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WO1994028021A1 - Proteines endometriales, compositions antigeniques et procedes de detection de l'endometriose - Google Patents

Proteines endometriales, compositions antigeniques et procedes de detection de l'endometriose Download PDF

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Publication number
WO1994028021A1
WO1994028021A1 PCT/US1994/006081 US9406081W WO9428021A1 WO 1994028021 A1 WO1994028021 A1 WO 1994028021A1 US 9406081 W US9406081 W US 9406081W WO 9428021 A1 WO9428021 A1 WO 9428021A1
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WIPO (PCT)
Prior art keywords
endometriosis
protein
endometrial
antigen
antibody
Prior art date
Application number
PCT/US1994/006081
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English (en)
Inventor
Subbi Mathur
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Medical University Of South Carolina
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Filing date
Publication date
Application filed by Medical University Of South Carolina filed Critical Medical University Of South Carolina
Priority to AU69606/94A priority Critical patent/AU6960694A/en
Publication of WO1994028021A1 publication Critical patent/WO1994028021A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57442—Specifically defined cancers of the uterus and endometrial
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/364—Endometriosis, i.e. non-malignant disorder in which functioning endometrial tissue is present outside the uterine cavity

Definitions

  • the invention relates to purified endometriosis-associated proteins (free of albumin), purified endometriosis-associated antigenic compositions, and methods of diagnosing endometriosis.
  • Endometriosis is a complex disease with possibly multiple causes frequently seen in association with infertility.
  • Dysmenorrhea menstrual abnormalities
  • endometriosis is a complex disease with possibly multiple causes frequently seen in association with infertility.
  • Dysmenorrhea menstrual abnormalities
  • endometriosis is a complex disease with possibly multiple causes frequently seen in association with infertility.
  • Dysmenorrhea menstrual abnormalities
  • Chihal et al. disclose the development of an endometrial antibody monitoring assay using whole endometrial extract (Chihal et al., 1986), which is expensive to obtain in reagent quantities.
  • Endometrial antigens and verifying their immunogenicity in an animal model are important steps for further purification and isolation.
  • Antigens immunogenic in humans sometimes may be weakly antigenic in animals, which may hinder attempts at purifying the relevant antigens.
  • Establishing an endometrial antigen specific antibody assay for noninvasive diagnosis of endometriosis requires that the target endometrial autoantigens exist and elicit antibody responses in patient populations regardless of geographical location.
  • the present invention overcomes the above-stated difficulties and provides noninvasive methods of diagnosing endometriosis.
  • a purified antigen composition having a molecular weight of about 64-66 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis is provided.
  • Purified antigenic endometrial proteins, separated from albumin and having a molecular weight of about 64 kilodaltons as determined by sodium dodecyl sulphate gel electrophoresis under reducing conditions and isoelectric focusing points (pi) of about 3.5, 4.0, 6.0, 6.5 and 8.0 are provided.
  • a purified antigenic endometrial protein separated from albumin and having a molecular weight of about 94-97 kilodaltons as determined by sodium dodecyl sulphate gel electrophoresis under reducing conditions and a pi of about 3.5 is also provided.
  • the presence of antibodies that specifically bind the antigen composition or antigenic protein is a marker for endometriosis in a subject.
  • a purified monoclonal antibody that specifically binds the antigenic composition or protein is also provide .
  • a method of diagnosing endometriosis in a subject comprises the steps of (a) contacting an antibody-containing sample from the subject with the 64-66 kDa antigen of the invention; and (b) detecting the reaction of the antigen with an antibody from the sample, the reaction indicating endometriosis in the subject.
  • the antigen of the above method can also be one or a combination of the present antigenic proteins.
  • the method comprises the steps of (a) contacting a sample from the subject with the monoclonal antibody; and (b) detecting the reaction of the antibody with the antigen in the sample, the reaction indicating endometriosis in the subject.
  • the detecting step in the above methods can be an enzyme linked immunosorbent assay (ELISA), an immunofluorescence assay, a western blot assay or a passive hemagglutination assay.
  • ELISA enzyme linked immunosorbent assay
  • An example of passive hemagglutination, immunofluorescence, ELISA and Western blot analyses are provided in the Examples.
  • Figure 1 is a schematic depiction of a two dimensional gel of unabsorbed endometrial and implant antigens from patients with endometriosis, including standards.
  • Figure 2 is a schematic depiction of a two dimensional gel of absorbed endometrial and implant antigens from patients with endometriosis, including standards.
  • Figure 3 is a schematic depiction of a Western blot of a two dimensional gel of endometrial and implant antigens from patients with endometriosis with serum from patients with endometriosis, including standards.
  • the invention further provides a purified antigenic endometrial protein, separated from albumin and having a molecular weight of about 64 kilodaltons as determined by sodium dodecyl sulphate gel electrophoresis under reducing conditions and an isoelectric focusing point of about 3.5.
  • the invention further provides a purified antigenic endometrial protein, separated from albumin and having a molecular weight of 94-97 kilodaltons as determined by sodium dodecyl sulphate gel electrophoresis under reducing conditions and an isoelectric focusing point of about 3.5.
  • the invention further provides a purified antigenic endometrial protein, separated from albumin and having a molecular weight of about 64 kilodaltons as determined by sodium dodecyl sulphate gel electrophoresis under reducing conditions and an isoelectric focusing point of about 6.5.
  • the invention provides a purified antigenic endometrial protein, separated from albumin and having a molecular weight of about 64 kilodaltons as determined by sodium dodecyl sulphate gel electrophoresis under reducing conditions and an isoelectric focusing point of about 8.0.
  • the invention further provides a purified antigenic endometrial protein, separated from albumin and having a molecular weight of about 64 kilodaltons as determined by sodium dodecyl sulphate gel electrophoresis under reducing conditions and an isoelectric focusing point of about 6.0.
  • the invention further provides a purified antigenic endometrial protein, separated from albumin and having a molecular weight of about 64 kilodaltons as determined by sodium dodecyl sulphate gel electrophoresis under reducing conditions and an isoelectric focusing point of about 4.0.
  • purified can describe an antigen composition that is sufficiently free of contaminants or cell components with which the antigen normally occurs to distinguish the antigen from the contaminants or components.
  • purified when used in reference to a “protein,” describes proteins that are separated from other proteins of the subject. Examples of purified proteins and purified antigen compositions are provided herein to further describe the invention.
  • the approximately 64 kDa endometrial proteins have been separated from each other and from albumin (about 66 kDa with a pi of about 5.5) by two dimensional gel electrophoresis, based their isoelectric focusing points, which are shown to be different from each other and from albumin as demonstrated in the Examples.
  • albumin about 66 kDa with a pi of about 5.5
  • isoelectric focusing points which are shown to be different from each other and from albumin as demonstrated in the Examples.
  • Prior to the present disclosure it was not known that more than one protein existed in the approximately 64-66 kDa band from endometrial tissue or endometrial implants of patients with endometriosis.
  • Example 3 describes an amino acid sequencing protocol that was used to obtain the N-terminal sequence of one of the present 64 kDa proteins.
  • a purified antigenic endometrial protein having the amino-terminal amino acid sequence defined in the Sequence Listing as SEQ ID N0:1 is provided. Having provided the partial sequence of the present protein, only routine skill is necessary to determine the remaining amino acid sequence of the protein (Schleisinger, 1988). Once this routine step is accomplished, the determination of a nucleotide sequence that encodes the protein is also routine.
  • Endometriosis-specific antigenic polypeptide fragments of the antigenic proteins or a fragment of a protein that binds an antibody that specifically binds the antigenic protein are provided.
  • the polypeptide fragments of the present invention can be recombinant proteins obtained by cloning the nucleic acids which encode antigenic endometrial proteins in an expression system capable of producing the antigenic polypeptide or fragments thereof.
  • An antigenic polypeptide fragment of the antigenic protein can also be isolated from the whole protein by chemical or mechanical disruption. The purified fragments thus obtained can be tested to determine their antigenicity (immunogenicity) and specificity by the methods taught herein. Antigenic fragments of the antigen can also be synthesized directly.
  • An immunoreactive fragment is defined as an amino acid sequence of at least about 5 consecutive amino acids derived from the antigen amino acid sequence.
  • the amino acid sequence of the antigen is provided, it is also possible to synthesize, using standard peptide synthesis techniques, polypeptide fragments chosen to be homologous to antigenic regions of the endometrial protein. These fragments can be modified if desired by inclusion, deletion or modification of particular amino acids residues in the derived sequences so long as the specificity of the native polypeptide is retained.
  • the antigen can include amino acid sequences in which one or more amino acids have been substituted with another amino acid to provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, alter enzymatic activity, or to increase antigenicity. Thus, synthesis or purification of an extremely large number of polypeptides derived from the antigen is possible.
  • the antigenicity or immunogenicity of a fragment can be readily ascertained by comparing it to an antigenic protein of the invention in, for example, an enzyme linked immunosorbent assay, an immunofluorescence assay, a western blot assay, a hemagglutination assay, etc. as provided herein or in other well known methods utilizing antibody-antigen interactions.
  • a composition comprising a purified antigenic endometrial protein, separated from albumin and having a molecular weight of about 64 kilodaltons as determined by sodium dodecyl sulphate gel electrophoresis under reducing conditions and an isoelectric focusing point of about 3.5 and a purified antigenic endometrial protein, separated from albumin and having a molecular weight of 94-97 kilodaltons as determined by sodium dodecyl sulphate gel electrophoresis under reducing conditions and an isoelectric focusing point of about 3.5 is provided.
  • the composition can be free of albumin.
  • a composition comprising a purified antigenic endometrial protein, separated from albumin and having a molecular weight of about 64 kilodaltons as determined by sodium dodecyl sulphate gel electrophoresis under reducing conditions and an isoelectric focusing point of about 8.0 and a purified antigenic endometrial protein, separated from albumin and having a molecular weight of about 64 kilodaltons as determined by sodium dodecyl sulphate gel electrophoresis under reducing conditions and an isoelectric focusing point of about 6.0 is provided.
  • the composition can be free of albumin.
  • the invention further provides a composition
  • a composition comprising a purified antigenic endometrial protein, separated from albumin and having a molecular weight of about 64 kilodaltons as determined by sodium dodecyl sulphate gel electrophoresis under reducing conditions and an isoelectric focusing point of about 8.0 and a purified antigenic endometrial protein, separated from albumin and having a molecular weight of about 64 kilodaltons as determined by sodium dodecyl sulphate gel electrophoresis under reducing conditions and an isoelectric focusing point of about 4.0.
  • the composition can be free of albumin.
  • the invention further provides a composition comprising a purified antigenic endometrial protein, separated from albumin and having a molecular weight of about 64 kilodaltons as determined by sodium dodecyl sulphate gel electrophoresis under reducing conditions and an isoelectric focusing point of about 8.0 and a purified antigenic endometrial protein, separated from albumin and having a molecular weight of about 64 kilodaltons as determined by sodium dodecyl sulphate gel electrophoresis under reducing conditions and an isoelectric focusing point of about 3.5.
  • the composition can be free of albumin.
  • a composition comprising a purified antigenic endometrial protein, separated from albumin and having a molecular weight of about 64 kilodaltons as determined by sodium dodecyl sulphate gel electrophoresis under reducing conditions and an isoelectric focusing point of about 6.0 and a purified antigenic endometrial protein, separated from albumin and having a molecular weight of about 64 kilodaltons as determined by sodium dodecyl sulphate gel electrophoresis under reducing conditions and an isoelectric focusing point of about 4.0 is also provided.
  • the composition can be free of albumin.
  • composition comprising a purified antigenic endometrial protein, separated from albumin and having a molecular weight of about 64 kilodaltons as determined by sodium dodecyl sulphate gel electrophoresis under reducing conditions and an isoelectric focusing point of about 8.0, a purified antigenic endometrial protein, separated from albumin and having a molecular weight of about 64 kilodaltons as determined by sodium dodecyl sulphate gel electrophoresis under reducing conditions and an isoelectric focusing point of about 6.0 and a purified antigenic endometrial protein, separated from albumin and having a molecular weight of about 64 kilodaltons as determined by sodium dodecyl sulphate gel electrophoresis under reducing conditions and an isoelectric focusing point of about 4.0.
  • endometriosis-associated antigens can form the antigenic composition of the invention, for example, including the 6.5 pi antigen or other combinations of 2, 3, 4 or all 5 or the above antigens.
  • the composition can be free of albumin.
  • the antigen compositions can be used to detect antibodies that specifically bind the endometriosis-associated antigen composition.
  • the concentration of each protein in the above described compositions relative to the other protein(s) in the composition is determined by determining the relative antigenicity and effectiveness of various concentrations in, for example, an enzyme linked immunosorbent assay, an immunofluorescence assay, a western blot assay, a hemagglutination assay, etc. as provided herein.
  • a composition comprising two or more of the antigenic polypeptide fragments of the present antigenic proteins is also provided.
  • concentration of each fragment in the composition relative to the other fragments is determined by determining the relative effectiveness of various concentrations in, for example, an enzyme linked immunosorbent assay, an immunofluorescence assay, a western blot assay, a hemagglutination assay, etc. as provided herein.
  • a purified antigen composition having a molecular weight of about 64 to 66 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis is also provided.
  • This antigen composition migrates as a band on an SDS-PAGE gel and, as is the nature of bands on a gel, does not necessarily contain a single protein species.
  • This composition is distinct from the other compositions having 64 kDa proteins recited herein, because it is expected to include human serum albumin, which migrates at approximately 64 kDa, along with the endometrial antigenic proteins in SDS-PAGE gels.
  • the presence of the antigen composition is a marker for endometriosis in a subject.
  • the purified antigenic endometrial proteins, the antigenic compositions and antigenic polypeptide fragments of the proteins are also referred to herein as "the antigen” or "the 64-66 kDa antigen.”
  • the antigen or "the 64-66 kDa antigen.”
  • the presence of the 64 kDa antigen is a marker for endometriosis in a subject as demonstrated in the Examples.
  • a purified antigen composition having a molecular weight of about 46 to 48 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis or an endometriosis specific antigen fragment thereof, or a fragment reactive with an antibody specifically reactive with the antigen is also provided.
  • This antigen is also referred to herein as "the antigen” or "the 46-48 kDa antigen.”
  • the presence of the 46-48 kDa antigen is a marker for endometriosis in a subject as demonstrated in the Examples. In the methods described below, any of the present antigens can used alternatively or together.
  • the purified proteins, fragments and compositions of the invention can be bound (immobilized) to a solid support.
  • the antigen can be immobilized for the present diagnostic tests according to well known methods.
  • the antigen can be fixed to the well of an ELISA plate simply by drying the antigen on to the surface of the plate.
  • the antigen can be bound to cyanogen bromide treated sepharose beads or other suitable neutral material.
  • the purified antigen bound to a solid support and a ligand specifically reactive with the antigen are also contemplated.
  • a purified ligand specifically reactive with the antigen can be an antibody or other moiety that binds the antigen.
  • the antibody can be a monoclonal antibody obtained by standard methods and as described herein.
  • the monoclonal antibody can be secreted by a hybridoma cell line specifically produced for that purpose (Harlow and Lane, 1988).
  • the ligand can be a purified polyclonal antibody or a fragment thereof that specifically binds the antigen.
  • a purified antibody that specifically binds the antigen is also provided.
  • the term “bind” includes nonrandom association with an antigen.
  • "Specifically binding” as used herein describes an antibody or other ligand that does not cross react substantially with any antigen other than the one specified, in this case, the 64 kDa antigenic endometrial proteins, the 64 kDa antigenic endometrial protein compositions, the 64-66 kDa antigenic endometrial composition or the 46-48 kDa antigen.
  • the antibody can be either polyclonal or monoclonal.
  • Antibodies can be made as described in the art (see e.g., Harlow and Lane, Antibodies; A Labora tory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1988). Briefly, purified antigen can be injected into an animal in an amount and in intervals sufficient to elicit an immune response. Antibodies can either be purified directly, or spleen cells can be obtained from the animal. The cells are then fused with an immortal cell line and screened for antibody secretion. The antibodies can be used to screen DNA clone libraries for cells secreting the antigen.
  • the antibody can be bound to a solid support or labeled with a detectable moiety or both bound and labeled.
  • detectable moieties contemplated with the composition of the present invention are those listed below in the description of the diagnostic methods, including fluorescent, enzymatic and radioactive markers.
  • the present invention provides an isolated nucleic acid encoding an antigenic endometrial protein or endometriosis specific fragment of the protein.
  • the sequence of the nucleic acid is readily determined from the purified antigen using routine methods described in the art (Schleisinger, 1988).
  • isolated is meant separated from other nucleic acids naturally occurring in the subject, for example separated from other genes or coding sequences of the subject.
  • the nucleic acid encoding the antigen is specific for the antigen.
  • specific is meant an isolated sequence which does not hybridize with other nucleic acids to prevent a detectable positive hybridization with the antigen-encoding nucleic acid.
  • This specific nucleic acid can be used to detect the antigen in methods such as polymerase chain reaction, ligase chain reaction and hybridization. Alternatively, the nucleic acid can be utilized to produce an antigenic protein.
  • An isolated nucleic acid capable of selectively hybridizing with or selectively amplifying a nucleic acid encoding the proteins or fragments thereof, under conditions of high stringency is also contemplated.
  • An isolated nucleic acid complementary to the above nucleic acid is also provided. The sequences can be selected based on the nucleotide sequence and the utility of the particular sequence.
  • the term "selectively hybridizes" excludes the occasional randomly hybridizing nucleic acids as well as nucleic acids that encode other known recombinases.
  • the selectively hybridizing nucleic acids can be used, for example, as probes or primers for detecting the presence of and location of a gene encoding a protein of the invention that has the nucleic acid to which it hybridizes.
  • the selectively hybridizing nucleic acid can encode a polypeptide, and, can thereby be placed in a vector and host to produce the antigen, a functionally similar antigen or an antigenic fragment.
  • the selectively hybridizing nucleic acids of the invention can have at least 70%, 80%, 85%, 90%, 95%, 97%, 98% and 99% complementarity with the segment and strand of the sequence to which it hybridizes.
  • the nucleic acids can be at least 18 and up to 4000 nucleotides in length.
  • the nucleic acid can be an alternative coding sequence for the protein, or can be used as a probe or primer for detecting the presence of the nucleic acid encoding the protein. If used as primers, the invention provides compositions including at least two nucleic acids which selectively hybridize with different regions of a nucleic acid so as to amplify a desired region.
  • the probe or primer can range between 70% complementary bases and full complementarity and still hybridize under high stringency conditions.
  • the degree of complementarity between the hybridizing nucleic acid (probe or primer) and the sequence to which it hybridizes (DNA from a sample) should be at least enough to exclude hybridization with a nucleic acid encoding an unrelated protein.
  • a nucleic acid that selectively hybridizes with a nucleic acid of the protein coding sequence will not selectively hybridize under stringent conditions with a nucleic acid for a different protein, and vice versa.
  • High stringency conditions refers to the washing conditions used in a hybridization protocol.
  • the washing conditions should be a combination of temperature and salt concentration chosen so that the denaturation temperature is approximately 5-20°C below the calculated T m of the hybrid under study.
  • the temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to the probe or protein coding nucleic acid of interest and then washed under conditions of different stringencies. For example, hybridizations with oligonucleotide probes shorter than 18 nucleotides in length are done at 5-10°C below the estimated T m in 6X SSPE, then washed at the same temperature in 2X SSPE as described in Sambrook et al.
  • T m of such an oligonucleotide can be estimated by allowing 2°C for each A or T nucleotide, and 4°C for each G or C.
  • An 18 nucleotide probe of 50% G+C would, therefore, have an approximate T m of 54°C.
  • nucleic acids of the present invention can readily obtain using routine methods to synthesize a full gene as well as shorter nucleotide fragments.
  • techniques for obtaining nucleic acids such as those provided in the Sequence Listing are specifically provided in the application.
  • additional methods are provided in the art that can be utilized without significant modification.
  • Ferretti et al. Proc. Natl . Acad. Sci . 82:599-603 (1986)
  • Wosnick et al. Gene 76:153-160 (1989) show routine methods to synthesize a gene of known sequence. More specifically, Ferretti et al.
  • nucleic acids of the invention are also contemplated as long as the essential structure and function of the polypeptide (antigen) encoded by the nucleic acids are maintained.
  • fragments used as primers or probes can have substitutions so long as enough complementary bases exist for selective hybridization (Kunkel et al. Methods Enzymol . 1987:154:367, 1987).
  • a vector comprising the nucleic acids of the present invention is also provided.
  • the vectors of the invention can be in a host capable of expressing the antigen. Once the nucleotide sequence of the nucleic acid encoding the antigen is determined, a cDNA library of endometrial DNA can be screened for the expression of the antigen using probes derived from the antigen coding sequence.
  • E. coli expression vectors known to one of ordinary skill in the art useful for the expression of the antigen in E. coli .
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.
  • bacilli such as Bacillus subtilus
  • enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species.
  • prokaryotic hosts one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication).
  • any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (Trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.
  • the promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences for example, for initiating and completing transcription and translation. If necessary an amino terminal methionine can be provided by insertion of a Met codon 5' and in-frame with the antigen. Also, the carboxy-terminal extension of the antigen can be removed using standard oligonucleotide mutagenesis procedures.
  • Mammalian cells permit the expression of proteins in an environment that favors important post-translational modifications such as folding and cysteine pairing, addition of complex carbohydrate structures, and secretion of active protein.
  • Vectors useful for the expression of antigen in mammalian cells are characterized by insertion of the antigen coding sequence between a strong viral promoter and a polyadenylation signal.
  • the vectors can contain genes conferring either gentamicin or methotrexate resistance for use as selectable markers.
  • the antigen and immunoreactive fragment coding sequence can be introduced into a Chinese hamster ovary cell line using a methotrexate resistance-encoding vector. Presence of the vector DNA in transformed cells can be confirmed by
  • RNA corresponding to the antigen coding sequence can be confirmed by Northern analysis.
  • suitable host cell lines capable of secreting intact human proteins have been developed in the art, and include the CHO cell lines, HeLa cells, myeloma cell lines, Jurkat cells, etc.
  • Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
  • Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus, etc.
  • the vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts.
  • a method of diagnosing endometriosis in a subject comprises the steps of (a) contacting an antibody-containing sample from the subject with the 64-66 kDa antigen composition of the invention; and (b) detecting the reaction of the antigen with an antibody from the sample, the reaction indicating endometriosis in the subject.
  • the antigen is a purified antigen composition having a molecular weight of about 46-48 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis or an endometriosis specific antigen fragment thereof, or a fragment reactive with an antibody specifically reactive with the antigen.
  • a fluid sample such as serum, peritoneal fluid, urine, saliva or cervical mucous.
  • a fluid sample such as serum, peritoneal fluid, urine, saliva or cervical mucous.
  • the above method can be used to confirm clinical diagnosis of endometriosis in endometrial biopsy specimens.
  • the detecting step in the above methods can be an enzyme linked immunosorbent assay (ELISA), an immunofluorescence assay, a western blot assay or a passive hemagglutination assay.
  • ELISA enzyme linked immunosorbent assay
  • An example of passive hemagglutination, immunofluorescence, ELISA and Western blot analyses are provided in the Examples.
  • the antibody can be bound to a substrate and reacted with the antigen. Thereafter, a secondary labeled antibody is bound to epitopes not recognized by the first antibody and the secondary antibody is detected. Since the present invention provides endometriosis specific antigens and antibodies for the diagnosis of endometriosis other serological methods such as competitive inhibition, flow cytometry, immunoprecipitation and other immunoblotting methods (e.g., dot blot) can also be used as detection methods.
  • the antigen can be bound to a substrate and contacted by a fluid sample such as serum, peritoneal fluid, urine, saliva or cervical mucous.
  • a fluid sample such as serum, peritoneal fluid, urine, saliva or cervical mucous.
  • This sample can be taken directly from the patient or in a partially purified form.
  • antibodies specific for the antigen (the primary antibody) will specifically react with the bound antigen.
  • a secondary antibody bound to, or labeled with, a detectable moiety can be added to enhance the detection of the primary antibody.
  • the secondary antibody or other ligand which is reactive either specifically with a different epitope of the antigen or nonspecifically with the ligand or reacted antibody, will be selected for its ability to react with multiple sites on the primary antibody.
  • several molecules of the secondary antibody can react with each primary antibody, making the primary antibody more detectable.
  • the detectable moiety will allow visual detection of a precipitate or a color change, visual detection by microscopy, or automated detection by spectrometry, radiometric measurement or the like.
  • detectable moieties include fluorescein and rhodamine (for fluorescence microscopy), horseradish peroxidase (for either light or electron microscopy and biochemical detection), biotin-streptavidin (for light or electron microscopy) and alkaline phosphatase (for biochemical detection by color change).
  • the detection methods and moieties used can be selected, for example, from the list above or other suitable examples by the standard criteria applied to such selections (Harlow and Lane, 1988).
  • the presence of the antigen and, thus endometriosis can also be determined by detecting the presence of a nucleic acid specific for the antigen.
  • the nucleic acid specific for the antigen can be detected utilizing a nucleic acid amplification technique, such as polymerase chain reaction or ligase chain reaction.
  • the nucleic acid is detected utilizing direct hybridization or by utilizing a restriction fragment length polymorphism.
  • the present invention provides a method of diagnosing endometriosis, comprising ascertaining the presence of a nucleotide sequence associated with a restriction endonuclease cleavage site.
  • PCR primers which hybridize only with nucleic acids specific for the antigen can be utilized. The presence of amplification indicates the
  • the ligands may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, topically, transdermally, or the like, although oral or topical administration is typically preferred.
  • parenterally e.g., intravenously
  • intramuscular injection e.g., intraperitoneal injection
  • topically e.g., transdermally
  • transdermally e.g., transdermally
  • oral or topical administration e.g., oral or topical administration is typically preferred.
  • the exact amount of such compounds required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the disease that is being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact amount. However, an appropriate amount may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
  • the ligands of the present invention can be in pharmaceutical compositions in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, lotions, creams, gels, or the like, preferably in unit dosage form suitable for single administration of a precise dosage.
  • the compositions will include, as noted above, an effective amount of the selected compound in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc.
  • pharmaceutically acceptable a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the diagnostic kit of the present invention can be used to detect the presence of a primary antibody specifically reactive with the present purified 64 kDa antigenic endometrial protein with an isoelectric focusing point of about 3.5.
  • the diagnostic kit of the present invention can be used to detect the presence of a primary antibody specifically reactive with the present purified 64 kDa antigenic endometrial protein with an isoelectric focusing point of about 4.0, the purified 64 kDa antigenic endometrial protein with an isoelectric focusing point of about 6.0, the purified 64 kDa antigenic endometrial protein with an isoelectric focusing point of about 8.0 or combinations thereof.
  • Antigenic endometriosis-specific polypeptide fragments of the present protein can also be used as reagents in the antibody detecting kit.
  • the diagnostic kit of the present invention can also detect the presence of a primary antibody specifically reactive with the present purified 64-66 kDa antigen composition.
  • the kit can include a detectable amount of the antigen bound to a solid support, a secondary antibody reactive with the antibody specifically reactive with the antigen and a reagent for detecting a reaction of the secondary antibody with the primary antibody.
  • a kit can be an ELISA kit and can comprise the substrate, antigen, primary and secondary antibodies when appropriate, and any other necessary reagents such as detectable moieties, enzyme substrates and color reagents as described above.
  • the diagnostic kit can, alternatively, be an immunoblot kit generally comprising the components and reagents described herein.
  • kits comprising a detectable amount of the monoclonal or purified polyclonal antibody of the invention bound to a solid support.
  • the kit can detect the presence of the 64-66 kDa antigen specifically reactive with the antibody or an immunoreactive fragment thereof.
  • the kit can include an antibody bound to a substrate, a secondary antibody reactive with the antigen and a reagent for detecting a reaction of the secondary antibody with the antigen.
  • a kit can be an ELISA kit and can comprise the substrate, primary and secondary antibodies when appropriate, and any other necessary reagents such as detectable moieties, enzyme substrates and color reagents as provided in the art.
  • the diagnostic kit can, alternatively, be an immunoblot kit generally comprising the components and reagents described herein.
  • Blood, PF and endometrium were collected from the subjects during proliferative phase of their menstrual cycle. Out of town specimens were received packed on dry ice and stored and were frozen at -70° C. Endometrium was obtained by curettage during laparoscopy and the implants were removed during laparoscopy or therapeutic surgical excision of endometriotic implants. The tissues were cleaned of blood by stringent washing procedures. The clinical diagnoses of the subjects were revealed only after the results of the present immunologic studies were obtained.
  • Passive hemagglutination assay for ovarian and endometrial antibodies. Passive hemagglutination assay was performed according to the established protocol (Mathur et al., 1982; Chihal et al. , 1986; Badawy et al. , 1990). Briefly, human 0, Rh-positive erythrocytes (RBCs) were coated washed and packed with pooled endometrial, implant or ovarian antigens in the presence of chromium chloride, diluted to a concentration of 0.01%, layered over serially diluted human or polyclonal antibody samples in V-bottom microtiter plates (Cooke Co., Alexandria, VA) and incubated at room temperature for 2 hr.
  • RBCs erythrocytes
  • Immunofluorescent antibody assay for ovarian and endometrial antibodies Immunofluorescent antibody assay for ovarian and endometrial antibodies.
  • Frozen 5 ⁇ m thick sections of normal ovaries, endometrium, implants or cultured endometrial epithelial cells were fixed lightly in a 3:1 methanol and acetic acid mixture, layered with patients' serum, PF or rabbit antiserum and incubated at 4°C for 30 min. The cells were then washed with ice-cold PBS, followed by cold distilled water and incubated with Fluorescein thiocyanate (FITC) conjugated F(ab)2 of anti-rabbit or anti-human IgG. Control cells without samples layered over them were similarly treated. Positive and negative controls were maintained.
  • FITC Fluorescein thiocyanate
  • Serum samples from 20 infertile patients with endometriosis from Chicago pre-screened for auto-antibodies to phospholipid, histone and nucleotide antigens (NG) and nine fertile women laparoscopically confirmed to be free of endometriosis were blindly tested for the presence of endometrial antibodies (SM) using the passive hemagglutination assay.
  • SM endometrial antibodies
  • 11 were untreated, two were on danazol, two received prednisone, two were on clomiphene citrate and one patient received either depo-medroxyprogesterone acetate, ZOVIRAX (acyclovir) or PREMARIN (conjugated estrogen).
  • Binding of the sera to intact histone fractions, ssDNA, dsDNA, poly 1 and poly (dT) or purified phospholipids (cardiolipin, phosphatidyl serine, phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl glycerol and phosphatidyl inositol) was measured (NG) by a routine enzyme linked immunosorbent assay (ELISA; Confino et al., 1990). A test was considered positive if the optical density exceeded the 99 percent confidence interval of 400 control sera (Confino et al., 1990). Sera from the fertile women were used in all assays as negative controls. Testing endometrial and implant extracts for WBC or nuclear antigens.
  • T lymphocyte antigens such as CD2 (T11), CD3 (T3), CD4 (T4), CD5 (T1 ) and CD8 (T8), B lymphocyte antigens and monocyte/macrophage antigen CD14 (Coulter Immunology, Hialeah, Florida) were tested for IgG against endometrial and implant extracts by Western blot analysis. Extracts of WBCs were the positive controls. The antisera and the monoclonal antibodies strongly reacted against gastric parietal cells (for nuclear antibodies) and whole WBCs (for WBC antibodies) by immunofluorescence.
  • WBC antigens Western blot analyses of endometrial and implant extracts against monoclonal antibodies to lymphocyte subsets and monocytes/macrophages demonstrated a lack of binding of the monoclonal antibodies with above antigens, showing that these extracts are uncontaminated with WBCs.
  • Nuclear antigens There was no correlation between the presence of endometrial and nuclear auto-antibodies in the sera of 20 endometriosis patients from Chicago (different from patients in Table 3). Serum from 9 fertile controls had negligible titers ( ⁇ 8 or log 2 3) of antibodies to endometrium and implants. None of them had nuclear antibodies by the immunofluorescence assay. Eight patients had endometrial antibody titers > 64. Two of these had IgA auto-antibodies to histone fraction H2A; one had IgG auto-antibodies to histone fraction H2B, while another was positive for IgA auto-antibodies to ssDNA. Eight other patients had endometrial antibody titers of 16 and 32.
  • Serum No. 1 Anti-human centromere
  • Serum No. 2 With speckled nuclear immune reactivity
  • Serum No. 3 Anti-human nuclear rim
  • Serum No. 4 homogenous anti-nuclear pattern
  • Serum No. 5 Anti-human nucleoli.
  • These sera were positive against nuclei of gastric parietal cells by immunofluorescence.
  • the lack of correlation between endometrial and nuclear antibodies in the present study demonstrates that the endometrial autoimmunity observed in patients with endometriosis is distinct from autoimmunity to phospholipid, histone or nucleotide antigen
  • the endometrial and implant proteins with MW of 29 to 68 and >68 kDa were eluted from gels containing separated endometrial and implant proteins of patients with endometriosis, using Bio-Rad Model 422 Electro-Eluter (Abramovitz et al., 1984). The eluate was later lyophilized in a spin-vacuum (Speed-Vac, Seavant Co., Farmington, New Jersey) and reconstituted in normal saline.
  • the hemagglutinating antibody titers, and the intensities of immunofluorescent antibody (IFA) reactions or the antigenic bands binding with antibodies in the Western blot analysis were highest in the serum IgG of rabbits immunized with eluted endometrial and implant proteins with MW 29 to 68 kDa, closely followed by those immunized with proteins of MW > 68 kDa.
  • the MW of endometrial and implant antigens binding with antisera from rabbits immunized with 29 to 68 or > 68 kDa proteins were similar (Table 4).
  • the IFA patterns and intensities were similar to those of the patients' serum or PF IgG.
  • the antigenic extracts used in the study were of comparable protein contents. Serum and P.F. from the controls failed to show significant IgG binding other than a light binding to antigens with MW 15, 18 and 30 kDa (in only a few subjects) and the endogenous immunoglobulin heavy (52/54 kDa) and light (25/27) chains already present in the endometrial or implant blanks from patients. On the contrary, endometrial and/or endometriosis implant antigens with MW of 34, 42, 46/48, 64/66, 84, 94/97 and 120 kDa were found in patients with endometriosis and elicited local and systemic IgG auto-antibody responses in > 25% of these patients.
  • MW Molecular weights (MW) : Antigens with MW of 34, 42, 46/48, 64, 84, 94 and 120 kDa in endometrium and implants of 76 patients with endometriosis from 4 cities bound with IgG in serum and PF of most patients, but not the controls, by Western blot analysis. Specificity: Endometrial and implant extracts were free of nuclear and white blood cell (WBC) antigens since they did not react with monoclonal antibodies to WBC subsets and 5 sera with nuclear antibodies.
  • WBC white blood cell
  • Endometrial antigens with MW of 34, 42, 46/48, 64, 84, 94 and 120 kDa are specific, immunogenic and are relevant to endometrial autoimmunity in patients with endometriosis irrespective of their city or race.
  • Table 1 Percentage of endometriosis patients from Charleston, Dallas and Boston, with the reactive antigens on their endometrium and implants. Endometrium from controls failed to have these antigens.
  • FPLC Fast protein liquid chromatography
  • FPLC molecular weight (MW) standards were used to help estimate the MW of the eluted proteins.
  • the endometrial and implant extracts were subjected to FPLC by using Superose columns. There are 4 important peaks. The eluates from each peak were pooled
  • Fraction numbers 2 and 3 contain the 34, 48, 64-66 and 72 kDa bands (See the FPLC and the Western blot analysis).
  • a 66 kDa protein common to normal and patients' endometrium is also present and shows up when Fast protein liquid chromatography was used, followed by separation of the endometrial proteins according to their molecular weights. As shown below, this protein is human albumin.
  • Endometrial Antigens from Patients with Endometriosis (pooled from the same four patients in Table 7): 200 ⁇ l sample; 0.1 v range, 0.3 Flow rate, 0.2 AUFS
  • Implant Antigens from patients 6-8 (Representative data of 8 runs); also from patients 9 , 3 and 8 (4 runs):
  • the endometrial protein with a mw of 66 kDa (66 kDa found in all fractions especially in 3 and 4), 72 and 43 kDa were glycoproteins.
  • the protein band at 64 kDa did not include glycoproteins.
  • the 64 and 66 kDa bands are present in both endometrium and implants of patients with endometriosis.
  • chromium chloride (0.05%) by mixing a drop of 0.1% stock plus 19 drops of gelatinized saline (250 mg of gelatine to 250 ml of saline (0.1%) and heat with stirring until dissolved). Add 2 drops of 1% CrCl3 stock solution, diluted 1:20, to each tube after 2 hr incubation. Shake for 5 min. Wash all RBCs 3 times with gelatinized saline as above. Suspend the final RBC button in 2 ml gelatinized saline. Prepare and load microtiter plates: Eight samples can be tested per microtiter plate. Add a drop of gelatinized saline in each well of the plates with Pasteur pipette.
  • an ELISA was performed to document that patients with endometriosis do not have antibodies to 5 human albumin in their serum or peritoneal fluid.
  • Endometrial antigens were absorbed with cyanogen bromide treated sepharose beads coated with anti-albumin and Protein A to eliminate albumin and IgG respectively, 5 briefly as described below.
  • the excess ligand then washed away with at least 5 volumes of coupling buffer.
  • the gel was then transferred to 0.1 M tris-HCl buffer (pH 8.0) and left standing for 2 hrs rotating end to end to block the excess active sites.
  • the products was then washed for 3 alternating pH cycles with at least 5 volumes each buffer. Each cycle consisted of a wash with 0.1 M acetate buffer (0.5 M NaCl) (pH 4) followed by a wash with 0.1 M Tris-HCl (0.5 M NaCR) (pH 8) containing 0.5 M NaCl.
  • the gel was then equilibrated in TBS.
  • PAGE gels of the endometrial extracts were performed after these absorptions. Coomasie blue-stained PAGE gels were performed, including MW standards, unabsorbed endometrial extracts, endometrial extracts absorbed with anti-human IgG (the albumin band is still intact in quantity) and endometrial extracts absorbed with anti- albumin antiserum (shows a thick band of protein, in about the same position as the albumin area, and the heavy chain of IgG) . There was also a thick band of reactivity with patients' serum irrespective of the absorption of IgG or albumin, showing that the reactivity was not directed against albumin, but against a protein masked by albumin in this region.
  • the extract with albumin absorbed out still presented a protein band in the 64-66 kDa area.
  • PAGE of the endometrial antigens preabsorbed and absorbed with protein A and anti-albumin was performed.
  • the endometrial protein band in the 64-66 kDa region is intact even after absorption with anti-albumin.
  • mRNAs from fresh endometrial and implant specimens are isolated, for example using poly dT, in preparation for the generation of a cDNA library.
  • cDNA and second strand DNA is generated using standard protocols.
  • the DNA can routinely be inserted into a vector and used to transfect cells, which can then be screened for the expression of endometriosis-associated antigens using, for example, the Western blot protocol described herein.
  • the absorbed implant extract was subject to two dimensional gel electrophoresis using the equipment from Pharmacia, according to the manufacturer's protocol. Isoelectric focusing is performed in the first dimension, separating the constituent proteins according to their pi values.
  • the Ph gradient that was used in the immobiline dry strips was pH 3 to 10.
  • Two dimensional electrophoresis shows that there are at least five proteins in addition to albumin in the 64-66 kDa range.
  • Amino acid sequencing was performed on the proteins from the 64-66 kDa region purified by 2-dimensional gel electrophoresis.
  • Amino acid sequencing is carried out on Applied Biosystems sequencers equipped with on-line HPLC Systems, according to the manufacturer's instructions. In general 0.05 to 1 nanomole is sufficient to sequence from 10 to 40 residues respectively. Samples can be submitted dry on PVDF membranes or in less than 0.1 ml of a suitable solvent such as water, 5 mM NH 4 HC0 3 , 0.05% trifluoroacetic acid (TFA), or 50% CH 3 CN/0.05% TFA.
  • a suitable solvent such as water, 5 mM NH 4 HC0 3 , 0.05% trifluoroacetic acid (TFA), or 50% CH 3 CN/0.05% TFA.
  • the amino acid sequencing analysis includes HPLC identification of the resulting PTH-amino acids, a computerized printout for each cycle and a summary table of the PTH-amino acid yields.
  • the resulting sequence was searched against the National Center for Biological Information's databases (i.e. Protein Identification Resource (PIR), Genpept and Swiss Protein databases) and no matches were found.
  • PIR Protein Identification Resource
  • Genpept Genpept and Swiss Protein databases
  • Fig. 3 shows the results of a Western blot of a two dimensional gel with endometriosis patient serum IgG, using the blotting protocol described above in Example 1.
  • the data indicate that the 64 kDa protein with a pl value of about 3.5 was reactive with serum, as was a 94-97 kDa protein with a pi value of about 3.5.
  • these 5 purified proteins are markers for endometriosis.
  • the 64 and 94-97 kDa proteins are both reactive with patient serum as shown in the Western blot (Fig. 3) and have the same pi, they may 10 constitute fragments of a high molecular weight protein that appear as a result of electrophoresis under reducing conditions. There is also an approximately 34 kDa protein that could be a part of such a larger protein.
  • Burnette, D. "Western blotting” Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radio-iodinated Protein A. Anal .
  • MOLECULE TYPE protein
  • FRAGMENT TYPE N-terminal
  • Xaa can be Ala, Tyr, He or Pro

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Abstract

L'invention concerne un antigène purifié présentant une masse molaire d'environ 64 à 66 kDa comme déterminé par l'électrophorèse sur gel au polyacrylamide de sulfate de dodécyle de sodium. Des protéines endométriales exemptes d'albumine et présentant une masse molaire d'environ 64 kDa ainsi que des points de concentration isoélectrique de 3,5, 4,0, 6,0, 6,5 et 8,0 sont décrites. Une protéine endométriale purifiée, exempte d'albumine et présentant une masse molaire de 94 à 97 kDa ainsi qu'un point de concentration isoélectrique de 3,5 est également prévue. La présence d'une composition antigénique ou d'une protéine endométriale associée à l'endométriose indique l'endométriose chez un sujet. Un anticorps monoclonal purifié spécifiquement réactif avec l'antigène est décrit ainsi qu'une méthode de diagnostic de l'endométriose chez un sujet. Ladite méthode consiste a) à mettre un échantillon contenant un anticorps prélevé sur le sujet en contact avec ladite composition antigénique ou les protéines endométriales purifiées selon l'invention; et b) à détecter la réaction de l'antigène avec l'anticorps dans l'échantillon, la réaction signalant une endométriose chez le sujet. Après la production d'un antigène et d'un anticorps associés à l'endométriose, une méthode de diagnostic de l'endométriose chez un sujet à l'aide d'un anticorps monoclonal est également prévue.
PCT/US1994/006081 1993-05-28 1994-05-27 Proteines endometriales, compositions antigeniques et procedes de detection de l'endometriose WO1994028021A1 (fr)

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998010291A1 (fr) * 1996-09-06 1998-03-12 Osteometer Biotech A/S Marqueurs biochimiques de l'endometre humain
WO1999000671A3 (fr) * 1997-06-26 1999-06-10 Univ Michigan Procede d'identification d'antigenes de proteines cellulaires et de detection de la presence d'anticorps diriges contre des proteines cellulaires specifiques du serum
WO1999063079A1 (fr) * 1998-05-29 1999-12-09 Starzinski Powitz Anna Gene associe a l'endometriose
WO2000006732A3 (fr) * 1998-07-31 2000-05-04 Diagnostic Products Corp Polynucleotide codant un auto-antigene associe a l'endometriose
WO2000047739A3 (fr) * 1999-02-09 2000-11-30 Dade Behring Inc Autoantigenes permettant de diagnostiquer l'endometriose
WO2001007616A1 (fr) * 1999-07-22 2001-02-01 Diagnostic Products Corporation Polynucleotide codant pour des auto-antigenes associes a l'endometriose
US6677128B1 (en) 1997-06-26 2004-01-13 Regents Of The University Of Michigan Method for identification of cellular protein antigens and presence of antibodies to specific cellular protein antigens in serum
US6743595B1 (en) 1999-01-25 2004-06-01 Metriogene Biosciences Inc. Method and diagnostic kit for diagnosis of endometriosis
EP1614692A3 (fr) * 2004-07-07 2006-04-05 Diagnostic Products Corporation ME-5, ME-2, and EPP2: protéines humaines antigéniques réactives contre des autoanticorps présents dans le serum de femmes atteintes d'endométriose
RU2334987C1 (ru) * 2007-04-12 2008-09-27 Людмила Васильевна Дикарева Способ диагностики патологии эндометрия у больных миомой матки
US7833729B2 (en) 1998-07-31 2010-11-16 Siemens Heathcare Diagnostics Inc. Method for detecting endometriosis in a patient sample
WO2018063767A1 (fr) 2016-09-27 2018-04-05 Exxonmobil Chemical Patents Inc. Processus de polymérisation
WO2018063764A1 (fr) 2016-09-27 2018-04-05 Exxonmobil Chemical Patents Inc. Procédé de polymérisation
WO2018063765A1 (fr) 2016-09-27 2018-04-05 Exxonmobil Chemical Patents Inc. Procédé de polymérisation
WO2018118155A1 (fr) 2016-12-20 2018-06-28 Exxonmobil Chemical Patents Inc. Procédé de polymérisation

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992018535A1 (fr) * 1991-04-09 1992-10-29 Eastman Kodak Company Antigene endometrial, composition, trousse pour epreuve et methode de determination d'anticorps endometriaux

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992018535A1 (fr) * 1991-04-09 1992-10-29 Eastman Kodak Company Antigene endometrial, composition, trousse pour epreuve et methode de determination d'anticorps endometriaux

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
AMERICAN FERTILITY SOCIETY MEETING PROGRAM SUPPLEMENT, issued November 1989, D. GARZA et al., "Antigenic Differences Between the Endometrium of Woman with and without Endometriosis", page S34, Abstract No. 0-080. *
AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Volume 27, Number 1/2, issued January-March 1992, S. MATHUR et al., "Endometrial Autoimmunity in Endometriosis: Immunogenicity and Specificity of the Target Antigens", page 53, Abstract No. 132. *

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998010291A1 (fr) * 1996-09-06 1998-03-12 Osteometer Biotech A/S Marqueurs biochimiques de l'endometre humain
WO1999000671A3 (fr) * 1997-06-26 1999-06-10 Univ Michigan Procede d'identification d'antigenes de proteines cellulaires et de detection de la presence d'anticorps diriges contre des proteines cellulaires specifiques du serum
US6677128B1 (en) 1997-06-26 2004-01-13 Regents Of The University Of Michigan Method for identification of cellular protein antigens and presence of antibodies to specific cellular protein antigens in serum
WO1999063079A1 (fr) * 1998-05-29 1999-12-09 Starzinski Powitz Anna Gene associe a l'endometriose
US7833729B2 (en) 1998-07-31 2010-11-16 Siemens Heathcare Diagnostics Inc. Method for detecting endometriosis in a patient sample
WO2000006732A3 (fr) * 1998-07-31 2000-05-04 Diagnostic Products Corp Polynucleotide codant un auto-antigene associe a l'endometriose
US6525187B1 (en) 1998-07-31 2003-02-25 Diagnostic Products Corporation Polynucleotide encoding autoantigens associated with endometriosis
US7368533B2 (en) 1998-07-31 2008-05-06 Siemens Medical Solutions Diagnostics Polypeptide autoantigens associated with endometriosis
US6743595B1 (en) 1999-01-25 2004-06-01 Metriogene Biosciences Inc. Method and diagnostic kit for diagnosis of endometriosis
WO2000047739A3 (fr) * 1999-02-09 2000-11-30 Dade Behring Inc Autoantigenes permettant de diagnostiquer l'endometriose
WO2001007616A1 (fr) * 1999-07-22 2001-02-01 Diagnostic Products Corporation Polynucleotide codant pour des auto-antigenes associes a l'endometriose
EP1106690A3 (fr) * 1999-11-23 2001-07-25 Diagnostic Products Corporation Polynucléotide codant pour des auto-antigènes associés à l'endométriose
EP1614692A3 (fr) * 2004-07-07 2006-04-05 Diagnostic Products Corporation ME-5, ME-2, and EPP2: protéines humaines antigéniques réactives contre des autoanticorps présents dans le serum de femmes atteintes d'endométriose
US7879562B2 (en) 2004-07-07 2011-02-01 Siemens Healthcare Diagnostics Inc. Methods of diagnosing endometriosis in human subjects using the ME-5 polypeptide
US7981626B2 (en) 2004-07-07 2011-07-19 Siemens Healthcare Diagnostics Inc. Method of detecting endometriosis in human subjects using SEQ ID No. 9 or an epitope thereof
US8030007B2 (en) 2004-07-07 2011-10-04 Siemens Healthcare Diagnostics Inc. Method for the detection of endometriosis using an ME-2 antigen
RU2334987C1 (ru) * 2007-04-12 2008-09-27 Людмила Васильевна Дикарева Способ диагностики патологии эндометрия у больных миомой матки
WO2018063767A1 (fr) 2016-09-27 2018-04-05 Exxonmobil Chemical Patents Inc. Processus de polymérisation
WO2018063764A1 (fr) 2016-09-27 2018-04-05 Exxonmobil Chemical Patents Inc. Procédé de polymérisation
WO2018063765A1 (fr) 2016-09-27 2018-04-05 Exxonmobil Chemical Patents Inc. Procédé de polymérisation
WO2018118155A1 (fr) 2016-12-20 2018-06-28 Exxonmobil Chemical Patents Inc. Procédé de polymérisation

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