standard sigmoidoscopic examinations appear normal in 30 to 50 percent of patients.
  • Tissue obtained at surgery often reveals chronic inflammatory involvement of the submucosal layers of the bowel wall, but, even when such surgical specimens reveal submucosal inflammation, these histopathological findings are not specific for Crohn's disease.
  • a biopsy often yields supportive evidence of Crohn's disease, but final differentiation from ulcerative colitis must rely heavily on an individual's history, the clinical course of one's symptoms, and on the pattern of information seen on barium contrast x-ray studies.
  • barium contrast x-ray examination is the single most important procedure for the diagnosis of Crohn's disease because it frequently reveals the characteristic changes associated with the disease, such as a marked distortion of the bowel wall contour or the appearance of intervening areas of normal mucosa between affected regions of tissue.
  • sigmoidoscopic examination may reveal important mucosal abnormalities such as a granularity to the mucosal surface, which indicates scarring and microulceration, and friability, which is the appearance of punctate blood spots along the mucosal surface.
  • Histopathological analysis of tissue biopsies may also aid in diagnosis of the disease, with the most characteristic signs of ulcerative colitis being atrophy of the mucosal glands, and the presence of crypt abscesses (i.e.. polymorphonuclear leukocytes in the crypts of Lieberkuhn) . Although such histopathologies are distinctive, they are not, however, definitively diagnostic of ulcerative colitis. Barium contrast x-ray examination is the most useful means by which to distinguish between the two types of inflammatory bowel diseases. Barium x- ray, for example, reveals a mottled and roughened barium appearance at the bariu -mucosa interface, which is highly characteristic of microulcerations seen with ulcerative colitis.
  • ulcerative colitis Neither the cause of ulcerative colitis nor that of Crohn's disease is known, but several potential explanations have been suggested. It has been suggested that there is a genetic and/or cultural causal component associated with ulcerative colitis (Farmer, R.J., 1989, Scand. J. Gastroenterol. Suppl. 170:64 ; Almy, T.P. et al.. 1966, Gastroenterol. 5_1:757), with a clustering of the disease among certain groups. Ulcerative colitis, for example, is between two and four times more common among Jews than non-Jews, and approximately four times more common among whites than non-whites. Genetic and/or cultural causal components involved in the development of Crohn's disease have also been postulated.
  • Crohn's disease may be caused by infection (Kirsner, J.B. and Shorter, R.G., 1982, N. Engl. J. Med. 306:837-848) . It has further been suggested that the resistance of the disease to medication and the long incubation time of 9 to 27 months in laboratory animals (Cave, D.R. et al. , 1978, Gastroenterol. J5 :632-637) of symptoms reminiscent of Crohn's disease, coupled with the slow, chronic course of the disease may indicate an unconventional causative agent for Crohn's disease.
  • RNA virus Gajdusek, D.C., 1985, in Subviral Pathogens of Plants and Animals: Viroids and Prions, Maramorosch, A. and McKelvey, J.J., eds., pp.
  • WO 91/04324 have suggested that the causative agent of Crohn's disease may have homology to sequences contained within the RNA genome of the hepatitis delta virus, and suggest the possibility that circular RNA species with lengths ranging from 300 to 800 nucleotides may be present in Crohn's disease tissue.
  • the present invention provides small RNA molecules, uniquely identifiable by the sequences disclosed herein, and by their anomalous electrophoretic mobilities, which are approximately 120-250 nucleotides in length and have no homology to hepatitis delta virus RNA. Furthermore, the nucleic acids of the present invention are useful for the diagnosis of not only Crohn's disease but also ulcerative colitis.
  • the present invention relates to the diagnosis of the human inflammatory bowel diseases, Crohn's disease and ulcerative colitis, and to reagents and detection schemes useful for such diagnosis.
  • nucleic acids and nucleic acid detection schemes are presented which may be used to detect the presence of small RNA molecules which are found in ulcerative colitis- and/or Crohn's-diseased tissue, but are absent from, or are present in greatly reduced amounts in, the corresponding non-diseased tissue.
  • antibody reagents and detection schemes are presented which may be utilized to detect the presence of peptides which may be encoded by such small RNA molecules.
  • the reagents presented here may be used to detect disease- specific RNA and peptide molecules, the diagnosis of Crohn's disease or ulcerative colitis can be made rapidly and unequivocally, often very early in the course of disease, using tissue that may not yet even manifest visual aspects of disease.
  • the reagents presented herein make it possible to perform diagnosis on bodily samples in addition to biopsied intestinal tissue, including body fluids, such as blood, lymph or joint fluid, and other bodily excretions.
  • body fluids such as blood, lymph or joint fluid, and other bodily excretions.
  • the nucleic acids of interest which are present in diseased tissue may either be absent from, or present in greatly reduced levels in, the corresponding normal, non-diseased tissue.
  • disease-specific as used herein is meant to refer to either nucleic acids which are completely absent from corresponding non-diseased tissues, or to nucleic acids which are present in corresponding normal, non-diseased tissues, but at greatly reduced levels.
  • non-diseased refers to non-Crohn's and non-ulcerative colitis diseased tissues.
  • non-diseased as used herein may refer to any normal tissue or any non-inflammatory bowel diseased tissue.
  • the method for diagnosing an inflammatory bowel disease comprises: (a) obtaining frozen or fixed sections or nucleic acids from a human tissue, fluid, or stool sample suspected of containing an inflammatory bowel disease-specific small RNA molecule; (b) treating the frozen or fixed tissue sections or nucleic acids of step (a) with at least one nucleic acid reagent under conditions such that cDNA is made from such inflammatory bowel disease- specific small RNA molecule present among such nucleic acids; (c) amplifying a target sequence within the cDNA of step (b) according to a nucleic acid amplification method using at least one nucleic acid reagent complementary to the cDNA; and (d) detecting such amplified cDNA target sequence; wherein the nucleic acid reagent(s) of steps (b) and (c) act(s) as a synthesis initiation reagent(s) and is/are selected from the group consisting of:
  • nucleic acid the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:l depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules;
  • nucleic acid (2) a nucleic acid, the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:2 depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules;
  • nucleic acid the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:3 depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules;
  • nucleic acid the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:4 depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules;
  • nucleic acid a nucleic acid, the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:5 depicted in FIG. 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules;
  • nucleic acid the sequence of which comprises a portion of the nucleotide sequence SEQ ID NO: 1;
  • nucleic acid depicted in Table 1, which nucleic acid is capable of specifically binding the complement of inflammatory bowel-specific small RNA molecules;
  • nucleic acid the sequence of which comprises a portion of the nucleotide sequence SEQ ID NO: 1;
  • nucleic acid depicted in Table 1, which nucleic acid is capable of specifically binding the complement of inflammatory bowel-specific small RNA molecules;
  • nucleic acid a nucleic acid, the sequence of which comprises a portion of the nucleotide sequence SEQ ID NO:3 depicted in Table 1, which nucleic acid is capable of specifically binding the complement of inflammatory bowel-specific small RNA molecules;
  • nucleic acid depicted in Table 1, which nucleic acid is capable of specifically binding the complement of inflammatory bowel-specific small RNA molecules;
  • nucleic acid is capable of specifically binding the complement of inflammatory bowel-specific small RNA molecules.
  • the method for diagnosing an inflammatory bowel disease comprises: (a) obtaining frozen or fixed tissue sections or nucleic acids from a human tissue, fluid, or stool sample suspected of containing an inflammatory bowel disease-specific small RNA molecule; (b) contacting the frozen or fixed tissue sections or nucleic acids of step (a) with a nucleic acid reagent selected from the group consisting of:
  • nucleic acid the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:l depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules;
  • nucleic acid (2) a nucleic acid, the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:2 depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules;
  • nucleic acid the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:3 depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules;
  • nucleic acid the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:4 depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules;
  • nucleic acid the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:5 depicted in FIG. 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules; under conditions favorable for the specific annealing of the nucleic acid reagent to the complementary sequence within the inflammatory bowel disease- specific small RNA molecule; (c) removing all non- annealed nucleic acid reagent from the nucleic acid reagent:inflammatory bowel disease-specific small RNA molecule hybrid; and (d) detecting the nucleic acid reagent specifically annealed to the inflammatory bowel disease-specific small RNA molecule.
  • the method for diagnosing a human inflammatory bowel disease may also comprise:
  • step (b) exposing the sample of step (a) to an antibody directed against an epitope present on the inflammatory bowel disease-specific peptide for a time sufficient for specific binding of the antibody and the peptide to occur so that an antibody:peptide hybrid is formed comprising the antibody and the inflammatory bowel disease-specific peptide;
  • the present invention further contemplates pre ⁇ packaged diagnostic kits, conveniently used, e.g.. in clinical settings, comprising at least one of the specific nucleic acid reagents described herein for use as primers and/or probes in conjunction with he diagnostic methodology described herein.
  • certain of the inflammatory bowel disease-specific small RNA molecules described herein may be produced by an infectious agent responsible for such inflammatory bowel diseases. Therefore, it is an object of this invention to identify and isolate such infectious agents and, further, to develop preventative and ameliorative inflammatory bowel disease treatments, such as vaccines directed against such infectious agents.
  • FIG. 1 Crohn's disease/ulcerative colitis - specific human rRNA diagram.
  • a fragment (SEQ ID NO: 5) of human 28s rRNA is depicted which corresponds to one of the Crohn's disease/ulcerative colitis-specific small RNAs of the invention.
  • the fragment is depicted in its predicted secondary structure form.
  • SEQ. ID NO:3 corresponds to the nucleotide sequence between the black arrows;
  • SEQ. ID NO:4 corresponds to the nucleotide sequence between the white arrows.
  • FIG. 2 Two dimensional polyacrylamide gel analysis of total nucleic acid populations extracted from intestinal tissue of: (A) Crohn's disease patient; (B) ulcerative colitis patient; (C) normal subject; (D) intestinal cancer patient.
  • Polyacrylamide gels were made according to Pechan et al.. 1987, Z. Naturforsch 42c:1006-1008. Acrylamide concentration is 10% in both directions. Origin is always located at the left-hand bottom corner. Nucleic acids were visualized by silver staining (see Section 6.1 for details).
  • the arrows 1 and 2 indicate inflammatory bowel disease-specific RNA. Species useful for orientation of the gel (i.e.. 5s RNA and 7s RNA species) are also indicated.
  • FIG. 5s RNA and 7s RNA species are also indicated.
  • FIG. 4 Northern analysis demonstrating the presence of a small RNA band in Crohn's disease and ulcerative colitis tissue RNA samples which is absent from control (colon cancer) tissue RNA samples.
  • Control RNA Pathogen. #21, 22; Crohn's RNA: Pathogen. #25, S93-5949, S93-6839, S93-10984; ulcerative colitis RNA: Pathogen. #3, 9, Boston #1.
  • nucleic acid reagents and detection procedures are presented which may be utilized to detect small RNA molecules with anomalous electrophoretic mobilities that are found in ulcerative colitis- and/or Crohn's-diseased tissue, but are absent from, or are present in greatly reduced levels in, the corresponding normal, non-diseased tissue.
  • Working Examples are presented describing the identification of these small, RNA molecules having anomalous electrophoretic mobilities, and further presenting a basis for the nucleic acid diagnostic technique.
  • peptide and antibody reagents and detection procedures are presented which may be utilized to detect peptides produced by the small RNA molecules, described above, which are found in ulcerative colitis- and/or Crohn's-diseased tissue.
  • inflammatory bowel disease-specific small RNA molecules described herein may be produced by infectious agents, methods are presented for the isolation and identification of such agents.
  • RNA molecules As demonstrated in the Working Example presented in Section 6, below, small RNA molecules, or partial sequences derived from them, have been identified which are specific to Crohn's or ulcerative colitis- diseased tissue, i.e.. are present in the diseased tissue, but are absent from, or present in greatly reduced levels in, the corresponding normal, non- diseased tissue. "Greatly reduced”, as used herein, refers to an amount of RNA that is too low to be visible when analyzed according to the 2-dimensional gel electrophoresis techniques described, below. While no information yet exists as to whether RNA molecules are causative disease agents or, alternatively, are a consequence of disease, the detection of such molecules may be used for the diagnosis of ulcerative colitis and Crohn's disease.
  • RNA molecules which are uniquely identifiable by the nucleic acid reagents disclosed herein, and by their anomalous electrophoretic mobilities, are approximately 120-250 nucleotides in length.
  • the "anomalous electrophoretic mobilities" of these inflammatory bowel-specific RNA molecules refers to the characteristic of these molecules, when analyzed via two dimensional gel electrophoresis techniques such as those described below, to migrate to a position within the gel separate from the "diagonal" formed by the bulk of the RNA species within any given population of molecules being electrophoresed. Examples of the migration characteristics of two such RNA species (marked by arrows 1 and 2) are depicted in FIGS. 2A-B and 3A, in which, first, the migration distinct from the diagonal is visible, and second, the position to which the molecules migrate may be easily oriented by comparing to the 5s, 7s, and tRNA species, as marked.
  • nucleic acid reagents which may be used in the diagnosis of ulcerative colitis and Crohn's disease include, but are not limited to, all or part of the nucleic acids comprising the sequences (depicted as RNA molecules) shown below in Table 1, and/or their complements, provided such nucleic acid reagents are capable of specifically hybridizing
  • nucleic acid reagents may also include nucleic acids that comprise all or a portion of the nucleic acid comprising the sequence (SEQ ID No. 5) depicted in FIG. 1 which is capable of specially hybridizing to inflammatory bowel disease - specific small RNA molecules or their complements. Additionally, such nucleic acid reagents may also include nucleic acid molecules having the nucleotide sequence of SEQ ID NOs: 1-5, which are capable of specifically binding inflammatory bowel disease- specific small RNA molecules or their complements.
  • the nucleic acid reagents are oligonucleotides about 15 to about 30 nucleotides in length, the sequences of which represent a contiguous stretch of the aforesaid length anywhere along the sequences depicted in Table 1, below.
  • portion will typically refer to portions of the nucleic acid molecules or their complements which are at least about 15-30 nucleotides in length.
  • N refers to any nucleotide
  • Y refers to a pyrimidine
  • A/U' in SEQ ID NO: 2, refers to a nucleotide position which may contain either an A or a U base.
  • nucleic acid reagents of the invention specifically hybridize to inflammatory bowel disease-specific molecules are standard hybridization conditions well known to those of skill in the art. See, for example, Ausubel et al. (Ausubel, M. et al. , ed. , 1987, "Current Protocols in Molecular Biology", Vol. 1-2) and Sambrook et al. (Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press) which are incorporated herein by reference in their entirety.
  • partial nucleotide sequences are known for the inflammatory bowel-specific RNA molecules of the invention
  • techniques which are well known to those of skill in the art exist whereby full length nucleotide sequences may be obtained.
  • the partial sequence may be used to synthesize an oligonucleotide which may be used as a primer for a reverse transcription reaction in order to produce a cDNA copy of the small RNA molecule of interest.
  • the cDNA may then be sequenced using standard DNA sequencing techniques, coupled, if necessary, with well known nucleic acid amplification procedures.
  • primers and the sequences obtained from such primers the entire nucleotide sequence of the RNA molecules of interest may be obtained.
  • Certain of the inflammatory bowel disease-specific small RNA molecules of the invention may represent portions of mRNA transcripts or RNA genomes. Techniques which are well known to those of skill in the art are available with which to use the nucleotide sequence of the small RNA molecules to identify and isolate nucleic acid molecules comprising the full length coding sequences of such mRNA transcripts, genomic material, or the complements thereof. See for example Ausubel, supra.. and Maniatis, supra.
  • Nucleic acid reagents comprising all or any portion of the sequences, such as those described above, which are capable of specifically hybridizing to inflammatory bowel disease-specific small RNA molecules or their complements may be utilized for the detection of Crohn's disease- or ulcerative colitis-specific nucleic acid molecules (e.g.. small RNA molecules) .
  • Nucleic acid molecules of either DNA or RNA of the same sequences as those listed above may be utilized for diagnostic reagents.
  • nucleic acid molecules, either DNA or RNA, of the complementary sequence to those listed above may be utilized for diagnostic reagents.
  • Such nucleic acid reagents will generally be single stranded molecules, although, in addition, double stranded molecules may also be utilized.
  • each U (uridine) base is replaced by T (thymidine) base.
  • any nucleic acid molecule exhibiting a nucleotide sequence so substantially similar to the nucleotide sequence of SEQ ID NOS: 1-5 or to the complement of SEQ ID NOS: 1-5 (e.g.. about 90% homology to the nucleotide sequence of SEQ ID NOS: 1-5, or to the complement of SEQ ID NOS: 1-5) , such that the nucleic acid will still specifically recognize (i.e.
  • nucleic acid reagents of the invention may contain one or more modifications within their phosphodiester "backbones" which will not appreciably diminish the molecules' ability to hybridize to Crohn's disease- or ulcerative colitis- specific nucleic acid molecules. Such modifications, may include, but are not limited to the use of phosphorothioate or phosphoroamidate groups, phosphate triesters, carbamates, or methyl phosphonates, the synthetic chemistries of which are well known to those in the art. Additionally, the nucleic acid reagents to be used for diagnosis may be labeled using standard radioactive, fluorescent, or chromogenic labeling techniques which are well known to those of ordinary skill in the art.
  • RNA molecules have been identified which are specific to Crohn's or ulcerative colitis diseased tissue.
  • Certain of these RNA molecules may represent m-RNA transcripts, or portions of m-RNA transcripts, which encode inflammatory bowel disease- specific proteins. It is possible that certain of these disease-specific RNA molecules may be produced by infectious agents responsible for the development of inflammatory bowel diseases.
  • the RNA molecules of the invention may represent portions of RNA genomic, as opposed to transcriptional, material.
  • inflammatory bowel disease specific proteins may be encoded by the complement of the RNA molecules described herein.
  • Such potential Crohn's disease and/or ulcerative colitis specific proteins may be comprised, for example, of peptides such as those listed, below, in Table 2. Each of these peptides is oriented with the amino terminus at the left and the carboxy terminus on the right.
  • SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 represent peptides that may be encoded by the portion of the disease- specific RNA molecule whose nucleotide sequence is listed in Table 1, above, as SEQ ID NO: 1.
  • SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 represent peptides that may be encoded by the complement of the portion of the disease-specific RNA molecule whose nucleotide sequence is listed in Table 1, above, as SEQ ID NO: 1.
  • SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14 represent peptides that may be encoded by the portion of the disease-specific RNA molecule whose nucleotide sequence is listed in Table 1, above, as SEQ ID NO: 2.
  • SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18 represent peptides that may be encoded by the complement of the portion of the disease-specific RNA molecule whose nucleotide sequence is listed in Table 1, above, as SEQ ID NO: 2.
  • the small RNA molecules of invention may represent non-coding portions of m-RNA transcripts or, alternatively, portions of an RNA genome that do not code for proteins.
  • the inflammatory bowel disease-specific proteins will be encoded by the coding portion of the mRNA transcripts, or, alternatively, portions of an RNA genome that encode proteins, of which the small RNA molecules of the invention are a part. Techniques are described, above, in Section 5.1, for the isolation of full length nucleic acids, which would include the peptide-coding portions of the molecules.
  • amino acid sequence encoding the inflammatory bowel disease-specific peptides of the invention may be produced by utilizing any of a variety of peptide synthesis and expression techniques which are well known to those of skill in the art. For example, methods such as those described, below, in Section 5.2.1 may be used.
  • antibodies may be raised which are directed against any of the inflammatory bowel disease-specific peptides.
  • Techniques for the production of antibodies are well known to those of skill in the art, and may include, for example, procedures such as those described, below, in Section 5.2.2.
  • Methods for producing Crohn's disease and/or ulcerative colitis-specific peptides by expressing nucleic acid encoding such are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing Crohn's disease and/or ulcerative colitis-specific peptide coding sequences and appropriate transcriptional/translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, for example, the techniques described in Maniatis et al., supra.. and Ausubel et al., supra. DNA and RNA synthesis may, additionally, be performed using an automated synthesizers. See, for example, the techniques described in "Oligonucleotide Synthesis", 1984, Gait, M.J. ed. , IRL Press, Oxford.
  • host-expression vector systems may be utilized to express the coding sequences of the Crohn's disease and/or ulcerative colitis-specific peptides of interest.
  • Such host-expression systems represent vehicles by which the coding sequences of interest may be produced, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, exhibit the Crohn's disease and/or ulcerative colitis-specific peptides of the invention.
  • These include but are not limited to microorganisms such as bacteria (e.g., E. coli. B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing Crohn's disease and/or ulcerative colitis-specific peptide coding sequences; yeast (e.g. Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the Crohn's disease and/or ulcerative colitis-specific peptide coding sequences; insect cell systems infected with recombinant virus expres ⁇ sion vectors (e.g., baculovirus) containing the Crohn's disease and/or ulcerative colitis-specific peptide coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the Crohn's disease and/or ulcerative colitis- specific peptide coding sequences; or mamma
  • COS COS, CHO, BHK, 293, 3T3 harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter) .
  • promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter) .
  • a number of expression vectors may be advantageously selected depending upon the use intended for the Crohn's disease and/or ulcerative colitis- specific peptide being expressed. For example, when large quantities of Crohn's disease and/or ulcerative colitis- specific peptides are to be produced for the generation of antibodies, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S- transferase (GST) .
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the Crohn's disease and/or ulcerative colitis-specific peptide can be released from the GST moiety.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the Crohn's disease and/or ulcerative colitis- specific peptide coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter) .
  • the Crohn's disease and/or ulcerative colitis-specific peptide coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non- essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing Crohn's disease and/or ulcerative colitis-specific peptides in infected hosts.
  • Specific initiation signals may also be required for efficient translation of inserted Crohn's disease and/or ulcerative colitis-specific peptide coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire Crohn's disease and/or ulcerative colitis-specific peptide gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed.
  • exogenous translational control signals including the ATG initiation codon
  • the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert.
  • exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153 . :516-544) .
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cells lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc.
  • cell lines which stably express Crohn's disease and/or ulcerative colitis-specific peptides may be engineered.
  • host cells can be transformed with the Crohn's disease and/or ulcerative colitis-specific peptide DNA independently or coordinately controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine 5 kinase (Wigler, et al., 1977, Cell .11:223), hypoxanthine- guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 45:2026), and adenine phosphoribosyltransferase (Lowy, et al. , 1980, Cell 22:817) genes can be employed in tk " , hgprt " or aprt " cells, 0 respectively.
  • antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 7_8:1527); gpt, which confers resistance to 5 mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 7_8:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1) ; and hygro, which confers resistance to hygromycin (Santerre, et al., 1984, Gene 3J):147) genes.
  • methotrexate Wang, et al., 1980, Natl. Acad. Sci. USA
  • Described herein are methods for the production of antibodies which are capable of specifically recognizing a 5 Crohn's disease and/or ulcerative colitis-specific peptide of an epitope thereof. It should be understood that techniques such as those described herein may also be utilized for the production of antibodies directed against infectious agents, or epitopes thereof, which are capable Q of causing inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis.
  • Antibodies may include, but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs) , humanized or chimeric antibodies, single chain antibodies, 5 Fab fragments, F(ab') 2 fragments, fragments produced by a FAb expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
  • mAbs monoclonal antibodies
  • Such antibodies may be used, for example, in the detection of a Crohn's disease and/or ulcerative colitis- specific peptide in a biological sample.
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as a Crohn's disease and/or ulcerative colitis-specific peptide, or an antigenic functional derivative thereof.
  • an antigen such as a Crohn's disease and/or ulcerative colitis-specific peptide, or an antigenic functional derivative thereof.
  • various host animals may be immunized by injection with the Crohn's disease and/or ulcerative colitis-specific peptide, including but not limited to rabbits, mice, rats, etc.
  • adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete) , mineral gels such as lysolecithin, pluronic polyols, polyanion ⁇ , peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriu parvu .
  • BCG Bacille Calmette-Guerin
  • a monoclonal antibody which is a substantially homogeneous population of antibodies to a particular antigen, may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to the hybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497: and U.S. Patent No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al.. 1983, Immunology Today 4.:72; Cole et al. , 1983, Proc. Natl. Acad. Sci. USA jBO . :2026-2030) , and the EBV-hybridoma technique (Cole et al.
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • the hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.
  • techniques described for the production of single chain antibodies U.S. Patent 4,946,778; Bird, 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci.
  • Single chain antibodies are formed by linking the heavy and light chain fragment of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
  • Antibody fragments which contain specific binding sites for a Crohn's disease and/or ulcerative colitis- specific peptide may be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries may be constructed (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity to the Crohn's disease and/or ulcerative colitis-specific peptide of interest.
  • a variety of methods may be employed, utilizing reagents such as those described in Sections 5.1 and 5.2, above, for the diagnosis of the inflammatory bowel diseases, ulcerative colitis and Crohn's disease.
  • such reagents may be used for the detection of the presence of peptides and/or nucleic acid molecules specific to these diseases, i.e. , molecules present in diseased tissue but absent from, or present in greatly reduced levels relative to, the corresponding non-diseased
  • tissue or alternatively, present in a biological sample or bodily fluid of an individual having such an inflammatory bowel disease, but absent from, or present in greatly reduced levels in, the corresponding sample or fluid of an individual in which the disease is not present.
  • the methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one of the specific nucleic acid or antibody reagents described herein, which may be conveniently used, e.g.. in clinical settings, to diagnose
  • 3Q detection scheme include any human tissue, including, but not limited to, tissue from any portion of the alimentary canal, with bowel or intestinal tissue being preferred. Ulcerative colitis does not visibly manifest itself outside of the colon, therefore, colon tissue is preferred for the
  • stool samples may be used in conjunction with any of the detection schemes of the invention for the detection of either Crohn's disease or ulcerative colitis.
  • bodily fluids such as blood for example, may also be used as the starting material for disease detection.
  • the diagnostic method involves nucleic acid amplification, such as those described below, the starting sample can be a frozen or fixed tissue section or can be quite crude, with very little sample preparation required.
  • RNA from the tissue to be analyzed may easily be isolated using procedures which are well known to those in the art.
  • the methods employed herein may, for example, be such as those described and cited in Pechan, R. et al.. 1987, Z. Naturforsch 42c:1006-1008. particularly an acid phenol purification procedure such as that described in Roe, B.A. 1975, Nucl. Acids Res. 2:21-42, which is incorporated herein by reference in its entirety. Diagnostic procedures may also be performed "in situ" directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no RNA purification is necessary. Nucleic acid reagents such as those described in Section 5.1 may be used as probes and/or primers for such in situ procedures (Nuovo, G.J. , 1992, PCR in situ hybridization: protocols and applications, Raven Press, NY) .
  • Preferred diagnostic methods for the detection of Crohn's disease- or ulcerative colitis-specific nucleic acid molecules may involve for example, contacting and incubating nucleic acids, derived from the target tissue being analyzed, with one or more labeled nucleic acid reagents as are described in Section 5.1, under conditions favorable for the specific annealing of these reagents to their complementary sequences within the target molecule.
  • the lengths of these nucleic acid reagents are at least 15 to 30 nucleotides. After incubation, all non- annealed nucleic acids are removed from the nucleic acid rinflammatory bowel disease-specific small RNA molecule hybrid.
  • the target tissue nucleic acid may be immobilized, for example, to a solid support such as a membrane, or a plastic surface such as that on a microtiter plate or polystyrene beads.
  • a solid support such as a membrane, or a plastic surface such as that on a microtiter plate or polystyrene beads.
  • non-annealed, labeled nucleic acid reagents of the type described in Section 5.1 are easily removed. Detection of the remaining, annealed, labeled nucleic acid reagents is accomplished using standard techniques well-known to those in the art.
  • Crohn's disease or ulcerative colitis specific nucleic acid molecules may involve their amplification, e.g., by PCR (the experimental embodiment set forth in Mullis, K.B., 1987, U.S. Patent No. 4,683,202), ligase chain reaction (Barany, F., 1991, Proc. Natl. Acad. Sci. USA 88:189-193) , self sustained sequence replication (Guatelli, J.C. et al.. 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878) , transcriptional amplification system (Kwoh, D.Y et al.. 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177) , Q-Beta Replicase (Lizardi, P.M. et al.. 1988, Bio/Technology
  • RNA amplification method or any other RNA amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of disease- specific RNA molecules if such molecules are present in very low numbers.
  • a cDNA molecule is obtained from the target RNA molecule (e.g. , by reverse transcription of the RNA molecule into cDNA) .
  • a target sequence within the cDNA is then used as the template for a nucleic acid amplification reaction, such as a PCR amplification reaction, or the like.
  • the nucleic acid reagents used as synthesis initiation reagents (e.g. , primers) in the reverse transcription and nucleic acid amplification steps of this method are chosen from among the nucleic acid reagents described in Section 5.1.
  • the preferred lengths of such nucleic acid reagents are at least 15-30 nucleotides.
  • the nucleic acid amplification may be performed using radioactively or non-radioactively labeled nucleotides.
  • enough amplified product may be made such that the product may be visualized by standard ethidium bromide staining or by utilizing any other suitable nucleic acid staining method.
  • the Crohn's disease and ulcerative colitis-specific small RNA molecules may be visualized via two dimensional nucleic acid gel electrophoresis techniques well known to those of skill in the art. Such techniques utilize one dimension run under non-denaturing conditions and one dimension run under denaturing conditions. See for example, Pechan et al. (Pechan, R.
  • RNA molecules may be visualized apart from the diagonal formed by the bulk of the RNA species in a gel containing a population of RNA molecules.
  • Protein from the tissue to be analyzed may easily be isolated using techniques which are well known to those of skill in the art.
  • the protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, D. , 1988,
  • Preferred diagnostic methods for the detection of Crohn's disease and/or ulcerative colitis-specific peptide molecules may involve, for example, immunoassays wherein Crohn's disease and/or ulcerative colitis-specific peptides are detected by their interaction with an anti-Crohn's disease and/or ulcerative colitis-specific peptide antibody.
  • immunoassays wherein Crohn's disease and/or ulcerative colitis-specific peptides are detected by their interaction with an anti-Crohn's disease and/or ulcerative colitis-specific peptide antibody.
  • antibodies, or fragments of antibodies, such as those described, above, in Section 5.2.2, useful in the present invention may be used to quantitatively or qualitatively detect the presence of cells which express Crohn's disease and/or ulcerative colitis-specific peptides.
  • the antibodies (or fragments thereof) useful in the present invention may, additionally, be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of Crohn's disease and/or ulcerative colitis-specific peptides.
  • In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody of the present invention.
  • the histological sample is taken from a tissue suspected of containing Crohn's disease and/or ulcerative colitis-specific peptides.
  • the antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample.
  • Immunoassays for Crohn's disease and/or ulcerative colitis-specific peptides typically comprise incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells, or cells which have been incubated in tissue culture, in the presence of a detectably labeled antibody capable of identifying Crohn's disease and/or ulcerative colitis-specific peptides, and detecting the bound antibody by any of a number of techniques well-known in the art.
  • the biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins.
  • the support may then be washed with suitable buffers followed by treatment with the detectably labeled Crohn's disease and/or ulcerative colitis-specific peptide-specific antibody.
  • the solid phase support may then be washed with the buffer a second time to remove unbound antibody.
  • the amount of bound label on solid support may then be detected by conventional means.
  • solid phase support or carrier any support capable of binding an antigen or an antibody.
  • supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • Preferred supports include polystyrene beads.
  • suitable carriers for binding antibody or antigen or will be able to ascertain the same by use of routine experimentation.
  • the binding activity of a given lot of anti-Crohn's disease and/or ulcerative colitis-specific peptide antibody may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
  • EIA enzyme immunoassay
  • the enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means.
  • Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • Detection may be accomplished using any of a variety of other immunoassays.
  • a radioimmunoassay RIA
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
  • fluorescent labeling compounds fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • the antibody can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA) .
  • DTPA diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the antibody also can be detectably labeled by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • inflammatory bowel disease-specific RNA molecules described herein may be produced by an infectious agent responsible for such inflammatory bowel diseases.
  • infectious agent responsible for such inflammatory bowel diseases.
  • a variety of well known methods may be utilized to identify and isolate such inflammatory bowel disease infectious agents.
  • an inflammatory bowel disease infectious agent i.e.. a Crohn's disease and/or ulcerative colitis infectious agent
  • a Crohn's disease and/or ulcerative colitis infectious agent may be identified and isolated by immunoaffinity chromatography methods using an immunoadsorbent column to which an antibody is immobilized which is capable of binding the infectious agent.
  • Such an antibody may be monoclonal or polyclonal in origin.
  • such an antibody is directed to an epitope on the surface of the Crohn's disease and/or ulcerative colitis infectious agent.
  • Surface refers to an epitope that is exposed to the environment external to the infectious agent.
  • the Crohn's disease and/or ulcerative colitis infectious agent may be biochemically purified from a Crohn's disease and/or ulcerative colitis diseased tissue sample.
  • tissue include, but are not limited to, inflammatory bowel diseased tissue from any portion of the alimentary canal, with bowel or intestinal tissue being preferred.
  • colon tissue is preferred.
  • the infectious agent may be isolated from bodily fluids, such as blood, of an individual exhibiting inflammatory bowel disease.
  • Crohn's disease and/or ulcerative colitis infectious agents may be isolated from stool samples of individuals manifesting inflammatory bowel disease.
  • a sample suspected of containing a Crohn's disease and/or ulcerative colitis infectious agent is passed over an immunoaffinity column such that the infectious agent binds to the column.
  • Conditions for optimal binding will be well known to those of skill in the art.
  • all unbound sample materials are washed out of the column.
  • the bound infectious agent is then eluted off the immunoaffinity column, using techniques well known to those of skill in the art.
  • the elution buffer may introduce a saturating amount of a small molecule that mimics the antigen present in the Crohn's disease and/or ulcerative colitis infectious agent, thus competing with the infectious agent itself for antibody binding, and causing the bound infectious agent to be released from the column.
  • such agent may be characterized according to standard procedures well known to those of skill in the art. For example, the ability to propagate such an agent may be studied. Such propagation characteristics may include, but are not limited to, the ability of the infectious agent to grow in cell-free media, and/or the ability of the infectious agent to infect and propagate within cells, preferably alimentary canal cells, and most preferably bowel, intestinal, or colon cells.
  • the infectious agent may be subjected to well known techniques for the amplification and sequencing of its constituent nucleic acids. See, for example, Ausubel supra. and Sambrook, supra.
  • a method for the isolation and identification of an inflammatory bowel disease infectious agent may comprise: (a) exposing a sample suspected of containing an inflammatory bowel disease infectious agent to an immobilized antibody directed against an epitope on the surface of the infectious agent for a time sufficient to allow binding of the infectious agent to the antibody; (b) removing all non-bound sample components; and (c) eluting the bound infectious agent from the immobilized antibody, thereby identifying and isolating an inflammatory bowel disease infectious agent.
  • RNA populations from Crohn's disease or ulcerative colitis tissue are isolated and partially sequenced.
  • Nucleic acid reagents comprising portions of the sequences presented here, and/or their complements, which are capable of specifically hybridizing to (i.e.. binding or annealing to) inflammatory bowel disease-specific small RNA molecules, or their complements, are well suited for use as extremely sensitive diagnostic tools for the identification of individuals with these diseases.
  • the resulting aqueous phase is loaded on a DEAE cellulose (Sigma) column, previously washed and equilibrated with 0.14 M Na Acetate (pH 4.5), at a ratio of approximately 1 ml packed DEAE cellulose per 3 mis aqueous phase.
  • the DEAE cellulose column is washed first with 0.14 M sodium acetate (pH 4.5) at a ratio of 20 is per ml DEAE cellulose, followed by 0.14 M sodium acetate (pH 4.5) that contains 0.3 M sodium chloride (NaCl) at a ratio of 20 mis per ml DEAE cellulose.
  • Nucleic acid is then eluted with 0.14 M Na Acetate, pH 4.5, containing 1.0 M NaCl, at a ratio of 2 mis per gram DEAE cellulose. 10-20 1-1.5 ml fractions are collected and their absorbances at 260 nm (A 260 ) are measured. Those fractions containing nucleic acid are combined and immediately precipitated with 2.5 volumes ice cold absolute ethanol. After standing overnight at -20°C, the precipitate is collected by centrifugation, washed twice with 95% ethanol, and then dried in vacuo.
  • RNAs were separated generally according to the two-dimensional gel methods of Pechan et al. (Pechan, R. et al.. 1987, Zeitschrift fur Naturforschung 42c:1006-1018) , with modifications. Briefly, 10% polyacrylamide gels (20cm x 40cm) were used for both directions. The first dimension (20cm length) was run under non-denaturing conditions at room temperature. The second dimension (40cm length) was run under the denaturing conditions of 7M urea and 50°C- 60°C. The second dimension gel of the two-dimensional gel electrophoretic procedure was stained with silver or ethidium bromide as described below. The latter was used if the RNA was to be extracted from the gel.
  • a gel to be silver stained is soaked successively at room temperature in a 50% ethanol, 10% acetic acid solution for 12 to 16 hours, a 10% ethanol, 1% acetic acid solution for 1 hour, and a 12 mM AgN0 3 (Silver Nitrate, Sigma, 2.04g/l) solution for 1 hour.
  • the gel is then rinsed with distilled water for 30 seconds and placed in a 0.28% HCHO (formaldehyde, Baker, 7.0 ml/1), 0.25M KOH (potassium Hydroxide, Baker, 14.03g/l) solution with gentle shaking for 1 to 2 minutes and further incubation for 8 to 9 minutes for a total exposure of 10 minutes.
  • the gel is then moved to a new tray and incubated in 0.06 M Na 2 C0 3 -H 2 0 (sodium carbonate monohydrate, Baker, 7.42g/l) for 15 to 20 minutes. (Because the gels used in this Example were 1.2 mm thick, this step was slow, which may cause background to appear.) After this incubation, the gel is exposed to air for 10 to 20 minutes then conserved in plastic wrap. For incubations, plastic trays and only Millipore-filtered water should be used. Examples of such silver stained gels are shown in FIGs. 2A-D and 3A-B,
  • a gel to be ethidium bromide (EtBr) stained is soaked in a solution of 500 ng EtBr/1 lx TBE (0.089 M Tris-borate, 0.089 M boric acid, 0.002 M EDTA) for 30 minutes. During incubation, the tray should periodically be gently moved. Following incubation, the gel is destained by soaking in 1 liter TBE (lx) for 10 minutes. The gel is then examined on a long-wave UV transilluminator and the appropriate RNA species is extracted using standard techniques as described by Pechan, R. et al.. 1987, Zeitschrift fur Naturforschung 4_2c:1006-1018.
  • RNA molecules which are specific for Crohn's disease or ulcerative colitis were separated using two- dimensional gel analysis according to Pechan, R. et al. (1987, Zeitschrift fur Natureforschung 42c: 1006-1018; see Section 6.1 for details).
  • An example of one such separation is shown in FIG. 2A-D.
  • RNA from the Crohn's disease and the ulcerative colitis tissue samples exhibit RNA species (as indicated by the arrows 1 and 2) which are absent from both non-disease tissue (FIG. 2C) and intestinal cancer tissue (FIG. 2D) .
  • RNAs were isolated from the species indicated by arrow 1 (FIG. 2A) utilizing the techniques described above, in Section 6.1. Isolated RNA molecules were then terminally labeled with 32 P, and subjected, utilizing well- known means, to RNA sequencing analysis. Partial sequence (SEQ ID NOs:l-2) information was obtained, as shown in Table 3, below. SEQ ID NO: 1 represents the sequence of the 5' end of the RNA molecule, while SEQ ID NO: 2 represents the sequence of the 3* end of the molecule. It is estimated that the entire RNA molecule is approximately 220 nucleotides in length. Thus, approximately 140 nucleotides lie between the sequence represented by SEQ ID NO: 1 and that represented by SEQ ID NO: 2. No sequences homologous to these were found in publicly accessible nucleotide databases. Databases were searched using BLASTIN (National Center for Biological Information) .
  • N refers to any nucleotide
  • Y refers to a pyrimidine
  • A/U' in SEQ ID NO: 2, refers to a nucleotide position which may contain either an A or a U base.
  • FIG 3A-B are the results of another study whereby inflammatory bowel disease small RNA molecules were identified and characterized.
  • Crohn's disease tissue FIG. 3A
  • colon cancer tissue FIG. 3B
  • the Crohn's disease-specific RNA species are indicated by the arrows 1 and 2 in FIG. 3A.
  • These analytical gels were loaded with 40 ⁇ g of Crohn's disease tissue RNA and 70 ⁇ g colon cancer tissue RNA.
  • RNA species migrates together with the 5s rRNA species in the first dimension, and migrates significantly slower than 5s rRNA in the second dimension.
  • This RNA species was isolated, from a preparative two- dimensional gel loaded with 300 ⁇ g Crohn's disease tissue RNA. The gel was stained with EtBr and the band containing the RNA of interest was cut out. The RNA was eluted from the gel by soaking the gel piece in 500 ⁇ l buffer (10 mM Tris-HCl, pH 7.5, 1 m M EDTA, 0.1 M NaCl, 0.1% sodium dodecyl sulfate (SDS)) at 37° overnight, followed by precipitation.
  • 500 ⁇ l buffer (10 mM Tris-HCl, pH 7.5, 1 m M EDTA, 0.1 M NaCl, 0.1% sodium dodecyl sulfate (SDS)
  • RNA was labelled. In the 5 absence of dephosphorylation, only the 5' end could be labeled, indicating that the 3' end is protected. The 3' end could be labeled after dephosphorylation, demonstrating that the 3' blocking group was a phosphate group. Portions of the RNA were either 3' or 5' labeled, and subjected to
  • RNA sequencing 15 approximately one half the full length size. Both RNAs were cut out and purified as described above, then were treated with nucleoside specific endoribonucleases (RNase T, (G) , RNase U 2 (A>G) , RNase Phy.M (A and U) , and RNase A (C and U) for RNA sequencing. All endoribonucleases were
  • RNA •20 purchased from Pharmacia. From the small RNA isolated after partial alkaline hydrolysis, 35 (SEQ ID NO: 3) and 33 (SEQ ID NO: 4) nucleotides were sequenced from the 5' and 3' labeled RNA, respectively. These sequences are shown, below, in Table 4.
  • N refers to any nucleotide
  • Y refers to a pyrimidine
  • nucleic acid reagents are successfully used to detect disease-specific RNA molecules, and thus to rapidly diagnose Crohn's disease and ulcerative colitis-affected tissues.
  • the membrane was air dried for two hours at room temperature and the RNA was crosslinked to the filter, using a Stratalinker (Stratagene) at 0.12J/cm 2 .
  • the filter was prehybridized for 3 hours at 42°C in 50% formamide, 5x SSPE (2Ox SSPE: 3M NaCl, 0.2M NaH 2 P0 4 -H 2 0 (sodium phosphate, monobasic, monohydrate), 200mM EDTA; pH 7.4), 5x Denhardt's (50x Denhardt's: 5g/0.51 Ficoll(Type 400; Pharmacia), 5g/0.51 polyvinylpyrrolidone, 5g/0.51 bovine serum albumin (Fraction V; Sigma)), 0.1% SDS, and 100 ⁇ g/ml salmon sperm DNA.
  • 5x SSPE 2Ox SSPE: 3M NaCl, 0.2M NaH 2 P0 4 -H 2 0 (sodium phosphate
  • FIG. 4 shows the results of a Northern analysis wherein RNA isolated from control (colon cancer), Crohn's disease, and ulcerative colitis tissue was probed with a sequence homologous to a portion of the RNA obtained from the RNA species indicated in FIG. 3A, arrow 2.
  • the arrow in FIG. 4 indicates a band of the same size as that of the FIG. 3A, arrow 2 species.
  • This band is present in 3 of the 4 Crohn's disease RNA samples, in 2 of the 3 ulcerative colitis RNA samples, and is absent from the control RNA samples.
  • the single Crohn's disease RNA sample ulcerative colitis RNA sample in which the diagnostic band is not readily visible may, for example, represent samples in which the diagnostic band is too faint to be detected by Northern analysis, or may represent samples in which the rRNA cleavage step(s) necessary to produce this size of species has not yet occurred.

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Abstract

The present invention relates to the diagnosis of the human inflammatory bowel diseases, Crohn's disease and ulcerative colitis, and to nucleic acid and peptide reagents, detection methods, and pre-packaged kits useful for such diagnosis. Specifically, nucleic acids are presented which may be used to detect the presence of small RNA molecules which are found in ulcerative colitis- and/or Crohn's-diseased tissue, but are absent from, or are present in greatly reduced levels in, the corresponding tissue exhibiting manifestations of these diseases. Further, antibody reagents are presented which may be used to detect the presence of peptides which are found in ulcerative colitis- and/or Crohn's-diseased tissue, but are absent from, or are present in greatly reduced levels in, the corresponding tissue exhibiting manifestations of these diseases.

Description

DIAGNOSIS OF HUMAN INFLAMMATORY BOWEL DISEASES AND NUCLEIC ACID REAGENTS THEREFOR
1. INTRODUCTION Detection methods are presented which may be used for the diagnosis of the human inflammatory bowel diseases, Crohn's disease and ulcerative colitis. First, nucleic acids and nucleic acid detection methods are presented herein which may be utilized to detect small RNA molecules found in ulcerative colitis- and/or Crohn's-diseased tissues, but which are absent from, or are present in greatly reduced levels in, the corresponding non-diseased tissues. Second, reagents and schemes are presented whereby peptides encoded by such RNA molecules may be identified.
Using such detection schemes, simple, rapid, and unequivocal diagnosis of Crohn's disease or ulcerative colitis is possible for the first time. In addition, diagnoses may be made very early in the course of the conditions, using tissue which does not yet even manifest visual aspects of disease. The diagnostic procedures presented here, therefore, represent a great improvement over the current diagnostic techniques which rely on imprecise visual observations and which may only be utilized once disease has become overtly established.
2. BACKGROUND OF THE INVENTION 2.1 HUMAN INFLAMMATORY BOWEL DISEASES Two types of nonspecific human inflammatory bowel diseases are distinguished on the basis of clinical and pathological findings: ulcerative colitis and Crohn's disease. Each of these conditions has its own prognosis and treatment, but both of the diseases may have uncertain diagnoses and may be confused with other diseases of the colon or small intestine.
2.1.1 ULCERATIVE COLITIS Ulcerative colitis is primarily an inflammatory disease which affects the superficial epithelial layer of the rectum and distal colon, although it may also extend to involve the entire colonic mucosa. The cause of the disease is unknown and exacerbations and remissions are common. Symptoms of ulcerative colitis usually develop in individuals between 25 and 45 years of age. Although the illness occurs less frequently in the elderly, attacks in patients older than 60 years of age may be severe, and mortality rates are very high for this group.
The most common clinical manifestations of ulcerative colitis are constipation and passage of blood or mucus with the stools. Such symptoms may continue for several months, or even years, before the disease causes the onset of diarrhea or systemic manifestations. The relative severity of the disease can differ among individuals. For example, ailments which are confined to the abdomen and perianal region usually indicate a mild or moderate manifestation of the disease, while prominent, generalized symptoms such as fatigue and/or weight loss often indicate a more severe disease manifestation. The majority of patients with ulcerative colitis manifest a mild form of the disease, which is confined to the distal colon and rectum. Such patients have intermittent diarrhea with no extracolonic or systemic manifestations. Physical examinations are usually negative, although a localized tenderness over the distal colon may be detected. Approximately 25 percent of ulcerative colitis cases are classified as moderately severe, with patients passing watery or pasty stools containing mucus and gross quantities of blood. Systemic manifestations, such as intermittent fatigue and an increased need for sleep, are often noted in these moderately severe cases, and, during exacerbations, low-grade fevers and mild weight loss may also occur. A small percentage of individuals experience severe ulcerative colitis with recurrent exacerbations which may lead to hospitalization. Patients with severe colitis typically exhibit temperatures of 100°F (38°C) or higher, and profuse, constant, bloody stools. If symptoms are allowed to persist, extensive weight loss may occur. In these cases, physical examination may be normal, but a distended abdomen with tympany and absent bowel sounds may indicate a full-blown flare up of the disease.
2.1.2 CROHN'S DISEASE
Crohn's disease, or granulomatous ileocolitis, is a chronic inflammatory disease of unknown etiology which slowly destroys the alimentary tract. The disease usually involves the terminal ileum and/or colon, but may manifest itself as a widespread illness involving any portion of the hollow gut from mouth to anus. The chronic inflammation of the disease occurs in all layers of the bowel. Crohn's disease is chiefly a disease of younger people. Nearly 60% of all cases begin between ages of ten to twenty-five, with peak incidence of onset occurring in teenagers and the majority of affected individuals being between 20 and 40 years of age. In the United States, the annual incidence is about 2 per 100,000 individuals, the world-wide incidence is approximately 6.1 per 100,000 individuals, and the world-wide prevalence is 60 per 100,000 individuals.
The most common clinical manifestations of Crohn's disease are pain, mild nonbloody diarrhea, anorexia, and mild anemia. It is not unusual for patients to exhibit severe, generalized fatigue which prevents them from performing ordinary daily activities. An abdominal mass, caused by a thickened bowel loop or transmural inflammation, may be palpated in as many as 25 percent of patients. Further, if the small intestine is extensively involved, there may be evidence of malnutrition, and tetany due to hypocalcemia. Complications of the disease may include such extracolonic conditions as arthritis, renal stones, and an increased prevalence of gallstones. (Stauffer, J.Q. et al.. 1973, Ann. Intern. Med. 7£:383; Bohles, H. et al.. 1988, Klin. Wochenschr. _56_:87; Andersson, H. et al.. 1987, Scand. J. Gastroenterol. 2J2:253). Unlike ulcerative colitis, which often initially presents itself as an acute, severe disease, Crohn's disease is an indolent, or so- called smoldering, disease. The disease often becomes chronic before a patient sees a physician, and the average individual has symptoms for approximately five years before the diagnosis of Crohn's disease is made.
2.2 DIAGNOSIS OF ULCERATIVE COLITIS AND CROHN'S DISEASE
The only diagnostic currently available for the human inflammatory bowel diseases, Crohn's disease and ulcerative colitis, involves conventional visual macroscopic and microscopic examination of bowel and colon tissue, and, as such, is only able to detect the existence of established disease rather than to anticipate the onset of the diseases. No truly early detection schemes exist for diagnosing ulcerative colitis or Crohn's disease before the onset of clinical symptoms.
In addition, because of its often generalized symptoms, there may be some difficulty in arriving at a diagnosis of Crohn's disease. The mild, aching pain which usually accompanies the disease, for example, is often misdiagnosed as being caused by an irritable bowel syndrome. In other cases, an initial diagnosis of Behcet's syndrome is made before the onset of the bowel symptoms which point to a correct diagnosis of Crohn's disease (Tolia, V. et al. , 1989, Am. J. Gastroenterol. j$4_:322). Sigmoidoscopy may reveal lower colon abnormalities such as ulceration, granularity, and nodule formation, but because Crohn's most often affects only the terminal ileum, cecu , and ascending colon, standard sigmoidoscopic examinations appear normal in 30 to 50 percent of patients. Tissue obtained at surgery often reveals chronic inflammatory involvement of the submucosal layers of the bowel wall, but, even when such surgical specimens reveal submucosal inflammation, these histopathological findings are not specific for Crohn's disease. A biopsy often yields supportive evidence of Crohn's disease, but final differentiation from ulcerative colitis must rely heavily on an individual's history, the clinical course of one's symptoms, and on the pattern of information seen on barium contrast x-ray studies. Currently, barium contrast x-ray examination is the single most important procedure for the diagnosis of Crohn's disease because it frequently reveals the characteristic changes associated with the disease, such as a marked distortion of the bowel wall contour or the appearance of intervening areas of normal mucosa between affected regions of tissue. In attempting to diagnose ulcerative colitis, sigmoidoscopic examination may reveal important mucosal abnormalities such as a granularity to the mucosal surface, which indicates scarring and microulceration, and friability, which is the appearance of punctate blood spots along the mucosal surface. Histopathological analysis of tissue biopsies may also aid in diagnosis of the disease, with the most characteristic signs of ulcerative colitis being atrophy of the mucosal glands, and the presence of crypt abscesses (i.e.. polymorphonuclear leukocytes in the crypts of Lieberkuhn) . Although such histopathologies are distinctive, they are not, however, definitively diagnostic of ulcerative colitis. Barium contrast x-ray examination is the most useful means by which to distinguish between the two types of inflammatory bowel diseases. Barium x- ray, for example, reveals a mottled and roughened barium appearance at the bariu -mucosa interface, which is highly characteristic of microulcerations seen with ulcerative colitis.
2.3 CAUSES OF ULCERATIVE COLITIS AND CROHN'S DISEASE
Neither the cause of ulcerative colitis nor that of Crohn's disease is known, but several potential explanations have been suggested. It has been suggested that there is a genetic and/or cultural causal component associated with ulcerative colitis (Farmer, R.J., 1989, Scand. J. Gastroenterol. Suppl. 170:64 ; Almy, T.P. et al.. 1966, Gastroenterol. 5_1:757), with a clustering of the disease among certain groups. Ulcerative colitis, for example, is between two and four times more common among Jews than non-Jews, and approximately four times more common among whites than non-whites. Genetic and/or cultural causal components involved in the development of Crohn's disease have also been postulated. For example, a clustering of the disease within families has been shown, the disease is more common among Jews of middle European origin than among non-Jews (Roth, M.P. et al.. 1989, Gastroenterol. £7:900; Roth, M.P. et al.. 1989, Gastroenterol. £6:1016), and it develops in twins and siblings at a much higher rate than predicted by chance (Purrmann, J. et al.. 1990, Hepatogastroenterol. J32:81). Kuster et al. (Kuster, W. et al.. 1989, Am. J. Med. Genet. 2:105) have postulated that a recessive gene exhibiting incomplete penetrance may be a causative Crohn's disease agent.
Alternatively, it has been suspected that Crohn's disease may be caused by infection (Kirsner, J.B. and Shorter, R.G., 1982, N. Engl. J. Med. 306:837-848) . It has further been suggested that the resistance of the disease to medication and the long incubation time of 9 to 27 months in laboratory animals (Cave, D.R. et al. , 1978, Gastroenterol. J5 :632-637) of symptoms reminiscent of Crohn's disease, coupled with the slow, chronic course of the disease may indicate an unconventional causative agent for Crohn's disease. Because the presence of antibodies against double- stranded RNA has been detected in Crohn's disease patients and their close personal contacts (Korsmeyer, S.J. et al.. 1976, Proc. Natl. Acad. Sci. USA 278:574- 585; DeHoratius, R.J. et al.. 1978, Lancet .1:1116- 1119) , it has been suggested that the unconventional agent could be an RNA virus (Gajdusek, D.C., 1985, in Subviral Pathogens of Plants and Animals: Viroids and Prions, Maramorosch, A. and McKelvey, J.J., eds., pp. 483-544, Academic Press, London) or virus-like (i.e.. viroid; Diener, T.O., 1979, Viroids and Viroid Diseases, John Wiley and Sons, New York; Riesner, D. and Gross, J. 1985, Ann. Rev. Biochem. 54:531-564) pathogen. Butcher et al. (Butcher, P.D. et al. , 1986, Arch. Virol. JB8:113-120; Butcher, P.D. et al. , 1984, Biochem. Soc. Trans. 1111-1112) compared low molecular-weight RNA populations in Crohn's derived and normal tissues but found no differences. Pechan et al. (Pechan, R. et al.. 1987, Z. Naturforsch 42c:1006-1008) . however, have reported the presence of at least three RNA species with electrophoretic mobilities consistent with those characteristic of circular RNA molecules approximately 150-300 nucleotides in length within Crohn's disease tissue which are absent in the corresponding normal tissue. Having detected these circular RNA species as part of a bulk RNA analysis which separated RNA molecules according to their differential physical properties, Pechan et al. (1987) did not provide information regarding the sequence identity of these potentially Crohn's disease-specific molecules. Robertson and Goldberg (Robertson, H.D. and Goldberg, A.R. , 1990, International Patent, Pub. No. WO 91/04324) have suggested that the causative agent of Crohn's disease may have homology to sequences contained within the RNA genome of the hepatitis delta virus, and suggest the possibility that circular RNA species with lengths ranging from 300 to 800 nucleotides may be present in Crohn's disease tissue.
The present invention provides small RNA molecules, uniquely identifiable by the sequences disclosed herein, and by their anomalous electrophoretic mobilities, which are approximately 120-250 nucleotides in length and have no homology to hepatitis delta virus RNA. Furthermore, the nucleic acids of the present invention are useful for the diagnosis of not only Crohn's disease but also ulcerative colitis.
3. SUMMARY OF THE INVENTION The present invention relates to the diagnosis of the human inflammatory bowel diseases, Crohn's disease and ulcerative colitis, and to reagents and detection schemes useful for such diagnosis. In particular, for the first time, nucleic acids and nucleic acid detection schemes are presented which may be used to detect the presence of small RNA molecules which are found in ulcerative colitis- and/or Crohn's-diseased tissue, but are absent from, or are present in greatly reduced amounts in, the corresponding non-diseased tissue. In addition, antibody reagents and detection schemes are presented which may be utilized to detect the presence of peptides which may be encoded by such small RNA molecules.
Because of the ease and sensitivity by which the reagents presented here may be used to detect disease- specific RNA and peptide molecules, the diagnosis of Crohn's disease or ulcerative colitis can be made rapidly and unequivocally, often very early in the course of disease, using tissue that may not yet even manifest visual aspects of disease. Indeed, the reagents presented herein make it possible to perform diagnosis on bodily samples in addition to biopsied intestinal tissue, including body fluids, such as blood, lymph or joint fluid, and other bodily excretions. Thus, such diagnostic procedures represent a great improvement over current diagnostic methods which rely on histopathological, gross, and/or microscopic observations which are imprecise and may only be utilized once disease has become overtly established. As noted above, the nucleic acids of interest which are present in diseased tissue may either be absent from, or present in greatly reduced levels in, the corresponding normal, non-diseased tissue. Thus, for ease of explanation, the term "disease-specific" as used herein is meant to refer to either nucleic acids which are completely absent from corresponding non-diseased tissues, or to nucleic acids which are present in corresponding normal, non-diseased tissues, but at greatly reduced levels.
Further, "non-diseased" refers to non-Crohn's and non-ulcerative colitis diseased tissues. Thus, the term "non-diseased" as used herein may refer to any normal tissue or any non-inflammatory bowel diseased tissue.
Typically, the method for diagnosing an inflammatory bowel disease comprises: (a) obtaining frozen or fixed sections or nucleic acids from a human tissue, fluid, or stool sample suspected of containing an inflammatory bowel disease-specific small RNA molecule; (b) treating the frozen or fixed tissue sections or nucleic acids of step (a) with at least one nucleic acid reagent under conditions such that cDNA is made from such inflammatory bowel disease- specific small RNA molecule present among such nucleic acids; (c) amplifying a target sequence within the cDNA of step (b) according to a nucleic acid amplification method using at least one nucleic acid reagent complementary to the cDNA; and (d) detecting such amplified cDNA target sequence; wherein the nucleic acid reagent(s) of steps (b) and (c) act(s) as a synthesis initiation reagent(s) and is/are selected from the group consisting of:
(1) a nucleic acid, the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:l depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules;
(2) a nucleic acid, the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:2 depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules;
(3) a nucleic acid, the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:3 depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules;
(4) a nucleic acid, the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:4 depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules;
(5) a nucleic acid, the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:5 depicted in FIG. 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules;
(6) a nucleic acid, the sequence of which comprises a portion of the nucleotide sequence SEQ ID
NO:l depicted in Table 1, which nucleic acid is capable of specifically binding the complement of inflammatory bowel-specific small RNA molecules;
(7) a nucleic acid, the sequence of which comprises a portion of the nucleotide sequence SEQ ID
NO:2 depicted in Table 1, which nucleic acid is capable of specifically binding the complement of inflammatory bowel-specific small RNA molecules;
(8) a nucleic acid, the sequence of which comprises a portion of the nucleotide sequence SEQ ID NO:3 depicted in Table 1, which nucleic acid is capable of specifically binding the complement of inflammatory bowel-specific small RNA molecules;
(9) a nucleic acid, the sequence of which comprises a portion of the nucleotide sequence SEQ ID
NO:4 depicted in Table 1, which nucleic acid is capable of specifically binding the complement of inflammatory bowel-specific small RNA molecules; and
(10) a nucleic acid, the sequence of which comprises a portion of the nucleotide sequence SEQ ID
NO:5 depicted in FIG. 1, which nucleic acid is capable of specifically binding the complement of inflammatory bowel-specific small RNA molecules.
In another embodiment, the method for diagnosing an inflammatory bowel disease comprises: (a) obtaining frozen or fixed tissue sections or nucleic acids from a human tissue, fluid, or stool sample suspected of containing an inflammatory bowel disease-specific small RNA molecule; (b) contacting the frozen or fixed tissue sections or nucleic acids of step (a) with a nucleic acid reagent selected from the group consisting of:
(1) a nucleic acid, the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:l depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules;
(2) a nucleic acid, the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:2 depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules;
(3) a nucleic acid, the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:3 depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules;
(4) a nucleic acid, the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:4 depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules; and
(5) a nucleic acid, the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:5 depicted in FIG. 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules; under conditions favorable for the specific annealing of the nucleic acid reagent to the complementary sequence within the inflammatory bowel disease- specific small RNA molecule; (c) removing all non- annealed nucleic acid reagent from the nucleic acid reagent:inflammatory bowel disease-specific small RNA molecule hybrid; and (d) detecting the nucleic acid reagent specifically annealed to the inflammatory bowel disease-specific small RNA molecule.
The method for diagnosing a human inflammatory bowel disease may also comprise:
(a) obtaining a human sample suspected of containing an inflammatory bowel disease-specific peptide;
(b) exposing the sample of step (a) to an antibody directed against an epitope present on the inflammatory bowel disease-specific peptide for a time sufficient for specific binding of the antibody and the peptide to occur so that an antibody:peptide hybrid is formed comprising the antibody and the inflammatory bowel disease-specific peptide;
(c) removing all peptide and antibody in solution not in the antibody:peptide hybrid; and (d) detecting the antibody:peptide hybrid. The present invention further contemplates pre¬ packaged diagnostic kits, conveniently used, e.g.. in clinical settings, comprising at least one of the specific nucleic acid reagents described herein for use as primers and/or probes in conjunction with he diagnostic methodology described herein.
It is still further contemplated that certain of the inflammatory bowel disease-specific small RNA molecules described herein may be produced by an infectious agent responsible for such inflammatory bowel diseases. Therefore, it is an object of this invention to identify and isolate such infectious agents and, further, to develop preventative and ameliorative inflammatory bowel disease treatments, such as vaccines directed against such infectious agents.
3.1. ABBREVIATIONS
Amino Acid Three Letter Code
Alanine Ala
Arginine Arg
Asparagine Asn
Aspartic Acid Asp
Cysteine Cys
Glutamic Acid Glu
Glutamine Gin
Glycine Gly
Histidine His
Isoleucine He Amino Acid Three Letter Code
Leucine Leu
Lysine Lys
Methionine Met
Phenylalanine Phe
Proline Pro
Serine Ser
Threonine Thr
Tryptophan Trp
Tyrosine Tyr
Valine Val
Not specified
4. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 Crohn's disease/ulcerative colitis - specific human rRNA diagram. A fragment (SEQ ID NO: 5) of human 28s rRNA is depicted which corresponds to one of the Crohn's disease/ulcerative colitis-specific small RNAs of the invention. The fragment is depicted in its predicted secondary structure form. SEQ. ID NO:3 corresponds to the nucleotide sequence between the black arrows; SEQ. ID NO:4 corresponds to the nucleotide sequence between the white arrows.
FIG. 2 Two dimensional polyacrylamide gel analysis of total nucleic acid populations extracted from intestinal tissue of: (A) Crohn's disease patient; (B) ulcerative colitis patient; (C) normal subject; (D) intestinal cancer patient. Polyacrylamide gels were made according to Pechan et al.. 1987, Z. Naturforsch 42c:1006-1008. Acrylamide concentration is 10% in both directions. Origin is always located at the left-hand bottom corner. Nucleic acids were visualized by silver staining (see Section 6.1 for details). The arrows 1 and 2 indicate inflammatory bowel disease-specific RNA. Species useful for orientation of the gel (i.e.. 5s RNA and 7s RNA species) are also indicated. FIG. 3 Two dimensional polyacrylamide gel analysis of total nucleic acid populations extracted from intestinal tissue of: (A) Crohn's disease patient (S93-10984) ; and (B) control, colon cancer patient (S92-21508) . The arrows 1 and 2 indicate inflammatory bowel disease-specific RNA. Species useful for orientation of the gel (i.e.. 5s RNA, 7s RNA, and tRNA species) are also indicated.
FIG. 4 Northern analysis demonstrating the presence of a small RNA band in Crohn's disease and ulcerative colitis tissue RNA samples which is absent from control (colon cancer) tissue RNA samples. Control RNA: Pathogen. #21, 22; Crohn's RNA: Pathogen. #25, S93-5949, S93-6839, S93-10984; ulcerative colitis RNA: Pathogen. #3, 9, Boston #1.
5. DETAILED DESCRIPTION OF THE INVENTION Reagents and detection schemes which may be used for the diagnosis of the human inflammatory bowel diseases, ulcerative colitis and Crohn's disease, are presented here. Using such reagents and detection schemes, simple, rapid, unequivocal, and early diagnosis of these diseases is possible for the first time.
First, nucleic acid reagents and detection procedures are presented which may be utilized to detect small RNA molecules with anomalous electrophoretic mobilities that are found in ulcerative colitis- and/or Crohn's-diseased tissue, but are absent from, or are present in greatly reduced levels in, the corresponding normal, non-diseased tissue. Working Examples are presented describing the identification of these small, RNA molecules having anomalous electrophoretic mobilities, and further presenting a basis for the nucleic acid diagnostic technique.
Second, peptide and antibody reagents and detection procedures are presented which may be utilized to detect peptides produced by the small RNA molecules, described above, which are found in ulcerative colitis- and/or Crohn's-diseased tissue.
Further, because it is possible that some of the inflammatory bowel disease-specific small RNA molecules described herein may be produced by infectious agents, methods are presented for the isolation and identification of such agents.
5.1 CROHN'S DISEASE AND ULCERATIVE COLITIS SPECIFIC NUCLEIC ACID REAGENTS
As demonstrated in the Working Example presented in Section 6, below, small RNA molecules, or partial sequences derived from them, have been identified which are specific to Crohn's or ulcerative colitis- diseased tissue, i.e.. are present in the diseased tissue, but are absent from, or present in greatly reduced levels in, the corresponding normal, non- diseased tissue. "Greatly reduced", as used herein, refers to an amount of RNA that is too low to be visible when analyzed according to the 2-dimensional gel electrophoresis techniques described, below. While no information yet exists as to whether RNA molecules are causative disease agents or, alternatively, are a consequence of disease, the detection of such molecules may be used for the diagnosis of ulcerative colitis and Crohn's disease. The small RNA molecules, which are uniquely identifiable by the nucleic acid reagents disclosed herein, and by their anomalous electrophoretic mobilities, are approximately 120-250 nucleotides in length. The "anomalous electrophoretic mobilities" of these inflammatory bowel-specific RNA molecules refers to the characteristic of these molecules, when analyzed via two dimensional gel electrophoresis techniques such as those described below, to migrate to a position within the gel separate from the "diagonal" formed by the bulk of the RNA species within any given population of molecules being electrophoresed. Examples of the migration characteristics of two such RNA species (marked by arrows 1 and 2) are depicted in FIGS. 2A-B and 3A, in which, first, the migration distinct from the diagonal is visible, and second, the position to which the molecules migrate may be easily oriented by comparing to the 5s, 7s, and tRNA species, as marked.
The nucleic acid reagents which may be used in the diagnosis of ulcerative colitis and Crohn's disease include, but are not limited to, all or part of the nucleic acids comprising the sequences (depicted as RNA molecules) shown below in Table 1, and/or their complements, provided such nucleic acid reagents are capable of specifically hybridizing
(i.e.. binding or annealing) to inflammatory bowel disease-specific small RNA molecules or their complements. Further, such nucleic acid reagents may also include nucleic acids that comprise all or a portion of the nucleic acid comprising the sequence (SEQ ID No. 5) depicted in FIG. 1 which is capable of specially hybridizing to inflammatory bowel disease - specific small RNA molecules or their complements. Additionally, such nucleic acid reagents may also include nucleic acid molecules having the nucleotide sequence of SEQ ID NOs: 1-5, which are capable of specifically binding inflammatory bowel disease- specific small RNA molecules or their complements. Typically, the nucleic acid reagents are oligonucleotides about 15 to about 30 nucleotides in length, the sequences of which represent a contiguous stretch of the aforesaid length anywhere along the sequences depicted in Table 1, below. In addition, therefore, the term "portion", as used herein will typically refer to portions of the nucleic acid molecules or their complements which are at least about 15-30 nucleotides in length.
TABLE 1
1. (SEQ ID N0:1)
5•GCAAAGCCGAGAUAGGCAUUACUGGACNGGGUACNCGGG3 '
2. (SEQ ID NO:2) 5'GAA/UUACAAACAAUCCCCACGCACCCCGACACA3 '
3. (SEQ ID NO:3) 5'GUCCGUCCGUCCGUCCUCCUCCUCCCCCGUCUCCG3
4. (SEQ ID NO:4) 5'CGGCGGCGGCGGYGGCGGCGGCGGCGGCGGCGG3
N refers to any nucleotide; Y refers to a pyrimidine; "A/U', in SEQ ID NO: 2, refers to a nucleotide position which may contain either an A or a U base.
The conditions under which the nucleic acid reagents of the invention specifically hybridize to inflammatory bowel disease-specific molecules are standard hybridization conditions well known to those of skill in the art. See, for example, Ausubel et al. (Ausubel, M. et al. , ed. , 1987, "Current Protocols in Molecular Biology", Vol. 1-2) and Sambrook et al. (Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press) which are incorporated herein by reference in their entirety.
In instances where partial nucleotide sequences are known for the inflammatory bowel-specific RNA molecules of the invention, techniques which are well known to those of skill in the art exist whereby full length nucleotide sequences may be obtained. For example, the partial sequence may be used to synthesize an oligonucleotide which may be used as a primer for a reverse transcription reaction in order to produce a cDNA copy of the small RNA molecule of interest. The cDNA may then be sequenced using standard DNA sequencing techniques, coupled, if necessary, with well known nucleic acid amplification procedures. By utilizing combinations of primers and the sequences obtained from such primers, the entire nucleotide sequence of the RNA molecules of interest may be obtained. Certain of the inflammatory bowel disease-specific small RNA molecules of the invention may represent portions of mRNA transcripts or RNA genomes. Techniques which are well known to those of skill in the art are available with which to use the nucleotide sequence of the small RNA molecules to identify and isolate nucleic acid molecules comprising the full length coding sequences of such mRNA transcripts, genomic material, or the complements thereof. See for example Ausubel, supra.. and Maniatis, supra. Nucleic acid reagents comprising all or any portion of the sequences, such as those described above, which are capable of specifically hybridizing to inflammatory bowel disease-specific small RNA molecules or their complements may be utilized for the detection of Crohn's disease- or ulcerative colitis-specific nucleic acid molecules (e.g.. small RNA molecules) . Nucleic acid molecules of either DNA or RNA of the same sequences as those listed above may be utilized for diagnostic reagents. Alternatively, nucleic acid molecules, either DNA or RNA, of the complementary sequence to those listed above may be utilized for diagnostic reagents. Such nucleic acid reagents will generally be single stranded molecules, although, in addition, double stranded molecules may also be utilized. It should be noted that when DNA molecules are being used, each U (uridine) base is replaced by T (thymidine) base. Further, any nucleic acid molecule exhibiting a nucleotide sequence so substantially similar to the nucleotide sequence of SEQ ID NOS: 1-5 or to the complement of SEQ ID NOS: 1-5 (e.g.. about 90% homology to the nucleotide sequence of SEQ ID NOS: 1-5, or to the complement of SEQ ID NOS: 1-5) , such that the nucleic acid will still specifically recognize (i.e. , hybridize, bind, or anneal to) Crohn's disease- or ulcerative colitis-specific nucleic acid molecules may also be used as a nucleic acid reagent for diagnosis of these diseases. Still further, the nucleic acid reagents of the invention may contain one or more modifications within their phosphodiester "backbones" which will not appreciably diminish the molecules' ability to hybridize to Crohn's disease- or ulcerative colitis- specific nucleic acid molecules. Such modifications, may include, but are not limited to the use of phosphorothioate or phosphoroamidate groups, phosphate triesters, carbamates, or methyl phosphonates, the synthetic chemistries of which are well known to those in the art. Additionally, the nucleic acid reagents to be used for diagnosis may be labeled using standard radioactive, fluorescent, or chromogenic labeling techniques which are well known to those of ordinary skill in the art.
5.2 CROHN'S DISEASE AND ULCERATIVE COLITIS SPECIFIC PEPTIDE AND ANTIBODY REAGENTS
As demonstrated in the Working Example presented in Section 6, below, small RNA molecules have been identified which are specific to Crohn's or ulcerative colitis diseased tissue. Certain of these RNA molecules may represent m-RNA transcripts, or portions of m-RNA transcripts, which encode inflammatory bowel disease- specific proteins. It is possible that certain of these disease-specific RNA molecules may be produced by infectious agents responsible for the development of inflammatory bowel diseases. In the case of infectious agents possessing RNA genomes, the RNA molecules of the invention may represent portions of RNA genomic, as opposed to transcriptional, material. As such, inflammatory bowel disease specific proteins may be encoded by the complement of the RNA molecules described herein.
Such potential Crohn's disease and/or ulcerative colitis specific proteins may be comprised, for example, of peptides such as those listed, below, in Table 2. Each of these peptides is oriented with the amino terminus at the left and the carboxy terminus on the right. In this table, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 represent peptides that may be encoded by the portion of the disease- specific RNA molecule whose nucleotide sequence is listed in Table 1, above, as SEQ ID NO: 1. Similarly, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 represent peptides that may be encoded by the complement of the portion of the disease-specific RNA molecule whose nucleotide sequence is listed in Table 1, above, as SEQ ID NO: 1. Further, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14 represent peptides that may be encoded by the portion of the disease-specific RNA molecule whose nucleotide sequence is listed in Table 1, above, as SEQ ID NO: 2. Similarly, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18 represent peptides that may be encoded by the complement of the portion of the disease-specific RNA molecule whose nucleotide sequence is listed in Table 1, above, as SEQ ID NO: 2.
TABLE 2
(SEQ ID NO: 6) Ala-Lys-Pro-Arg
(SEQ ID NO: 7) Gln-Ser-Arg-Asp-Arg-His-Tyr-Trp-Thr-Gly- Tyr-Ser/Pro/Thr/Ala
(SEQ ID NO: 8) Lys-Ala-Glu-Ile-Gly-Ile-Thr-Gly- Leu/Pro/Gln/Arg-Gly-Thr-Arg
(SEQ ID NO: 9) Pro-Val/Ala/Glu/Gly-Tyr-Pro-Val-Gln
(SEQ ID NO: 10) Pro-Cys/Arg/Ser/Gly-Thr-Leu/Pro/Gln/Arg- Ser-Ser-Asn-Ala-Tyr-Leu-Gly-Phe
(SEQ ID NO: H) Arg-Val-Pro-Cys/Arg/Gly/Ser-Pro-Val-Met- Pro-Ile-Ser-Ala-Leu
(SEQ ID NO: 12) Glu/Asp-Tyr-Lys-Gln-Ser-Pro-Arg-Thr-Pro- Thr
(SEQ ID NO: 13) Asn/Ile-Thr-Asn-Asn-Pro-His-Ala-Pro-Arg- His
(SEQ ID NO: 14) Ile/Leu-Gln-Thr-Ile-Pro-Thr-His-Pro-Asp- Thr
Figure imgf000026_0001
It is possible that the small RNA molecules of invention may represent non-coding portions of m-RNA transcripts or, alternatively, portions of an RNA genome that do not code for proteins. As such, the inflammatory bowel disease-specific proteins will be encoded by the coding portion of the mRNA transcripts, or, alternatively, portions of an RNA genome that encode proteins, of which the small RNA molecules of the invention are a part. Techniques are described, above, in Section 5.1, for the isolation of full length nucleic acids, which would include the peptide-coding portions of the molecules.
Once the amino acid sequence encoding the inflammatory bowel disease-specific peptides of the invention is known, such peptides may be produced by utilizing any of a variety of peptide synthesis and expression techniques which are well known to those of skill in the art. For example, methods such as those described, below, in Section 5.2.1 may be used.
Further, antibodies may be raised which are directed against any of the inflammatory bowel disease-specific peptides. Techniques for the production of antibodies are well known to those of skill in the art, and may include, for example, procedures such as those described, below, in Section 5.2.2.
5.2.1 SYNTHESIS AND EXPRESSION OF INFLAMMATORY BOWEL DISEASE-SPECIFIC PEPTIDES
Methods for the synthesis of polypeptides or fragments thereof, of Crohn's disease and/or ulcerative colitis- specific peptides are well-known to those of ordinary skill in the art. See, for example, Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman and Co., NY, which is incorporated herein, by reference, in its entirety.
Methods for producing Crohn's disease and/or ulcerative colitis-specific peptides by expressing nucleic acid encoding such are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing Crohn's disease and/or ulcerative colitis-specific peptide coding sequences and appropriate transcriptional/translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, for example, the techniques described in Maniatis et al., supra.. and Ausubel et al., supra. DNA and RNA synthesis may, additionally, be performed using an automated synthesizers. See, for example, the techniques described in "Oligonucleotide Synthesis", 1984, Gait, M.J. ed. , IRL Press, Oxford.
A variety of host-expression vector systems may be utilized to express the coding sequences of the Crohn's disease and/or ulcerative colitis-specific peptides of interest. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, exhibit the Crohn's disease and/or ulcerative colitis-specific peptides of the invention. These include but are not limited to microorganisms such as bacteria (e.g., E. coli. B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing Crohn's disease and/or ulcerative colitis-specific peptide coding sequences; yeast (e.g. Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the Crohn's disease and/or ulcerative colitis-specific peptide coding sequences; insect cell systems infected with recombinant virus expres¬ sion vectors (e.g., baculovirus) containing the Crohn's disease and/or ulcerative colitis-specific peptide coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the Crohn's disease and/or ulcerative colitis- specific peptide coding sequences; or mammalian cell systems (e.g. COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter) .
In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the Crohn's disease and/or ulcerative colitis- specific peptide being expressed. For example, when large quantities of Crohn's disease and/or ulcerative colitis- specific peptides are to be produced for the generation of antibodies, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the Crohn's disease and/or ulcerative colitis- specific peptide coding sequence may be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509) ; and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S- transferase (GST) . In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the Crohn's disease and/or ulcerative colitis-specific peptide can be released from the GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The Crohn's disease and/or ulcerative colitis- specific peptide coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter) . Successful insertion of the Crohn's disease and/or ulcerative colitis- specific peptide coding sequence will result in inactivation of the polyhedrin gene and production of non- occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene) . These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. (E.g., see Smith et al., 1983, J. Viol. 46:584; Smith, U.S. Patent No. 4,215,051). In mammalian host cells, a number of viral based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the Crohn's disease and/or ulcerative colitis-specific peptide coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non- essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing Crohn's disease and/or ulcerative colitis-specific peptides in infected hosts. (E.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655- 3659) . Specific initiation signals may also be required for efficient translation of inserted Crohn's disease and/or ulcerative colitis-specific peptide coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire Crohn's disease and/or ulcerative colitis-specific peptide gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of the Crohn's disease and/or ulcerative colitis-specific peptide coding sequence is inserted, exogenous translational control signals, including the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153.:516-544) .
In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cells lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc.
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express Crohn's disease and/or ulcerative colitis-specific peptides may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with the Crohn's disease and/or ulcerative colitis-specific peptide DNA independently or coordinately controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine 5 kinase (Wigler, et al., 1977, Cell .11:223), hypoxanthine- guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 45:2026), and adenine phosphoribosyltransferase (Lowy, et al. , 1980, Cell 22:817) genes can be employed in tk", hgprt" or aprt" cells, 0 respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 7_8:1527); gpt, which confers resistance to 5 mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 7_8:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1) ; and hygro, which confers resistance to hygromycin (Santerre, et al., 1984, Gene 3J):147) genes. 0
5.2.2 ANTIBODIES TO CROHN'S DISEASE AND/OR ULCERATIVE COLITIS-SPECIFIC PEPTIDES
Described herein are methods for the production of antibodies which are capable of specifically recognizing a 5 Crohn's disease and/or ulcerative colitis-specific peptide of an epitope thereof. It should be understood that techniques such as those described herein may also be utilized for the production of antibodies directed against infectious agents, or epitopes thereof, which are capable Q of causing inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis.
Antibodies may include, but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs) , humanized or chimeric antibodies, single chain antibodies, 5 Fab fragments, F(ab')2 fragments, fragments produced by a FAb expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. Such antibodies may be used, for example, in the detection of a Crohn's disease and/or ulcerative colitis- specific peptide in a biological sample. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as a Crohn's disease and/or ulcerative colitis-specific peptide, or an antigenic functional derivative thereof. For the production of polyclonal antibodies, various host animals may be immunized by injection with the Crohn's disease and/or ulcerative colitis-specific peptide, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete) , mineral gels such as lysolecithin, pluronic polyols, polyanionε, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriu parvu .
A monoclonal antibody, which is a substantially homogeneous population of antibodies to a particular antigen, may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to the hybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497: and U.S. Patent No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al.. 1983, Immunology Today 4.:72; Cole et al. , 1983, Proc. Natl. Acad. Sci. USA jBO.:2026-2030) , and the EBV-hybridoma technique (Cole et al. , 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.
In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci., 8JL:6851-6855; Neuberger et al., 1984,
Nature, 312:604-608; Takeda et al., 1985, Nature, 314:452- 454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. Alternatively, techniques described for the production of single chain antibodies (U.S. Patent 4,946,778; Bird, 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA £5:5879-5883; and Ward et al., 1989, Nature 334:544-546) can be adapted to produce anti-Crohn's disease and/or ulcerative colitis-specific peptide single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragment of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Antibody fragments which contain specific binding sites for a Crohn's disease and/or ulcerative colitis- specific peptide may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity to the Crohn's disease and/or ulcerative colitis-specific peptide of interest.
5.3 DIAGNOSIS OF CROHN'S DISEASE AND ULCERATIVE COLITIS
A variety of methods may be employed, utilizing reagents such as those described in Sections 5.1 and 5.2, above, for the diagnosis of the inflammatory bowel diseases, ulcerative colitis and Crohn's disease.
10 Specifically, such reagents may be used for the detection of the presence of peptides and/or nucleic acid molecules specific to these diseases, i.e. , molecules present in diseased tissue but absent from, or present in greatly reduced levels relative to, the corresponding non-diseased
15 tissue, or alternatively, present in a biological sample or bodily fluid of an individual having such an inflammatory bowel disease, but absent from, or present in greatly reduced levels in, the corresponding sample or fluid of an individual in which the disease is not present.
20 The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one of the specific nucleic acid or antibody reagents described herein, which may be conveniently used, e.g.. in clinical settings, to diagnose
__ patients exhibiting inflammatory bowel disease.
Because Crohn's disease can affect the entire esophageal tract and possibly other tissues in addition to bowel and intestinal tissue, the tissues which may be analyzed for evidence of this disease using any such
3Q detection scheme include any human tissue, including, but not limited to, tissue from any portion of the alimentary canal, with bowel or intestinal tissue being preferred. Ulcerative colitis does not visibly manifest itself outside of the colon, therefore, colon tissue is preferred for the
35 analysis of evidence of this disease. Additionally, stool samples may be used in conjunction with any of the detection schemes of the invention for the detection of either Crohn's disease or ulcerative colitis. Further, bodily fluids such as blood for example, may also be used as the starting material for disease detection. When the diagnostic method involves nucleic acid amplification, such as those described below, the starting sample can be a frozen or fixed tissue section or can be quite crude, with very little sample preparation required.
5.3.1 DETECTION OF INFLAMMATORY BOWEL DISEASE-SPECIFIC NUCLEIC ACIDS
RNA from the tissue to be analyzed may easily be isolated using procedures which are well known to those in the art. The methods employed herein may, for example, be such as those described and cited in Pechan, R. et al.. 1987, Z. Naturforsch 42c:1006-1008. particularly an acid phenol purification procedure such as that described in Roe, B.A. 1975, Nucl. Acids Res. 2:21-42, which is incorporated herein by reference in its entirety. Diagnostic procedures may also be performed "in situ" directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no RNA purification is necessary. Nucleic acid reagents such as those described in Section 5.1 may be used as probes and/or primers for such in situ procedures (Nuovo, G.J. , 1992, PCR in situ hybridization: protocols and applications, Raven Press, NY) .
Preferred diagnostic methods for the detection of Crohn's disease- or ulcerative colitis-specific nucleic acid molecules may involve for example, contacting and incubating nucleic acids, derived from the target tissue being analyzed, with one or more labeled nucleic acid reagents as are described in Section 5.1, under conditions favorable for the specific annealing of these reagents to their complementary sequences within the target molecule. Preferably, the lengths of these nucleic acid reagents are at least 15 to 30 nucleotides. After incubation, all non- annealed nucleic acids are removed from the nucleic acid rinflammatory bowel disease-specific small RNA molecule hybrid. The presence of nucleic acids from the target tissue which have hybridized, if any such molecules exist, is then detected. Using such a detection scheme, the target tissue nucleic acid may be immobilized, for example, to a solid support such as a membrane, or a plastic surface such as that on a microtiter plate or polystyrene beads. In this case, after incubation, non-annealed, labeled nucleic acid reagents of the type described in Section 5.1 are easily removed. Detection of the remaining, annealed, labeled nucleic acid reagents is accomplished using standard techniques well-known to those in the art. Alternative diagnostic methods for the detection of
Crohn's disease or ulcerative colitis specific nucleic acid molecules may involve their amplification, e.g., by PCR (the experimental embodiment set forth in Mullis, K.B., 1987, U.S. Patent No. 4,683,202), ligase chain reaction (Barany, F., 1991, Proc. Natl. Acad. Sci. USA 88:189-193) , self sustained sequence replication (Guatelli, J.C. et al.. 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878) , transcriptional amplification system (Kwoh, D.Y et al.. 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177) , Q-Beta Replicase (Lizardi, P.M. et al.. 1988, Bio/Technology
.5:1197), or any other RNA amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of disease- specific RNA molecules if such molecules are present in very low numbers.
In one embodiment of such a detection scheme, a cDNA molecule is obtained from the target RNA molecule (e.g. , by reverse transcription of the RNA molecule into cDNA) . A target sequence within the cDNA is then used as the template for a nucleic acid amplification reaction, such as a PCR amplification reaction, or the like. The nucleic acid reagents used as synthesis initiation reagents (e.g. , primers) in the reverse transcription and nucleic acid amplification steps of this method are chosen from among the nucleic acid reagents described in Section 5.1. The preferred lengths of such nucleic acid reagents are at least 15-30 nucleotides. For detection of the amplified product, the nucleic acid amplification may be performed using radioactively or non-radioactively labeled nucleotides. Alternatively, enough amplified product may be made such that the product may be visualized by standard ethidium bromide staining or by utilizing any other suitable nucleic acid staining method. In addition to the detection methods described above, the Crohn's disease and ulcerative colitis-specific small RNA molecules may be visualized via two dimensional nucleic acid gel electrophoresis techniques well known to those of skill in the art. Such techniques utilize one dimension run under non-denaturing conditions and one dimension run under denaturing conditions. See for example, Pechan et al. (Pechan, R. et al., 1987, Z. Naturforsch 42c:1006- 1008) . Because of their anomalous electrophoretic behavior when electrophoresed via such procedures, the inflammatory bowel specific small RNA molecules may be visualized apart from the diagonal formed by the bulk of the RNA species in a gel containing a population of RNA molecules.
5.3.2 DETECTION OF INFLAMMATORY BOWEL DISEASE SPECIFIC PEPTIDES
Protein from the tissue to be analyzed may easily be isolated using techniques which are well known to those of skill in the art. The protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, D. , 1988,
"Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press) , which is incorporated herein by reference in its entirety.
Preferred diagnostic methods for the detection of Crohn's disease and/or ulcerative colitis-specific peptide molecules may involve, for example, immunoassays wherein Crohn's disease and/or ulcerative colitis-specific peptides are detected by their interaction with an anti-Crohn's disease and/or ulcerative colitis-specific peptide antibody. For example, antibodies, or fragments of antibodies, such as those described, above, in Section 5.2.2, useful in the present invention may be used to quantitatively or qualitatively detect the presence of cells which express Crohn's disease and/or ulcerative colitis-specific peptides. This can be accomplished by im unofluorescence techniques employing a fluorescently labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorimetric detection. Such techniques are especially preferred if the Crohn's disease and/or ulcerative colitis- specific peptides are expressed on the cell surface.
The antibodies (or fragments thereof) useful in the present invention may, additionally, be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of Crohn's disease and/or ulcerative colitis-specific peptides. In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody of the present invention. The histological sample is taken from a tissue suspected of containing Crohn's disease and/or ulcerative colitis-specific peptides. The antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the Crohn's disease and/or ulcerative colitis-specific peptides, but also their distribution in the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.
Immunoassays for Crohn's disease and/or ulcerative colitis-specific peptides typically comprise incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells, or cells which have been incubated in tissue culture, in the presence of a detectably labeled antibody capable of identifying Crohn's disease and/or ulcerative colitis-specific peptides, and detecting the bound antibody by any of a number of techniques well-known in the art. The biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled Crohn's disease and/or ulcerative colitis-specific peptide-specific antibody. The solid phase support may then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on solid support may then be detected by conventional means.
By "solid phase support or carrier" is intended any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation. The binding activity of a given lot of anti-Crohn's disease and/or ulcerative colitis-specific peptide antibody may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
One of the ways in which the Crohn's disease and/or ulcerative colitis-specific peptide-specific- antibody can be detectably labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA) (Voller, A. , "The Enzyme Linked Immunosorbent Assay (ELISA)", Diagnostic Horizons 2:1-7, 1978)) (Microbiological Associates Quarterly Publication, Walkersville, MD) ; Voller, A. et al . , J. Clin . Pathol . 31:507-520 (1978); Butler, J.E., Meth . Enzymol . 73:482-523 (1981); Maggio, E. (ed.), ENZYME IMMUNOASSAY, CRC Press, Boca Raton, FL, 1980; Ishikawa, E. et al . , (eds.) ENZYME IMMUNOASSAY, Kgaku Shoin, Tokyo, 1981) . The enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
Detection may be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect Crohn's disease and/or ulcerative colitis-specific peptides through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoasεays , Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein) . The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
The antibody can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA) .
The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
5.4 IDENTIFICATION AND ISOLATION OF INFLAMMATORY BOWEL DISEASE INFECTIOUS AGENTS
It is possible that certain of the inflammatory bowel disease-specific RNA molecules described herein may be produced by an infectious agent responsible for such inflammatory bowel diseases. A variety of well known methods may be utilized to identify and isolate such inflammatory bowel disease infectious agents.
For example, an inflammatory bowel disease infectious agent, i.e.. a Crohn's disease and/or ulcerative colitis infectious agent may be identified and isolated by immunoaffinity chromatography methods using an immunoadsorbent column to which an antibody is immobilized which is capable of binding the infectious agent. Such an antibody may be monoclonal or polyclonal in origin. Preferably, such an antibody is directed to an epitope on the surface of the Crohn's disease and/or ulcerative colitis infectious agent. "Surface", as used in this context, refers to an epitope that is exposed to the environment external to the infectious agent. The Crohn's disease and/or ulcerative colitis infectious agent may be biochemically purified from a Crohn's disease and/or ulcerative colitis diseased tissue sample. Such tissues include, but are not limited to, inflammatory bowel diseased tissue from any portion of the alimentary canal, with bowel or intestinal tissue being preferred. In the case of an ulcerative colitis infectious agent, colon tissue is preferred. Alternatively, the infectious agent may be isolated from bodily fluids, such as blood, of an individual exhibiting inflammatory bowel disease. Still further, Crohn's disease and/or ulcerative colitis infectious agents may be isolated from stool samples of individuals manifesting inflammatory bowel disease.
A sample suspected of containing a Crohn's disease and/or ulcerative colitis infectious agent is passed over an immunoaffinity column such that the infectious agent binds to the column. Conditions for optimal binding will be well known to those of skill in the art. After binding, all unbound sample materials are washed out of the column. The bound infectious agent is then eluted off the immunoaffinity column, using techniques well known to those of skill in the art. For example, the elution buffer may introduce a saturating amount of a small molecule that mimics the antigen present in the Crohn's disease and/or ulcerative colitis infectious agent, thus competing with the infectious agent itself for antibody binding, and causing the bound infectious agent to be released from the column.
Upon isolation of the Crohn's disease and/or ulcerative colitis infectious agent, such agent may be characterized according to standard procedures well known to those of skill in the art. For example, the ability to propagate such an agent may be studied. Such propagation characteristics may include, but are not limited to, the ability of the infectious agent to grow in cell-free media, and/or the ability of the infectious agent to infect and propagate within cells, preferably alimentary canal cells, and most preferably bowel, intestinal, or colon cells. In addition, the infectious agent may be subjected to well known techniques for the amplification and sequencing of its constituent nucleic acids. See, for example, Ausubel supra. and Sambrook, supra.
A method for the isolation and identification of an inflammatory bowel disease infectious agent may comprise: (a) exposing a sample suspected of containing an inflammatory bowel disease infectious agent to an immobilized antibody directed against an epitope on the surface of the infectious agent for a time sufficient to allow binding of the infectious agent to the antibody; (b) removing all non-bound sample components; and (c) eluting the bound infectious agent from the immobilized antibody, thereby identifying and isolating an inflammatory bowel disease infectious agent.
6. EXAMPLE: IDENTIFICATION OF NUCLEIC ACIDS
DIAGNOSTIC OF CROHN'S DISEASE AND ULCERATIVE COLITIS
In this example, small RNA populations from Crohn's disease or ulcerative colitis tissue are isolated and partially sequenced. Nucleic acid reagents comprising portions of the sequences presented here, and/or their complements, which are capable of specifically hybridizing to (i.e.. binding or annealing to) inflammatory bowel disease-specific small RNA molecules, or their complements, are well suited for use as exquisitely sensitive diagnostic tools for the identification of individuals with these diseases.
6.1 MATERIALS AND METHODS Crude nucleic acid extracts from Crohn•s disease and control samples were prepared from tissues generally according to the acid phenol purification procedure of Roe, B.A., 1975, Nucl. Acids. Res. 2.:21-42. All steps are performed at 4°C, unless otherwise noted, and glass distilled water is used throughout. Briefly, 0.9 mis of liquid phenol (90%), 0.1 ml chloroform and 1.0 mis of 0.14 M sodium acetate (pH 4.5) are added per gram of tissue and homogenized. The mixture is then centrifuged at high speed for 15 minutes using a table top clinical centrifuge with swinging bucket rotors. The upper aqueous phase is removed and kept on ice. To the lower organic phase is added one volume of 0.14 M sodium acetate (pH 4.5) and the mixture is vortexed for two minutes and then centrifuged and described above. The resulting aqueous phase is removed and combined with the previous aqueous phase on ice. The total aqueous phase is combined with two volumes liquid phenol, vortexed for two minutes and centrifuged as described above. The resulting aqueous phase is removed, mixed with one volume phenol and one volume chloroform, vortexed two minutes, and centrifuged as above. The resulting aqueous phase is loaded on a DEAE cellulose (Sigma) column, previously washed and equilibrated with 0.14 M Na Acetate (pH 4.5), at a ratio of approximately 1 ml packed DEAE cellulose per 3 mis aqueous phase. The DEAE cellulose column is washed first with 0.14 M sodium acetate (pH 4.5) at a ratio of 20 is per ml DEAE cellulose, followed by 0.14 M sodium acetate (pH 4.5) that contains 0.3 M sodium chloride (NaCl) at a ratio of 20 mis per ml DEAE cellulose. Nucleic acid is then eluted with 0.14 M Na Acetate, pH 4.5, containing 1.0 M NaCl, at a ratio of 2 mis per gram DEAE cellulose. 10-20 1-1.5 ml fractions are collected and their absorbances at 260 nm (A260) are measured. Those fractions containing nucleic acid are combined and immediately precipitated with 2.5 volumes ice cold absolute ethanol. After standing overnight at -20°C, the precipitate is collected by centrifugation, washed twice with 95% ethanol, and then dried in vacuo.
Small RNAs were separated generally according to the two-dimensional gel methods of Pechan et al. (Pechan, R. et al.. 1987, Zeitschrift fur Naturforschung 42c:1006-1018) , with modifications. Briefly, 10% polyacrylamide gels (20cm x 40cm) were used for both directions. The first dimension (20cm length) was run under non-denaturing conditions at room temperature. The second dimension (40cm length) was run under the denaturing conditions of 7M urea and 50°C- 60°C. The second dimension gel of the two-dimensional gel electrophoretic procedure was stained with silver or ethidium bromide as described below. The latter was used if the RNA was to be extracted from the gel. A gel to be silver stained is soaked successively at room temperature in a 50% ethanol, 10% acetic acid solution for 12 to 16 hours, a 10% ethanol, 1% acetic acid solution for 1 hour, and a 12 mM AgN03 (Silver Nitrate, Sigma, 2.04g/l) solution for 1 hour. The gel is then rinsed with distilled water for 30 seconds and placed in a 0.28% HCHO (formaldehyde, Baker, 7.0 ml/1), 0.25M KOH (potassium Hydroxide, Baker, 14.03g/l) solution with gentle shaking for 1 to 2 minutes and further incubation for 8 to 9 minutes for a total exposure of 10 minutes. The gel is then moved to a new tray and incubated in 0.06 M Na2C03-H20 (sodium carbonate monohydrate, Baker, 7.42g/l) for 15 to 20 minutes. (Because the gels used in this Example were 1.2 mm thick, this step was slow, which may cause background to appear.) After this incubation, the gel is exposed to air for 10 to 20 minutes then conserved in plastic wrap. For incubations, plastic trays and only Millipore-filtered water should be used. Examples of such silver stained gels are shown in FIGs. 2A-D and 3A-B,
A gel to be ethidium bromide (EtBr) stained is soaked in a solution of 500 ng EtBr/1 lx TBE (0.089 M Tris-borate, 0.089 M boric acid, 0.002 M EDTA) for 30 minutes. During incubation, the tray should periodically be gently moved. Following incubation, the gel is destained by soaking in 1 liter TBE (lx) for 10 minutes. The gel is then examined on a long-wave UV transilluminator and the appropriate RNA species is extracted using standard techniques as described by Pechan, R. et al.. 1987, Zeitschrift fur Naturforschung 4_2c:1006-1018.
Conventional methods were used for RNA sequencing.
6.2 RESULTS Small RNA molecules which are specific for Crohn's disease or ulcerative colitis were separated using two- dimensional gel analysis according to Pechan, R. et al. (1987, Zeitschrift fur Natureforschung 42c: 1006-1018; see Section 6.1 for details). An example of one such separation is shown in FIG. 2A-D. As demonstrated in FIG. 2A-D, RNA from the Crohn's disease and the ulcerative colitis tissue samples exhibit RNA species (as indicated by the arrows 1 and 2) which are absent from both non-disease tissue (FIG. 2C) and intestinal cancer tissue (FIG. 2D) .
Small RNAs were isolated from the species indicated by arrow 1 (FIG. 2A) utilizing the techniques described above, in Section 6.1. Isolated RNA molecules were then terminally labeled with 32P, and subjected, utilizing well- known means, to RNA sequencing analysis. Partial sequence (SEQ ID NOs:l-2) information was obtained, as shown in Table 3, below. SEQ ID NO: 1 represents the sequence of the 5' end of the RNA molecule, while SEQ ID NO: 2 represents the sequence of the 3* end of the molecule. It is estimated that the entire RNA molecule is approximately 220 nucleotides in length. Thus, approximately 140 nucleotides lie between the sequence represented by SEQ ID NO: 1 and that represented by SEQ ID NO: 2. No sequences homologous to these were found in publicly accessible nucleotide databases. Databases were searched using BLASTIN (National Center for Biological Information) .
TABLE 3
1. (SEQ ID N0:1)
5'GCAAAGCCGAGAUAGGCAUUACUGGACNGGGUACNCGGG3 '
2. (SEQ ID NO:2) 5'GAA/UUACAAACAAUCCCCACGCACCCCGACACA3 '
N refers to any nucleotide; Y refers to a pyrimidine; "A/U', in SEQ ID NO: 2, refers to a nucleotide position which may contain either an A or a U base.
In FIG 3A-B are the results of another study whereby inflammatory bowel disease small RNA molecules were identified and characterized. In this study, Crohn's disease tissue (FIG. 3A) and colon cancer tissue (FIG. 3B) were electrophoresed by two-dimensional gel analysis and silver-stained as described, above, in Section 6.1. The Crohn's disease-specific RNA species are indicated by the arrows 1 and 2 in FIG. 3A. These analytical gels were loaded with 40 μg of Crohn's disease tissue RNA and 70 μg colon cancer tissue RNA.
One major Crohn's disease-specific RNA species, indicated by arrow 2 (FIG. 3A) migrates together with the 5s rRNA species in the first dimension, and migrates significantly slower than 5s rRNA in the second dimension. This RNA species was isolated, from a preparative two- dimensional gel loaded with 300 μg Crohn's disease tissue RNA. The gel was stained with EtBr and the band containing the RNA of interest was cut out. The RNA was eluted from the gel by soaking the gel piece in 500 μl buffer (10 mM Tris-HCl, pH 7.5, 1 m M EDTA, 0.1 M NaCl, 0.1% sodium dodecyl sulfate (SDS)) at 37° overnight, followed by precipitation.
A small portion of this RNA was labelled. In the 5 absence of dephosphorylation, only the 5' end could be labeled, indicating that the 3' end is protected. The 3' end could be labeled after dephosphorylation, demonstrating that the 3' blocking group was a phosphate group. Portions of the RNA were either 3' or 5' labeled, and subjected to
10 partial alkaline hydrolysis (5 min. at 95° in 50 μg NaC03, pH 9.2, 1 mM EDTA, then precipitated and subsequently separated on an 8% polyacrylamide gel containing 7 M urea. After a short exposure, two prominent RNA bands were visible, corresponding to the full length RNA and to a band
15 approximately one half the full length size. Both RNAs were cut out and purified as described above, then were treated with nucleoside specific endoribonucleases (RNase T, (G) , RNase U2 (A>G) , RNase Phy.M (A and U) , and RNase A (C and U) for RNA sequencing. All endoribonucleases were
•20 purchased from Pharmacia. From the small RNA isolated after partial alkaline hydrolysis, 35 (SEQ ID NO: 3) and 33 (SEQ ID NO: 4) nucleotides were sequenced from the 5' and 3' labeled RNA, respectively. These sequences are shown, below, in Table 4.
25 TABLE 4
3. (SEQ ID NO:3) 5'GUCCGUCCGUCCGUCCUCCUCCUCCCCCGUCUCCG3
4. (SEQ ID NO:4) 5'CGGCGGCGGCGGYGGCGGCGGCGGCGGCGGCGG3
N refers to any nucleotide; Y refers to a pyrimidine
30
Publicly accessible nucleotide databases were searched for sequences homologous to those of Table 4, above, using BLASTN (National Center for Biological Information) . The 35 5' sequence (SEQ ID NO: 3) showed 100% homology with a portion of human 28s rRNA, while the 3' sequence (SEQ ID NO: 4) showed a 96% homology with a different portion of the human 28s rRNA. The two sequences lie 44 nucleotides apart, as shown in the diagram in FIG. 1.
7. EXAMPLE: DIAGNOSIS OF CROHN'S
AND ULCERATIVE COLITIS In this example, nucleic acid reagents are successfully used to detect disease-specific RNA molecules, and thus to rapidly diagnose Crohn's disease and ulcerative colitis-affected tissues.
7.1 MATERIAL AND METHODS Tissue extracts were prepared as described in Section 6.1, above.
Northern blot analysis was performed using 10 μg RNA from: two colon cancer tissue samples (Pathogen. #21, 22) , which acted as the controls; four Crohn's disease tissue samples (Pathogen. #25, S93-5949, S93-6389, and S93-10984) ; and three ulcerative colitis tissue samples (Pathogen. #3, 9, and Boston #1). Samples were electrophoresed on a 10% polyacrylamide gel containing 7 M urea. RNA was transferred to a nylon membrane by electroblotting overnight in a BioRad transblotter at 250 mA, using 10 mM Tris-HCl, pH 7.8, 5 mM Na Acetate, 1 mM EDTA buffer. The membrane was air dried for two hours at room temperature and the RNA was crosslinked to the filter, using a Stratalinker (Stratagene) at 0.12J/cm2. The filter was prehybridized for 3 hours at 42°C in 50% formamide, 5x SSPE (2Ox SSPE: 3M NaCl, 0.2M NaH2P04-H20 (sodium phosphate, monobasic, monohydrate), 200mM EDTA; pH 7.4), 5x Denhardt's (50x Denhardt's: 5g/0.51 Ficoll(Type 400; Pharmacia), 5g/0.51 polyvinylpyrrolidone, 5g/0.51 bovine serum albumin (Fraction V; Sigma)), 0.1% SDS, and 100 μg/ml salmon sperm DNA. 20 pmol of a 5' labeled probe (18 nucleotides; 5' AGGAGGACGGACGGACGG 3'; SEQ ID NO: 19) and hybridized overnight. The membrane was washed once in 0.2xSSC, 0.1% SDS at room temperature, and once in the same buffer at 42°C for 20 minutes. The washed membrane was dried and exposed to film overnight at -70°C.
7.2 RESULTS FIG. 4 shows the results of a Northern analysis wherein RNA isolated from control (colon cancer), Crohn's disease, and ulcerative colitis tissue was probed with a sequence homologous to a portion of the RNA obtained from the RNA species indicated in FIG. 3A, arrow 2.
The arrow in FIG. 4 indicates a band of the same size as that of the FIG. 3A, arrow 2 species. This band is present in 3 of the 4 Crohn's disease RNA samples, in 2 of the 3 ulcerative colitis RNA samples, and is absent from the control RNA samples. The single Crohn's disease RNA sample ulcerative colitis RNA sample in which the diagnostic band is not readily visible may, for example, represent samples in which the diagnostic band is too faint to be detected by Northern analysis, or may represent samples in which the rRNA cleavage step(s) necessary to produce this size of species has not yet occurred.
It is apparent that many modifications and variations of this invention as set forth hereinabove may be made without departing from the spirit and scope thereof. The specific embodiments described above are given by way of example only and the invention is limited only by the terms of the appended claims.

Claims

CLAIMSWhat is claimed is:
1. A method for diagnosing a human inflammatory bowel disease, comprising:
(a) obtaining a human sample suspected of containing an inflammatory bowel disease-specific small RNA molecule;
(b) treating the sample of step (a) with at least one nucleic acid reagent under conditions such that cDNA is made from such inflammatory bowel disease-specific small RNA molecule present among such nucleic acids;
(c) amplifying a target sequence within the cDNA of step (b) according to a nucleic acid amplification method using at least one nucleic acid reagent complementary to the cDNA; and
(d) detecting such amplified cDNA target sequence; wherein the nucleic acid reagent(s) of steps (b) and (c) act(s) as a synthesis initiation reagent(s) and is/are selected from the group consisting of (1) a nucleic acid, the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID N0:1 depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel- specific small RNA molecules; (2) a nucleic acid, the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:2 depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel- specific small RNA molecules; (3) a nucleic acid, the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:3 depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel- specific small RNA molecules; (4) a nucleic acid, the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:4 depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel- specific small RNA molecules;
(5) a nucleic acid, the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:5 depicted in FIG. 1, which nucleic acid is capable of specifically binding inflammatory bowel- specific small RNA molecules;
(6) a nucleic acid, the sequence of which comprises a portion of the nucleotide sequence SEQ ID N0:1 depicted in Table 1, which nucleic acid is capable of specifically binding the complement of inflammatory bowel- specific small RNA molecules;
(7) a nucleic acid, the sequence of which comprises a portion of the nucleotide sequence SEQ ID NO:2 depicted in Table 1, which nucleic acid is capable of specifically binding the complement of inflammatory bowel- specific small RNA molecules;
(8) a nucleic acid, the sequence of which comprises a portion of the nucleotide sequence SEQ ID NO:3 depicted in Table 1, which nucleic acid is capable of specifically binding the complement of inflammatory bowel- specific small RNA molecules;
(9) a nucleic acid, the sequence of which comprises a portion of the nucleotide sequence SEQ ID NO:4 depicted in Table 1, which nucleic acid is capable of specifically binding the complement of inflammatory bowel- specific small RNA molecules; and
(10) a nucleic acid, the sequence of which comprises a portion of the nucleotide sequence SEQ ID NO:5 depicted in FIG. 1, which nucleic acid is capable of specifically binding the complement of inflammatory bowel- specific small RNA molecules.
2. The method of Claim 1 wherein the sample is a frozen or a fixed human tissue section.
3. The method of Claim 1 wherein the sample comprises nucleic acids from a human tissue, human fluid, or human stool sample.
4. The method of Claim 1 wherein the nucleic acid amplification method of step (c) is the polymerase chain reaction.
5. The method of Claim 1 wherein the human inflammatory bowel disease is Crohn's disease.
6. The method of Claim 1 wherein the human inflammatory bowel disease is ulcerative colitis.
7. The method of Claim 1 wherein the inflammatory bowel disease-specific nucleic acid reagent is about 15 nucleotides to about 30 nucleotides in length.
8. The method of Claim 1 wherein the inflammatory bowel disease-specific nucleic acid reagent is a DNA molecule.
9. The method of Claim 1 wherein the inflammatory bowel disease-specific nucleic acid reagent is a RNA molecule.
10. The method of Claim 1 wherein the inflammatory bowel disease-specific nucleic acid reagent contains one or more modified nucleotides which do not affect the ability of the reagent to bind inflammatory bowel disease-specific small RNA molecules or complements thereof.
11. The method of Claim 1 wherein the detection of step (d) is performed with an inflammatory bowel disease- specific nucleic acid reagent according to Claim 1 which is labeled.
12. The method of Claim 1 wherein the sample is taken from any part of the human alimentary canal.
13. The method of Claim 12 wherein the sample is taken from a human bowel or a human intestinal tissue.
14. The method of Claim 6 wherein the sample is taken from a human colon tissue.
15. A method for diagnosing a human inflammatory bowel disease, comprising:
(a) obtaining a human sample suspected of containing an inflammatory bowel disease-specific small RNA molecule;
(b) contacting the sample of step (a) with a nucleic acid reagent selected from the group consisting of
(1) a nucleic acid, the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID N0:1 depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel- specific small RNA molecules;
(2) a nucleic acid, the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:2 depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel- specific small RNA molecules;
(3) a nucleic acid, the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:3 depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel- specific small RNA molecules; (4) a nucleic acid, the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:4 depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel- specific small RNA molecules; and
(5) a nucleic acid, the sequence of which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:5 depicted in FIG. 1, which nucleic acid is capable of specifically binding inflammatory bowel- specific small RNA molecules; under conditions favorable for the specific annealing of the nucleic acid reagent to the complementary sequence within the inflammatory bowel disease-specific small, circular RNA molecule; (c) removing all non-annealed nucleic acid reagent from the nucleic acid reagent:inflammatory bowel disease- specific small RNA molecule hybrid; and (d) detecting the nucleic acid reagent specifically annealed to the inflammatory bowel disease-specific small RNA molecule.
16. The method of Claim 15 wherein the sample is a frozen or fixed human tissue section.
17. The method of Claim 15 wherein the sample comprises nucleic acids from a human tissue, human fluid, or human stool sample.
18. The method of Claim 15 wherein the human inflammatory bowel disease is Crohn's disease.
19. The method of Claim 15 wherein the human inflammatory bowel disease is ulcerative colitis.
20. The method of Claim 15 wherein the inflammatory bowel disease-specific nucleic acid reagent is about 15 nucleotides to about 30 nucleotides in length.
21. The method of Claim 15 wherein the inflammatory bowel disease-specific nucleic acid reagent is a DNA molecule.
22. The method of Claim 15 wherein the inflammatory bowel disease-specific nucleic acid reagent is a RNA molecule.
23. The method of Claim 15 wherein the inflammatory bowel disease-specific nucleic acid reagent contains one or more modified nucleotides which do not affect the sequence's ability to bind inflammatory bowel disease- specific small RNA molecules or their complements.
24. The method of Claim 15 wherein the inflammatory bowel disease-specific nucleic acid reagent is labeled.
25. The method of Claim 15 wherein the human tissue sample is taken from any part of the human alimentary canal.
26. The method of Claim 25 wherein the sample is taken from a human bowel or a human intestinal tissue.
27. The method of Claim 19 wherein the sample is taken from a human colon tissue.
28. An isolated nucleic acid, the sequence of which is selected from the group consisting of
(1) the sequence which comprises the complement of a portion of the nucleotide sequence SEQ ID N0:1 depicted in Table 1, which portion is capable of specifically binding inflammatory bowel-specific small RNA molecules;
(2) the sequence which comprises the complement of a portion of the nucleotide sequence SEQ ID NO:2 depicted in Table 1, which portion is capable of specifically binding inflammatory bowel-specific small RNA molecules;
(3) the sequence which comprises a portion of the nucleotide sequence SEQ ID NO:l depicted in Table 1, which portion is capable of specifically binding the complement of inflammatory bowel-specific small RNA molecules; and
(4) the sequence which comprises a portion of the nucleotide sequence SEQ ID NO:2 depicted in Table 1, which portion is capable of specifically binding the complement of inflammatory bowel-specific small RNA molecules.
29. The isolated nucleic acid of Claim 28 wherein the nucleic acid is a DNA molecule.
30. The isolated nucleic acid of Claim 28 wherein the nucleic acid is a RNA molecule.
31. The isolated nucleic acid of Claim 28 wherein the nucleic acid contains one or more modified nucleotides which do not affect the ability of the reagent to bind inflammatory bowel disease-specific small RNA molecules or complements thereof.
32. An isolated nucleic acid, the sequence of which comprises the complement of the nucleotide sequence SEQ ID NO: 1 or SEQ ID NO: 2, depicted in Table 1. 33. An isolated nucleic acid, the sequence of which is selected from the group consisting of
(1) the complement of the nucleotide sequence SEQ ID N0:1 depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules;
(2) the complement of the nucleotide sequence SEQ ID NO:2 depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules;
(3) the complement of the nucleotide sequence SEQ ID NO:3 depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules; (4) the complement of the nucleotide sequence
SEQ ID NO:4 depicted in Table 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules;
(5) the complement of the nucleotide sequence SEQ ID NO:5 depicted in FIG. 1, which nucleic acid is capable of specifically binding inflammatory bowel-specific small RNA molecules;
(6) the nucleotide sequence SEQ ID NO:l depicted in Table 1, which nucleic acid is capable of specifically binding the complement of inflammatory bowel-specific small RNA molecules;
(7) the nucleotide sequence SEQ ID NO:2 depicted in Table 1, which nucleic acid is capable of specifically binding the complement of inflammatory bowel-specific small RNA molecules;
(8) the nucleotide sequence SEQ ID NO:3 depicted in Table 1, which nucleic acid is capable of specifically binding the complement of inflammatory bowel-specific small RNA molecules; (9) the nucleotide sequence SEQ ID NO:4 depicted in Table 1, which nucleic acid is capable of specifically binding the complement of inflammatory bowel-specific small RNA molecules; and (10) the nucleotide sequence SEQ ID NO:5 depicted in FIG. 1, which nucleic acid is capable of specifically binding the complement of inflammatory bowel-specific small RNA molecules.
33. A pre-packaged kit for the diagnosis of human inflammatory bowel disease comprising at least one of the isolated nucleic acids of Claim 28.
34. An isolated inflammatory bowel disease-specific peptide encoded by the nucleic acid of Claim 28, and comprising the amino acid sequence SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18, as depicted in Table 2.
35. An isolated antibody directed against an epitope present on the peptide of Claim 34.
36. The isolated antibody of Claim 34 wherein the isolated antibody is a monoclonal antibody.
37. A method for diagnosing a human inflammatory bowel disease, comprising: (a) obtaining a human sample suspected of containing an inflammatory bowel disease-specific peptide; (b) exposing the sample of step (a) to an antibody according to Claim 35 for a time sufficient for specific binding of the antibody and the peptide to occur so that an antibody:peptide hybrid is formed comprising the antibody according to Claim 35 and the inflammatory bowel disease- specific peptide;
(c) removing all peptide and antibody in solution not in the antibody:peptide hybrid; and (d) detecting the antibody:peptide hybrid.
38. The method of Claim 37 wherein the sample comprises peptides from a human tissue, human fluid, or human stool sample.
39. The method of Claim 37 wherein the human inflammatory bowel disease is Crohn's disease.
40. The method of Claim 37 wherein the human inflammatory bowel disease is ulcerative colitis.
41. The method of Claim 37 wherein the sample is taken from any part of the human alimentary canal.
42. The method of Claim 41 wherein the human sample is taken from a human bowel or a human intestinal tissue.
43. The method of Claim 40 wherein the sample is taken from a human colon tissue.
44. The method of Claim 37 wherein the antibody is a monoclonal antibody.
45. The method of Claim 37 wherein the human sample is immobilized on a solid support.
46. The method of Claim 37 wherein the antibody is immobilized on a solid support.
47. A pre-packaged kit for the diagnosis of human inflammatory bowel disease comprising at least one of the isolated antibodies of Claim 35.
48. A method for the isolation of an inflammatory bowel disease infectious agent may comprise:
(a) exposing a sample suspected of containing an inflammatory bowel disease infectious agent to an immobilized antibody according to Claim 35 for a time sufficient to allow binding of the infectious agent to the antibody;
(b) removing all non-bound sample components; and
(c) eluting the bound infectious agent from the immobilized antibody, thereby isolating an inflammatory bowel disease infectious agent.
49. The method of Claim 48 wherein the inflammatory bowel disease is Crohn's disease.
50. The method of Claim 48 wherein the inflammatory bowel disease is ulcerative colitis.
51. An isolated inflammatory bowel disease infectious agent produced by the method of Claim 48.
PCT/US1994/002806 1993-03-15 1994-03-15 Diagnosis of human inflammatory bowel diseases and nucleic acid reagents therefor WO1994021662A1 (en)

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WO2015067913A1 (en) 2013-11-07 2015-05-14 Diagnodus Limited Biomarkers
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AP750A (en) * 1995-01-25 1999-07-06 Aventis Pharma Sa Method for preparing 4,10-diacetoxy-2a-benzoyloxy-5b, 20-epoxy-1, 7b-dihydroxy-9-oxo-tax-11-en-13a-y) (2r,3s) -3-benzoylamono-2-hydroxy-3-Phenylpropionate trihydrate.
WO2015067913A1 (en) 2013-11-07 2015-05-14 Diagnodus Limited Biomarkers
CN110432924A (en) * 2019-08-06 2019-11-12 杭州智团信息技术有限公司 Borborygmus sound detection device, method and electronic equipment
CN110432924B (en) * 2019-08-06 2021-10-22 杭州智团信息技术有限公司 Bowel sound detection device and method and electronic equipment

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