WO2000047739A2

WO2000047739A2 - Autoantigens for the diagnostic of endometriosis

Info

Publication number: WO2000047739A2
Application number: PCT/US2000/002710
Authority: WO
Inventors: Dipty Desai; Nurith Kurn
Original assignee: Dade Behring Inc.
Priority date: 1999-02-09
Filing date: 2000-02-02
Publication date: 2000-08-17
Also published as: WO2000047739A3

Abstract

The invention pertains to a technique to discover auto-antigens found in patients having endometriosis. Furthermore, the invention comprises polypeptide auto-antigens which were discovered using this technique, as well as cDNA clones that encode these auto-antigens. Finally, the invention pertains to methods and diagnostic kits which utilize these polypeptides to detect endometriosis.

Description

AUTOANTIGENS DIAGNOSTIC OF ENDOMETRIOSIS

FIELD OF THE INVENTION

The present invention relates to polypeptide antigens which detect auto-antibodies in endometriosis patients and polynucleotides that encode these polypeptide antigens. The invention further relates to immunoassays and diagnostic kits which utilize the antigens to bind auto-antibodies. Finally, the invention relates to a method of isolating cDNA clones which encode antigens that are recognized by auto-antibodies of endometriosis patients.

Several publications are referenced in this application. Full citation to these references is found at the end of the specification immediately preceding the claims or where the publication is mentioned. Each of these publications is incorporated herein by reference These publications relate to the art to which this invention pertains.

BACKGROUND OF THE INVENTION

Endometriosis affects women of reproductive age and is characterized by altered growth regulation of the endometriotic tissue and extra uterine implantation of endometriotic tissue. The disease is a major contributor to female infertility and is wide spread. Only invasive methods are currently available for diagnosing this disease. The most common

method is laparoscopy.

A large body of evidence, accumulated by various researchers, indicates the existence of an auto-immune component in the pathogenesis or manifestation of endometriosis. although this conclusion is not universally accepted. If they could be unambiguously shown to exist, the identification of auto-antigens specifically related to endometriosis would be valuable for the development of serum-based diagnostic tests for the disease. The evidence for immunological abnormalities in patients with endometriosis is discussed in Martinez- Roman S., et al, "Immunological Factors In Endometriosis-Associated Reproductive Failure: Studies In Fertile And Infertile Women With And Without Endometriosis," Hum. Reprod., 12, 1794-1799, (1997).

A number of published reports claim detection of anti-endometrial auto-antibodies in patients with endometriosis (Hatayama H., et ah, "Detection of anti-endometrial antibodies in patients with endometriosis by cell ELISA," Am. J. Reprod. Immunol., 35 1 18-122, (1996); Kim J.G., et al., "Detection of anti-endometrial antibodies in sera of patients with endometriosis by dual labeling immunohistochemical method and Western blot," Am. J. Reprod. Immunol, 34, 80-87, (1995); Gorai I., et al, "Anti-endometrial auto-antibodies are generated in patients with endometriosis," Am. L. Reprod. Immunol, 29, 116-123, (1993); Fernandez-Shaw S., et al, "Anti-endometrial and anti-endothelial auto-antibodies in women with endometriosis," Hum. Reprod. 8, 310-315, (1993); O'Cruz O. J., et al, "Antibodies to carbonic anhydrase in endometriosis: prevalence, specificity and relationship to clinical and laboratory parameters," Fertil Steril, 66, 547-556, (1996); Mathur S., et al, "Endometrial antigens involved in the autoimmunity of endometriosis," Fertil Steril, 50, 860-863, (1988); Badaway S. Z., et al, "Endometrial antibodies in serum and peritoneal fluid of infertile patients with and without endometriosis," Fertil Steril 53, 930-932, (1990); Mathur S., et al, "Target antigens in endometrial autoimmunity of endometriosis," Autoimmunity, 20, 211 - 222, (1995); Pillai S., et al, "Antibodies to endometrial transferrin and alpha 2-Heremans Schmidt (HS) glycoprotein in pateints with endometriosis," Am. J. Reprod. Immunol, 35, 483-494, (1996); Chilhal H. J., et al, "An endometrial antibody assay in the clinical diagnosis and management of endometriosis," Fertil Steril, 46, 408-41 1, (1986); Kennedy S., et al, "Localization of anti-endometrial antibody binding in women with endometriosis using a double labeling immunohistochemical method," Br. J. Obstet. Gynaecol, 97, 671-674, (1990); Rajkumar K., Malliah V., Simpson C. W., J., "Identifying the presence of antibodies against endometrial antigens. A preliminary study," Reprod. Med. 37, 552-556, (1992)). Others have reported the lack of reproducibility of the various methods used for the detection of anti-endometrial antibodies in patients with endometriosis (Switchenko A. C, Kauffman R. S., Becker M., Fertil. Steril. 56, 235-241, (1991), "Are there anti-endometrial antibodies in sera of women with endometriosis?"; Fernandez- Shaw S., et al, "Anti-endometrial antibodies in women measured with an enzyme-linked immunosorbent assay," Hum. Reprod. 11, 1 180- 1184, (1996)).

One method which has been successfully used for identifying auto-antigens for some diseases is probing tissue-specific or cell-specific copy DNA (cDNA) libraries with patient sera (Dong Q., et al,, "Cloning and sequencing of a novel 64-kDa auto-antigen recognized by patients with autoimmune thyroid disease." J. Clinc. Endocrinol Metab., 72, 1375-1381 , (1991); Lafyatis R. and Capron A., "Expression cloning using antibodies from a patient with rheumatoid arthritis of an auto-antigen homologous to the Drosophila splicing regulator," NATO ASI Ser., Ser. H 80, 59-73, (1994); Pietropaolo M., et_al, "Islet cell auto-antigen 69 kD (ICA69). Molecular cloning and characterization of a novel diabetes-associated auto- antigen," J. Clin. Invest. 92, 359-371, (1993); Szoostecki C, et al, "Isolation and characterization of cDNA encoding a human nuclear antigen predominantly recognized by auto-antibodies from patients with primary biliary cirrhosis," J. Immunol 145, 4338-4347, (1990)). However, this method has not been used to identify auto-antigens of endometriosis.

There remains a need for a non-invasive assay that is useful in the diagnosis of endometriosis and useful in monitoring endometriosis patients. Furthermore, there is a need for a diagnostic kit that contains the components required to perform such an assay. Additionally, there remains a need for a method which can be used to successfully identify polypeptides that are auto-antigens in patients with endometriosis and a need to isolate DNA sequences encoding these polypeptides. Finally, there is a need to identify such autoantigens as well as novel proteins and genes that are associated with endometriosis.

SUMMARY OF THE INVENTION

The invention relates to a method for detecting endometriosis comprising forming a complex between auto-antibodies associated with endometriosis and one or more polypeptides comprising amino acid sequences SEQ ID NO: 13-25 and detecting the complex. The invention includes polypeptide fragments which have the same immunological specificity toward auto-antibodies as those of amino acid sequences SEQ ID NOJ3-25. Preferably the polypeptides used in the method are one or more of the polypeptides comprising the amino acid sequence of SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO:23 and SEQ ID NO:25 or fragments thereof having the same immunological specificity toward auto-antibodies associated with endometriosis.

Typically, the method is selected from the group consisting of radioimmunoassay, immunoprecipitation assay, enzyme-linked immunoassay (ELISA), depletion enzyme-linked immunoassay (DELISA), Western Blot, particle agglutination assay, Luminescent oxygen- channelling immunoassay, proximity-based immunoassay, and biosensor-based immunoassay. Typically, the complex formed using the method is detected using a labelled detection molecule. The label is typically selected from the group consisting of radioactive molecules, fluorescent molecules, chemiluminescers, dyes, oligonucleotides, enzymes, coenzymes, enzyme substrates, small organic molecules, and solid surfaces.

In one embodiment, the polypeptide is bound to a solid support for the method of

RECTIFIED SHEET (RULE 91) detecting auto-antibodies and detection is achieved by the use of a detection molecule which comprises a labeled antibody molecule which binds to human antibodies. In one embodiment the labeled antibody molecule comprises an enzyme conjugate of an anti-human Ig antibody. In this embodiment the method further comprises applying a substrate for the enzyme to the conjugate, the enzyme catalyzing the modification of the substrate into a detectable product. In a preferred embodiment, the enzyme is alkaline phosphatase and the substrates are methylumbellifeiyl phosphate (MUP) or p-nitrophenyl phosphate (PNPP). Typically, the auto-antibodies are detected as a component of plasma, sweat, urine, saliva, and preferably serum.

Another aspect of the current invention is a method for diagnosing endometriosis comprising conducting a serological assay for auto-antibodies capable of binding one or more polypeptides of SEQ ID NO: 13-25 and detecting the complex. This aspect of the invention includes polypeptide fragments which have the same immunological specificity toward auto- antibodies as those of amino acid sequences SEQ ID NO: 13-25. Preferably, the polypeptides used in this method are one or more of the polypeptides comprising the amino acid sequence of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:23 and SEQ ID NO:25 or fragments thereof having the same immunological specificity toward auto-antibodies associated with endometriosis.

Another aspect of the current invention is a method for detecting endometriosis comprising contacting a fluid from a patient with one or more polypeptides comprising amino acid sequences SEQ ID NO: 13-25 and capturing the complex, applying a detection molecule to the captured antibodies bound to the polypeptide, and detecting the bound detection molecule. Typically, the detection molecule has the capacity to specifically bind to the

RECTIFIED SHEET (RULE 91) captured complex through an interaction with the auto antibodies.

This aspect of the invention includes polypeptide fragments which have the same immunological specificity toward auto-antibodies as those of amino acid sequences SEQ ID NO: 13-25. Preferably the polypeptides used in the method are one or more of the polypeptides comprising the amino acid sequence of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO:23 and SEQ ID NO:25 or fragments thereof having the same immunological specificity toward auto-antibodies associated with endometriosis. In one preferred embodiment of this aspect of the invention, the method is used for monitoring an endometriosis patient by periodically conducting the assay.

In one embodiment, the polypeptide is bound to a solid support for this method of detecting auto-antibodies and the detection molecule comprises a labeled antibody molecule which binds to human antibodies. In one embodiment the labeled antibody molecule comprises a polyclonal antibody, a monoclonal antibody, or fragments thereof. In this embodiment the method further comprises applying a substrate for the enzyme to the conjugate, the enzyme in the conjugate catalyzes the modification of the substrate into a detectable product. In a preferred embodiment, the enzyme is alkaline phosphatase and the substrates are methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate (PNPP).

Another aspect of the invention is an endometriosis detection kit which contains one or more polypeptides comprising amino acid sequences SEQ ID NO: 13-25 and a detection molecule capable of binding to human antibodies. This aspect of the invention includes polypeptide fragments which have the same immunological specificity toward auto- antibodies as those of amino acid sequences SEQ ID NOJ3-25. Preferably the polypeptides used in the method are one or more of the polypeptides comprising the amino acid sequence

RECTIFIED SHEET (RULE 91) of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:23 and SEQ ID NO:25, or fragments thereof having the same immunological specificity toward auto-antibodies associated with endometriosis.

In one embodiment, the polypeptide is bound to a solid support as part of the kit and the detection molecule comprises a labeled antibody molecule which binds to human antibodies. In one embodiment the labeled antibody molecule comprises a polyclonal antibody, a monoclonal antibody, or fragments thereof.. In this embodiment the kit further comprises a substrate for the enzyme. In a preferred embodiment, the enzyme is alkaline phosphatase and the substrates are methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate (PNPP).

Another aspect of the current method is a reagent for detecting endometriosis comprising one or more polypeptides comprising amino acid sequences SEQ ID NO: 13-25. This aspect of the invention includes polypeptide fragments which have the same immunological specificity toward auto-antibodies as those of amino acid sequences SEQ ID NO: 13-25. Preferably the polypeptide reagents are one or more of the polypeptides comprising the amino acid sequence of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:23 and SEQ ID NO:25 or fragments thereof having the same immunological specificity toward auto-antibodies associated with endometriosis. In one preferred embodiment the polypeptide is labeled with a detectable moiety.

Another aspect of the current invention are isolated polypeptides comprising the amino acid sequence of one or more of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23 and fragments of any of these polypeptides having the same immunological specificity.

RECTIFIED SHEET (RULE 91) Another aspect of the current invention is a method for detecting auto-antigens found in endometriosis patients comprising screening an endometriotic cDNA expression library with pooled fluid from normal and endometriosis patients. Typically, the pooled fluid is selected from the group consisting of plasma, sweat, urine, and saliva, and preferably serum.

Another aspect of the current invention is an isolated nucleic acid molecule comprising a polynucleotide of SEQ ID NOJ-12 or a polynucleotide which hybridizes to SEQ ID NOJ-12, a polynucleotide encoding a polypeptide of a translation of SEQ ID NO: 1-12 or a polypeptide encoded by the cDNA sequence which hybridizes to SEQ ID NOJ-12, a polynucleotide encoding a polypeptide with the same immunological specificity as any of the polypeptides encoded by SEQ ID NO: 1-12, or a polynucleotide which is a variant of SEQ ID NOJ-12.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS We used a technique that is novel with respect to endometriosis to discover auto- antigens found in patients having this disease. Using this method, we isolated and characterized copy DNA (cDNA) clones which encode polypeptides that are recognized by auto-antibodies found in the serum of endometriosis patients. The method for isolating the cDNAs and polypeptides, as well as the cDNAs and polypeptides that have been discovered using the method are described below. These cDNAs and especially the polypeptides they encode can be used in assays and as components in kits for diagnosing or monitoring endometriosis. These assays and kits are described in detail below.

METHOD OF ISOLATING AUTO-ANTIBODIES RELATED TO ENDOMETRIOSIS

In general, endometriotic auto-antigens were discovered by screening of endometriotic cDNA expression libraries with sera from affected and normal individuals. The process permitted the probing of a large number of phage expressed gene products displayed on nitrocellulose membranes. cDNA was prepared from the diseased tissue and cloned in a lambda expression vector. The cloned cDNA did not necessarily contain the full- length genes but often contained representative expressed fragments from the 3 ' end of the genes closer to the poly A tail. Bacteria infected with the phage were plated on agar and then transferred onto nitrocellulose membranes. Replicate membrane preparations from the same plate were exposed either to pooled sera from healthy donors or pooled sera from patients having endometriosis. Only those clones that were differentially bound by antibodies from the endometriosis pool, as compared to antibodies from healthy controls pool, were isolated and expanded. The expressed sequences from the expanded clones were determined by nucleic acid sequencing and the amino acid sequences of the open reading frames giving rise to a protein product were determined. These sequences were compared with public databases to identify known sequences. Not all of the sequences that were identified could be found in public databases and may or may not have been previously identified. None of the amino acid segments that were identified as portions of known proteins were previously known to bind to auto-antibodies from endometriosis patients.

The antigens isolated by this method have the particular advantage that the phage population probed with the patients sera is random. That is, the method allows for identification of auto-antigens without prior assumption of function, cellular localization, and role in the pathogenesis of the disease or abundance. The use of these auto-antigens, individually or preferably in combination with each other, in assays for auto-antibodies provides the opportunity to achieve high clinical sensitivity and specificity as well as a means for assessing disease progression, prognosis and therapeutic efficacy. The clones containing the recombinant proteins thus identified were individually adsorbed onto nitrocellulose strips and individual serum samples from patients having endometriosis, healthy donors, and patients with other diseases were separately tested with each of the strips. The extent of binding by antibody was determined using an enzyme labeled anti -human antibody to probe for bound immunoglobulins. These results demonstrate the utility of these cloned polypeptides to identify auto-antibodies from endometriosis patients. These peptides and the proteins in which they are normally expressed can be used separately or in various combinations for the differential diagnosis of endometriosis.

More detailed descriptions of the methods used to isolate the cDNA clones encoding autoantigens of endometriosis are found in the specific examples.

ISOLATED NUCLEIC ACIDS

The present invention relates to the polynucleotides which were isolated by the above- described method which correspond to SEQ ID NO: 1-12 and polynucleotides which encode similar polypeptides to those encoded by SEQ ID NO: 1-12 and which may include additional coding and/or non-coding sequences.

The polynucleotides of the present invention may be in the form of ribonucleic acid (RNA) or in the form of deoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence of polynucleotides of the present invention may be identical to the coding sequence of any of SEQ ID NO: 1-12 or may be a different coding sequence which, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptide as that encoded by SEQ ID NO: 1-12. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. The polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.

The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of SEQ ID NO: 1-12, SΞQ ID NOJ3-25, respectively. The variants of the polynucleotides may be naturally occurring allelic variants of the polynucleotides or non-naturally occurring variants of the polynucleotides.

The polynucleotides of the present invention may be used to express the polypeptide auto-antigens they encode by inserting the polynucleotides into a vector and operatively linking them to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lacZ, trp, phoA, and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for

RECTIFIED SHEET (RULE 91) culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and

Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQΕ70, pQE60 and pQE-9, (QIAGEN, Inc., Valencia, CA); pBluescript vectors, phage lambda gtl 1, Phagescript vectors, pNH8A, PNHlόa, pNH18A, pNH46A, (Stratagene Cloning Systems, Inc., Lo Jolla, CA); and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5, (Pharmacia Biotech, Inc., Piscataway, NJ). Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG (Stratagene Cloning Systems, Inc., Lo Jolla, CA); and ρSVK3, pBPV, pMSG and pSVL (Pharmacia Biotech, Inc., Piscataway, NJ). Other suitable vectors will be readily apparent to the skilled artisan.

ISOLATED POLYPEPTIDES

The present invention further relates to polypeptides that are useful for the detection of auto-antibodies present in endometriosis patients which have the amino acid sequences of SEQ ID NO: 13-25, the deduced amino acid sequences of DNA SEQ D NOJ-12 respectively, as well as fragments, analogs and derivatives of such polypeptides.

The terms "fragment," "derivative" and "analog" when referring to any of the polypeptides which have the deduced amino acid sequences of SEQ ID NO: 13-25 mean a polypeptide which retains essentially the same biological function and preferably mean a polypeptide which retains essentially the same immunological specificity as such

RECTIFIED SHEET (RULE 91) polypeptides. Thus, an analog includes a polypeptide of different length than any of those which have the deduced amino acid sequences of SEQ ID NO: 1-12 but which retains the same immunological specificity of those polypeptides. In this application, "polypeptides with the same immunological specificity" refers to polypeptides that contain at least one common epitope that is recognized by auto-antibodies.

The fragment, derivative or analog of a polypeptide which has the deduced amino acid sequences of any of SEQ ID NO: 13-25 may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the epitopes recognized by auto-antibodies to endometriosis are retained, or (iii) one in which the polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence, or (v) one in which the polypeptide is fused to a detection molecule such as an enzyme, a member of a binding pair, or another type of label. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

"Member of a binding pair" refers to one of two different molecules, having an area on the surface or in a cavity which specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of the other molecule. The members of the specific binding pair are referred to as ligand and receptor (antiligand). These

RECTIFIED SHEET (RULE 91 will usually be members of an immunological pair such as antigen-antibody, although other specific binding pairs such as biotin-avidin, hormones-hormone receptors, nucleic acid duplexes, IgG-protein A, polynucleotide pairs such as DNA-DNA, DNA-RNA, and the like are not immunological pairs but are included in the invention and the definition of sbp member.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide. The polypeptides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

The polypeptides detected by auto-antibodies in endometriosis patients can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.

The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.

The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments. Many of these procedures are discussed in Harlow E. and Lane D., "Antibodies, A Laboratory Manual," Cold Spring Harbor Laboratory, Cold Spring Harbor, HY (1988), incorporated herein by reference. These antibodies can be used as controls in diagnostic kits which incorporate the polypeptides.

USE OF POLYPEPTIDES IN ASSAYS TO DETECT ENDOMETRIOSIS

The invention also provides diagnostic assays for the detection of auto-antibodies in patient fluid. These assays are based on the binding of the polypeptides of the invention by auto-antibodies in the fluid of a patient and detection of those bound antibodies. The fluid may be plasma, sweat, blood, saliva, urine, or preferably serum. Examples of preferred methods which are well-known in the art that can be used to carry out the assays include ELISA (Enzyme-linked immunosorbent assay), RIA (Radioimmuno assay), and Western blot analysis. Another preferred method for carrying out the assays is the DELISA (Depletion enzyme-linked immunosorbent assay) described in Void et al. U.S. Pat No. 5,561,049. Other methods for carrying out the invention include the use of magnetic-separation technologies, many of which are know in the art. see eg., Rao et al, U.S. Pat. No. 5,660,990: "Surface immobilization of magnetically collected materials" (1997), incorporated herein by reference. Certain methods of the present invention require washing steps following interaction of the sample with the antigens and in between the steps leading to signal formation. In other methods of the present invention, the wash steps are not required in so far as the methods are homogenous, i.e. no wash steps are required for signal production. Examples for the homogenous methods are the luminescent oxygen channeling immunoassay as described by Ullman et al. ("Luminescent oxygen channeling assay LOCI™: sensitive, broadly applicable homogenous immunoassay method" E. F. Ullman et al. Clinical Chemistry, 42:9, 1518-1526 (1996).), the scintillation proximity assay (SPA) described by Linace et al. (Methodol Surv. Biochem. Anal. 22, 3425-6 (1992)), and the method described in U.S. Patent No. 5,731,147 in which electrochemiluminscent organometalic compounds are used for detection and quantification of analytes in homogenous binding assays. A number of other types of assays that are known to one of ordinary skill in the art can be used to carry out the binding of auto- antibodies to the polypeptides of the present invention.

For the above-described assay the polypeptide may be utilized in a carrier-bound form. The carrier may be coupled to the polypeptide by any suitable technique known in the art. The term "carrier" should be interpreted broadly and includes a solid surface or support. A solid surface or support is a porous or non-porous water insoluble material. The support can be hydrophilic or capable or being rendered hydrophilic and includes inorganic powders such as silica, magnesium sulfate, and alumina; natural polymeric materials, particularly cellulosic materials and materials derived from cellulose, such as fiber containing papers, e.g., filter paper, chromatographic paper, etc.; synthetic or modified naturally occurring polymers, such as nitrocellulose, cellulose acetate, poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose, polyacrylate, polyethylene, polypropylene, poly (4- methylbutene), polystryrene, polymethacrylate, poly (ethylene terephthalate), nylon, poly(vinyl butyrate), etc; either used by themselves or in conjunction with other materials; glass available as Bioglass, cermics, metals, and the like. Natural or synthetic assemblies such as liposomes, phospholipid vesicles, and cells can also be employed.

The bound complex containing the polypeptide and auto-antibodies can be detected by many techniques which are known in the art. For example, after the antibody-antigen binding, the captured auto-antibodies can be bound by a detection molecule. Typically, the detection molecule contains a labeled effective antibody molecule which binds human antibodies. The effective antibody molecule may be for example a whole antibody molecule, a single-chain antibody molecule, or a fragment of an antibody molecule such as, for example, an Fab fragment. Alternatively, the antigen itself may be labeled. Many types of labels are known in the art. For example, the label may be a radioisotope, such as, but not limited to, iodine (¹²⁵I, ¹²ⁱI), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹²In), and technetium (^99mTc) or a dye such as, but not limited to,_Cascade Blue^® (Molecular Probes, Eugene, OR) or Lucifer Yellow (Molecular Probes, Eugene, OR).. The label may be a fluorescent molecule, such as, but not limited to, fluorescein or rhodamine.

Alternatively, the label may be a chemiluminescer, an oligonucleotide, a solid surface, or a small organic molecule. A small organic molecule is a compound of molecular weight less than about 1500, preferably 100 to 1000, more preferably 300 to 600 such as biotin, digoxin, fluorescein, rhodamine and other dyes tetracycline and other protein binding molecules, and haptens, etc. The small organic molecule can provide a means for attachment of a protein, polypeptide or a nucleotide sequence to a label or to a support.

Another label that may be used with the current invention is an enzyme, for example horseradish peroxidase or preferably alkaline phosphatase. When an enzyme is used as the label, typically a substrate is used in a buffer in which the enzyme is active. The chemical change in the substrate is detected. Substrates to be employed when horseradish peroxidase is the enzyme label include chloronapthol, aminoethylcarbazole (AEC), diaminobenzidine (DAB), and preferably tetramethylbenzidine (TMB). Examples of substrates which are employed when alkaline phosphatase is the enzyme label include p-nitrophenyl phosphate (PNPP), methylumbelliferyl phosphate (MUP), 5-bromo-4-chloro-3-indoyl phosphate (BCIP), 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT, Molecular Probes, Inc., Eugene, OR) and nitroblue tetrazolium (NBT) Alternatively, the enzyme and substrate can be reversed so that the substrate is the label.

Especially preferred instruments for carrying out the assays are biosensors. In a preferred embodiment the biosensors detect antibody binding to an immobilized antigen. Especially preferred are biosensors that utilize evanescent wave interactions. Details describing various embodiments and methods of fabricating evanescent wave biosensors can be found in Tran, T.A. et al, "Real-time immunoassays using fiber-optic long-period grating sensors," Proc. SPIE-Int. Soc. Opt. Eng., 2676, 165-170 (1996), Christensen, D.A. et al, "Optical Immunoassay systems based upon evanescent wave interactions," Proc. SPIE-Int. Soc. Opt. Eng.. 2680, 58-67 (1996), Mahoney, W.C. et al, "Real-time immunodiagnostics employing optical immunobiosensors," Immunoassay Autom., Academic, San Diego, Calif, 231-52, (1996), and Golden, J. P., et al, "A near IR biosensor for evanescent wave immunoassays," Proc. SPIE-Int. Soc. Opt. Eng.,2138, 241-45, (1994). These references are incorpated herein by reference.

The invention also includes a diagnostic kit comprising one or more containers filled with one or more of the polypeptides of the invention or one or more of the isolated polynucleotides which encode these polypeptides. In addition to these container(s) the kit may contain several other items. These items include, but are not limited to: a governmental notice reflecting approval by an appropriate governmental agency for the manufacture and sale of the kit; instructions for carrying out an assay using the kit, an opitonally-labeled detection molecule capable of binding to human antibodies, a substrate for the label; a positive and negative control comprising a solution containing and not containing, respectively, antibodies which recognize the one or more polypeptides of the invention, and buffers for performing rinse steps. Alternatively, the one or more polypeptide in the kit may be bound to a solid support such as a bead, microparticle, microtiter plate, or filter. The kit components are used to perform any of the assays described above.

If the kit comprises the isolated polynucleotides of the present invention, these polynucleotides are preferably provided for the production of the polypeptide auto-antigens that they encode. The polynucleotides may be provided in an isolated form, as inserts in a vector, or as cells that have been transfected or infected with the vectors containing the polynucleotides of the present invention.

Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.

EXAMPLE 1 Preparation of a cDNA Library from Endometriotic Tissue

The following steps are involved in the construction and immunoscreening of a tissue specific cDNA library using patient serum samples as specific probes.

Tissue Sample The tissue samples were obtained from Dr. Kiechle (Beaumont Hospital). The tissues were obtained from hysterectomies of patients suffering from a number of uterine abnormalities including endometriosis. The excised tissue was immediately rinsed in phosphate buffered saline (PBS) to remove contaminating blood and mucous and was flash frozen in liquid nitrogen.

mRNA isolation

A two step isolation procedure was adopted to maximize the purity of messenger RNA (mRNA) from endometrial tissue. The frozen tissues were homogenized in a guanidinium iso-thiocyanate buffer and lithium chloride (LiCl) was used to precipitate the total RNA. The mRNA was isolated from total RNA by affinity chromatography using an oligo-dT cellulose column. The oligo-dT bound mRNA was washed to remove other nonspecifically bound cellular materials and an elution step was used to obtain pure poly adenylated mRNA. The mRNA isolated from patient tissue using these procedures was subsequently used to synthesize a cDNA library. Construction of a cDNA Library cDNA Libraries were prepared by using completely denatured mRNA as the starting material and by using oligo-dT primers that bind to the poly A tail. The primers were designed such that they hybridize to the junction of a poly A tail and the encoded transcript. First strand synthesis was performed using reverse transcriptase. Eco Rl adapters were then ligated onto the end of the reverse transcribed first strand and DNA polymerase was used to synthesize the second strand. After second strand synthesis was completed, low molecular weight cDNAs were removed by gel filtration, and the remaining cDNAs were selected and cloned into a lambda vector. A modified version of the lambda gtl 1 vector (Stratagene Cloning Systems, Inc., Lo Jolla, CA) was used to construct the cDNA library. The library was packaged using Gigapack^® III (Stratagene Cloning Systems, Inc., Lo Jolla, CA) and was amplified once, on a solid support, to obtain a stable undistorted representation of the library.

The amplified library was subsequently stored at -70°C in 7% v/v DMSO.

Characterization of the cDNA Library Characterization of the library was performed at various levels. The cDNA library was probed with a microglobulin specific antibody. Approximately 0.1% of the clones were demonstrated to be immunoreactive, indicating that the library is representative of the tissue and that it contains cDNAs corresponding to both abundant and rare transcripts. The library was also characterized for insert size. PCR amplification of the clone inserts using lambda specific primers just outside the cloning site showed that the pilot library of 50 clones had a size distribution from 300 bp to 3000 bp with an average size of approximately 0.8 to 1 kb.

The amplified phage lysate also had a high titer of approximately 1 O^pfu/ml (plaque forming units per ml), once again indicating that the library is comprehensive and representative of the tissue. The ratio of the nonrecombinant to recombinant clones was found to be 1 : 100. Identification of auto-antigens

Display of Phage Expressed Recombinant Gene Products on Nitrocellulose Membranes

Cloning directly into the lacZ gene of lambda vectors β-galactosidase (β-gal) system

results in the expression of the foreign DNA as a β-gal fusion protein. Bacterial host cells

were infected with the phage library and were plated on a solid support. Infection was allowed to proceed, and the proteins released by the lysed cells were immobilized onto a nitrocellulose filter overlay saturated with isopropylthiogalactoside (IPTG) an inducer of protein expression. Patient or normal serum (primary antibody) was used as a probe to detect phage recombinants containing protein sequences of interest. Primary antibody binding was visualized by the use of an anti-human Ig antibody conjugated to alkaline phosphatase (secondary antibody) followed by colorometric detection using 5-bromo-4-chloro-3-indoyl phosphate (BCIP) and nitroblue tetrazolium (NBT) as substrates.

Freshly grown host cells (E. coli strain XLI-Blue MRF') were infected with a dilution of the lambda library. The infected cells were mixed with molten soft agar and were plated onto large LB plates. After a few hours of growth, protein production was induced in the phage infected cells by overlaying a nitrocellulose filter presoaked in IPTG. IPTG is a

gratuitous inducer which is used to enhance the expression of the β-gal fusion gene product at

37°C. A dry sterile nitrocellulose filter previously saturated with 10 mM IPTG was placed

onto the top agar. The filters were air-dried and forceps were used to place the membranes onto the top agar so as to avoid trapping air bubbles between the nitrocellulose and the agar.

The filters were marked in three asymmetric locations to orient the filter by stabbing through the filter into the agar with an 18-gauge needle. The plates containing the filters were

incubated at 37°C for 4 hours. The plates were then transferred to 4°C for a few hours, and

the filters were carefully removed and rinsed in Tris Buffered Saline containing Tween 20

(TBST).

Screening of the Phage Expressed Library with Pooled Patient Sera The filters were incubated in TBST containing a blocking agent. A blocking agent is used to saturate the nonspecific protein binding sites on the nitrocellulose paper and reduce background. Either 4% w/v Non-Fat Dry Milk (NFDM), 20% v/v fetal calf serum, or 20% v/v horse serum was used. Most of the work was done with NFDM. The filters were kept in

this solution for a minimum of 4 hours at room temperature (RT) or longer at 4°C. After the

blocking step was over, the filters were removed and rinsed in TBST. Once the blocking step was completed, the filters were incubated with primary antibody. A pool of human patient serum was used as a probe. By pooling patient serum samples, immunoreactivities that are common to all patient samples will have an additive effect and immunoreactivities that are variable from one patient to another will be diluted and will, therefore, have a negligible effect. Six different patient serum samples were pooled and diluted in TBST-containing sodium azide. The final dilution of human serum used as a probe was 1 :10 and each individual serum sample was diluted to 1 :60. Prior to use in the assay, the human serum samples were preadsorbed with bacterial and phage lysate proteins. Preadsorption results in the removal of those cross-reactive antibody species that would result in the generation of a high background. The filters were incubated in the preadsorbed diluted

serum pool overnight at 4°C. Filter washes were performed to remove nonspecifically bound serum proteins, and the blots were incubated with the anti-human alkaline phosphatase (AP) conjugated secondary antibody for two hours at RT. The immunoreactivities were visualized by a colorimetric reaction involving BCIP and NBT as substrates.

Isolation and Purification of the immunoreactive Phage Clones

The immunoreactive positive signals were identified, marked, and their location

pinpointed on the master plate. A 0.5 cm^ area in the vicinity of the positive signal was cored out, the included phage lysate was dispersed and the suspension was replated at a lower dilution. The replating and reprobing serves as a means of purification and as a means of replicating the immunoreactive recombinant phage clones. A 10% rate of relplication of signal-producing clones was observed. The signal-producing clones that were replicated were further purified to homogeneity, and purification of the recombinant clone was confirmed by both immunoreactivity profile of the clone and by polymerase chain reaction (PCR) amplification of the recombinant insert. PCR Amplification

Various primer sets were used for the amplification of the recombinant inserts. The primers that proved most effective include the Long Distance (LD) amplimers and sequencing primers from Clontech (Clontech, Inc., Palo Alto, CA). The recombinant inserts were amplified using the sequencing primers located upstream and downstream of the EcoRl sites. 5 μl of the high titer lysate was used for amplification. The target DNA was released

from the phage coats by a long initial denaturation step at 94°C. The primers were allowed to

anneal to the target at 62°C for 30s, and the extension was allowed to proceed at 72°C for 1

minute. After 25 cycles, the amplified material was analyzed by gel electrophoresis for confirming clone purity and for identifying the size of the insert. The amplified material was further purified away from the primers and dNTPs using a PCR purification column, and the purified material was subsequently used for sequencing. The sequences that have been identified as comprising endometriosis auto-antigens are listed in Table 1.

TABLE 1. Endometriosis Antigen Sequences And Relationships With Known Proteins

Clone Clone Sequence Homology With Known Sequences No. Size

1 1026 bp Sequ ID 1 Hu Supervillin mRNA

2 351 bp Seq ID 2 no significant homology

3 504 bp Seq ID 3 no significant homology

4 990 bp Seq ID 4 64 kd auto-antigen found in patients with auto- immune thyroid disease

5 1134 bp Seq ID 5 Hu AHNAK protein desmoyokin

7 1076 bp Seq ID 6 64 kd auto-antigen found in patients with auto- immune thyroid disease

(different than clone # 4 in immunoreactivity profile)

1536 bp Seq ID 7 microfibril associated glycoprotein has multiple stop codons at the 3' terminus

9 1318 bp Seq ID 8 no significant homology

10 406 bp Seq ID 9 No significant homology no signifcant protein associated homology homology to a bac-clone

11 508 bp Seq ID 10 no significant homology

12 567 bp Seq ID 11 Hu leucine rich protein

13 1226 bp Seq ID 12 Hu ribosomal protein S6 kinase EXAMPLE 2 Demonstration of Disease Specificity

Each of the purified clones was titered and plated out at a low density. Protein production was induced with IPTG and the expressed protein was transferred to nitrocellulose. Each filter was cut into multiple strips and the strips were probed with individual serum samples. By means of these strips, the recombinant clones (#1 to #13) were screened with a panel of four (4) endometriosis patient sera (E4, E12, E15, and E21), four (4) nonendometriosis patient sera (UC3, UC6, UC7, and UC14), and four (4) healthy control sera (HCl, HC2, HC3, and HC4) to determine immunoreactive differences between the patient and control serum samples. The intensity of reactivity of the different sera samples with phage expressed auto-antigen clones was assessed visually and assigned an arbitrary intensity level ranging from 1 to 5 denoting increasing intensity, respectively (Table 2). Cutoff values above, which binding was considered indicative of the presence of endometrial antibodies, were determined as a single value either for all clones, as shown in Table 3, or for each auto-antigen, as given in Table 4. The sum total of reactivity using multiple antigens was particularly informative for diagnosis. Moreover, several antigens, marked with "_*", were particularly informative when used individually.

TABLE 2.

The recombinant clones (# 1 to # 13) were screened with a panel of patient (four) unhealthy (four) and healthy (four) individual serum samples to determine immunoreactive differences between the patient and control serum samples. The intensity of reactivity of the different sera samples with phage expressed auto-antigen clones was assessed visually and assigned an arbitrary intensity level ranging from 1 to 5 denoting increasing intensity, respectively.

TABLE 3.

The reactivity data presented in Table 2 was used for demonstration of utility of the disclosed auto- antigens for the diagnosis of endometriosis. Reactivity values above 2 arbitrary units were designated "+" and those below 2 as "-".

TABLE 4. Binding To Endometriosis Clones By Patient Samples

— Patient Samples- Unhealthy Controls Healthy Controls

Clone Cutoff E4 E12 E15 E21 UC3 UC6 UC7 UC14 HCl HC2 HC3 HC4

(0-5 range)

2* 2.5 + + + + - - .

3 2.5 + + + + + . -

4* 3.5 + + + + - - -

7* 3.5 + + + + - _ -.

8 3.5 + + - + - -. -.

9 3.5 + + - + - -. -

10 2.5 + + + + - + -

11* 1.5 + + + + - . -.

13* 3.5 + + + + - - -.

Score 100 100 78 100 89 100 89 100 100 100 100 100

*Preferred auto-antigens for use in the assay of endometrial auto-antibodies are clones #2, #4. #7, #13, and #11. These antigens may be used individually or, preferably, in combinations of two or more antigens.

Table of abbreviations used in the s ecification

Claims

What is claimed is:

1. A method for detecting endometriosis in a patient comprising detecting the presence of auto antibodies in said patient which bind to one or more polypeptides selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO: 13-25, and fragments thereof having the same immunological specificity toward auto-antibodies associated with endometriosis, wherein the presence of said antibodies is indicative of endometriosis in said patient.

2. The method of claim 1 wherein said detection of said auto antibodies in said patient comprises:

(a) combining body fluid from said pateint with said one or more polypeptides;

(b) forming a complex between said auto-antibodies and said one or more polypeptides;

(c) detecting the complex.

3. The method of claim 2 wherein said one or more polypeptides are selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO: 14, 16, 18, 23, and 25, and fragments thereof having the same immunological specificity toward auto- antibodies associated with endometriosis.

4. The method of claim 1 wherin the method is selected from the group consisting of radioi munoassay, immunoprecipitation assay, enzyme-linked immunoassay (ELISA), depletion enzyme-linked immunoassay (DELISA), Western Blot, particle agglutination assay, luminescent oxygen-channelling immunoassay, proximity-based immunoassay, and biosensor-based immunoassay.

RECTIFIED SHEET (RULE 91)

5. A method for monitoring an endometriosis patient comprising periodically conducting the assay of claim 4.

6. The method of claim 5 wherein the fluid is selected from the group consisting of sera, plasma, sweat, urine, and saliva.

7. The method of claim 2 wherein the one or more polypeptides are bound to a solid support.

8. The method of claim 7 where the solid support is selected from the group consisting of beads, microparticles, microtiter plates, and membranes.

9. The method of claim 2 wherein said complex is detected using a detection molecule, said detection molecule comprising an antibody molecule that is labeled with a label, said antibody molecule binds to human antibodies.

10. The method of claim 9 wherein said labeled antibody molecule is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a single-chain antibody, and fragments thereof.

11. The method of claim 9 wherein said label is selected from the group consisting of radioactive molecules, fluorescent molecules, chemiluminescers, dyes, oligonucleotides, enzymes, coenzymes, enzyme substrates, small organic molecules, and solid surfaces.

12. The method of claim 2 wherein said one or more polypeptides are labeled.

RECTIFIED SHEET (RULE 91)

13. The method of claim 12 wherein said label is selected from the group consisting of radioactive molecules, fluorescent molecules, chemiluminescers, dyes, oligonucleotides, enzymes, coenzymes, enzyme substrates, small organic molecules and solid surfaces.

14. The method of claim 1 wherein said autoantibodies are found in patient fluid selected from the group consisting of sera, plasma, sweat, urine, and saliva.

15. A method for detecting endometriosis comprising:

(a) contacting a fluid from a patient with one or more polypeptides selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID

NO: 13-25, and fragments thereof having the same immunologicial specificity to autoantibodies of endometriosis, to capture antibodies in the fluid that bind to the polypeptide which are immobilized on a solid surface;

(b) applying a detection molecule to the captured antibodies bound to the polypeptide, the detection molecule having the capacity to specifically bind to the captured antibodies;

(c) detecting the bound detection molecule.

16. The method of claim 15 wherein the one or more polypeptides are selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO: 14,

16, 18, 23, and 25, and fragments thereof having the same immunologicial specificity toward autoantibodies associated with endomitriosis.

17. A kit for detecting endometriosis comprising: (a) one or more polypeptides selected from the group consisting of a polypeptide

RECTIFIED SHEET RULE 91 comprising the amino acid sequence of SEQ ID NO: 13-25, and fragments thereof having the same immunologicial specificity to autoantibodies of endometriosis; (b) a detection molecule capable of binding to human antibodies.

18. The kit of claim 17 wherein the one or more polypeptides are selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO: 14, 16, 18, 23, and 25, and fragments thereof having the same immunologicial specificity toward autoantibodies associated with endomitriosis.

19. The kit according to claim 18 wherein the one or more polypeptide are bound to a solid support.

20. The kit according to claim 19 wherein the detection molecule comprises a labeled antibody molecule which binds to human antibodies.

21. A reagent for detecting endometriosis comprising one or more polypeptides selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO: 13-25, and fragments thereof having the same immunologicial specificity to autoantibodies of endometriosis.

22. The reagent of claim 21 wherein the polypeptide is labeled.

23. The reagent of claim 22 wherein said label is selected from the group consisting of radioactive molecules, fluorescent molecules, chemiluminescers, dyes, oligonucleotides, enzymes, coenzymes, enzyme substrates, small organic molecules and solid surfaces.

RECTIFIED SHEET (RULE 91)

24. A reagent for detecting endometriosis comprising one or more polypeptides selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NOJ4, 16, 18, 23, and 25, and fragments thereof having the same immunologicial specificity toward autoantibodies associated with endomitriosis.

25. The reagent of claim 24 wherein the polypeptide is labeled.

26. The reagent of claim 25 wherein said label is selected from the group consisting of radioactive molecules, fluorescent molecules, chemiluminescers, dyes, oligonucleotides, enzymes, coenzymes, enzyme substrates, small organic molecules and solid surfaces.

27. A method for detecting auto-antigens found in endometriosis patients comprising screening an endometriotic cDNA expression library with pooled fluid from normal and endometriosis patients.

28. The method of claim 27 wherein the pooled fluid is selected from the group consisting of sera, plasma, sweat, urine, and saliva.

29. The method of claim 27 wherein the pooled fluid is sera.

30. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 13, and fragments thereof having the same immunologicial specificity toward autoantibodies associated with endomitriosis.

RECTIFIED SHEET (RULE 91)

31. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, and fragments thereof having the same immunologicial specificity toward autoantibodies associated with endomitriosis.

32. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 15, and fragments thereof having the same immunologicial specificity toward autoantibodies associated with endomitriosis.

33. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 16, and fragments thereof having the same immunologicial specificity toward autoantibodies associated with endomitriosis.

34. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 17, and fragments thereof having the same immunologicial specificity toward autoantibodies associated with endomitriosis.

35. An isolated polypeptide comprising an amino acid sequence selected from the group consistmg of SEQ ID NOJ8, and fragments thereof having the same immunologicial specificity toward autoantibodies associated with endomitriosis.

36. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOSJ9 and 20, and fragments thereof having the same immunologicial specificity toward autoantibodies associated with endomitriosis.

37. An isolated polypeptide comprising an amino acid sequence selected from the group

RECTIFIED SHEET RULE 91 consisting of SEQ ID NO:21, and fragments thereof having the same immunologicial specificity toward autoantibodies associated with endomitriosis.

38. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:22, and fragments thereof having the same immunologicial specificity toward autoantibodies associated with endomitriosis.

39. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:23, and fragments thereof having the same immunologicial specificity toward autoantibodies associated with endomitriosis.

40. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:24, and fragments thereof having the same immunologicial specificity toward autoantibodies associated with endomitriosis.

41. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:25, and fragments thereof having the same immunologicial specificity toward autoantibodies associated with endomitriosis.

42. An isolated nucleic acid molecule comprising a polynucleotide selected from the group consisting of:

(a) a polynucleotide of SEQ ID NO: 1 or a polynucleotide which h> bridizes to SEQ IDNOJ;

(b) a polynucleotide encoding a polypeptide of a translation of SEQ ID NO: 1 or a polypeptide encoded by a cDNA sequence which hybridizes to SEQ ID NO: 1 ;

(c) a polynucleotide encoding a polypeptide with similar immunological specificity to the polypeptide encoded by one or more of SEQ ID NO: 1;

RECTIFIED SHEET (RULE 91) (d) a polynucleotide which is a variant of SEQ ID NO: 1.

43. An isolated nucleic acid molecule comprising a polynucleotide selected from the group consisting of: (a) a polynucleotide of SEQ ID NO:2 or a polynucleotide which hybridizes to SEQ

ID NO:2;

(b) a polynucleotide encoding a polypeptide of a translation of SEQ ID NO: 2 or a polypeptide encoded by a cDNA sequence which hybridizes to SEQ ID NO:2;

(c) a polynucleotide encoding a polypeptide with similar immunological specificity to the polypeptide encoded by one or more of SEQ ID NO: 2;

(d) a polynucleotide which is a variant of SEQ ID NO:2.

44. An isolated nucleic acid molecule comprising a polynucleotide selected from the group consisting of: (a) a polynucleotide of SEQ ID NO:3 or a polynucleotide which hybridizes to SEQ

ID NO:3;

(b) a polynucleotide encoding a polypeptide of a translation of SEQ ID NO: 3 or a polypeptide encoded by a cDNA sequence which hybridizes to SEQ ED NO:3;

(c) a polynucleotide encoding a polypeptide with similar immunological specificity to the polypeptide encoded by one or more of SEQ ID NO: 3 ;

(d) a polynucleotide which is a variant of SEQ ID NO:3.

45. An isolated nucleic acid molecule comprising a polynucleotide selected from the group consisting of: (a) a polynucleotide of SEQ ID NO:4 or a polynucleotide which hybridizes to SEQ

ID NO:4;

(b) a polynucleotide encoding a polypeptide of a translation of SEQ ID NO: 4 or a polypeptide encoded by a cDNA sequence which hybridizes to SEQ ID NO:4;

(c) a polynucleotide encoding a polypeptide with similar immunological specificity to the polypeptide encoded by one or more of SEQ ID NO: 4;

(d) a polynucleotide which is a variant of SEQ ID NO:4.

RECTIFIED SHEET (RULE 91)

46. An isolated nucleic acid molecule comprising a polynucleotide selected from the group consisting of:

(a) a polynucleotide of SEQ ID NO:5 or a polynucleotide which hybridizes to SEQ ID NO-5;

(b) a polynucleotide encoding a polypeptide of a translation of SEQ ID NO: 5 or a polypeptide encoded by a cDNA sequence which hybridizes to SEQ ED NO:5;

(c) a polynucleotide encoding a polypeptide with similar immunological specificity to the polypeptide encoded by one or more of SEQ ID NO: 5; (d) a polynucleotide which is a variant of SEQ ID NO : 5.

47. An isolated nucleic acid molecule comprising a polynucleotide selected from the group consisting of:

(a) a polynucleotide of SEQ ID NO:6 or a polynucleotide which hybridizes to SEQ ID NO:6;

(b) a polynucleotide encoding a polypeptide of a translation of SEQ ID NO: 6 or a polypeptide encoded by a cDNA sequence which hybridizes to SEQ ID NO:6;

(c) a polynucleotide encoding a polypeptide with similar immunological specificity to the polypeptide encoded by one or more of SEQ ID NO: 6; (d) a polynucleotide which is a variant of SEQ ID NO:6.

48. An isolated nucleic acid molecule comprising a polynucleotide selected from the group consisting of:

(a) a polynucleotide of SEQ ID NO:7 or a polynucleotide which hybridizes to SEQ ID NO:7;

(b) a polynucleotide encoding a polypeptide of a translation of SEQ ID NO: 7 or a polypeptide encoded by a cDNA sequence which hybridizes to SEQ ID NO:7;

(c) a polynucleotide encoding a polypeptide with similar immunological specificity to the polypeptide encoded by one or more of SEQ ID NO: 7; (d) a polynucleotide which is a variant of SEQ ED NO:7.

RECTIFIED SHEET (RULE 91)

49. An isolated nucleic acid molecule comprising a polynucleotide selected from the group consisting of:

(a) a polynucleotide of SEQ ID NO: 8 or a polynucleotide which hybridizes to SEQ ID NO:8;

(b) a polynucleotide encoding a polypeptide of a translation of SEQ ID NO: 8 or a polypeptide encoded by a cDNA sequence which hybridizes to SEQ ID NO: 8;

(c) a polynucleotide encoding a polypeptide with similar immunological specificity to the polypeptide encoded by one or more of SEQ ID NO: 8;

(d) a polynucleotide which is a variant of SEQ ID NO: 8.

50. An isolated nucleic acid molecule comprising a polynucleotide selected from the group consisting of:

(a) a polynucleotide of SEQ ID NO:9 or a polynucleotide which hybridizes to SEQ ID NO:9; (b) a polynucleotide encoding a polypeptide of a translation of SEQ ID NO: 9 or a polypeptide encoded by a cDNA sequence which hybridizes to SEQ ID NO:9;

(c) a polynucleotide encoding a polypeptide with similar immunological specificity to the polypeptide encoded by one or more of SEQ ID NO: 9;

(d) a polynucleotide which is a variant of SEQ ID NO:9.

51. An isolated nucleic acid molecule comprising a polynucleotide selected from the group consisting of:

(a) a polynucleotide of SEQ ED NO: 10 or a polynucleotide which hybridizes to SEQ ID NOJ; (b) a polynucleotide encoding a polypeptide of a translation of SEQ ID NO: 10 or a polypeptide encoded by a cDNA sequence which hybridizes to SEQ ID NO: 10;

(c) a polynucleotide encoding a polypeptide with similar immunological specificity to the polypeptide encoded by one or more of SEQ ED NO: 10;

(d) a polynucleotide which is a variant of SEQ ID NO: 10.

52. An isolated nucleic acid molecule comprising a polynucleotide selected from the group consisting of:

RECTIFIED SHEET (RULE 91) (a) a polynucleotide of SEQ ID NO: 11 or a polynucleotide which hybridizes to SEQ ID OJl;

(b) a polynucleotide encoding a polypeptide of a translation of SEQ ID NO: 11 or a polypeptide encoded by a cDNA sequence which hybridizes to SEQ D NO:l 1; (c) a polynucleotide encoding a polypeptide with similar immunological specificity to the polypeptide encoded by one or more of SEQ ID NO: 11 ;

(d) a polynucleotide which is a variant of SEQ ID NO: 11.

53. An isolated nucleic acid molecule comprising a polynucleotide selected from the group consisting of:

(a) a polynucleotide of SEQ ID NO: 12 or a polynucleotide which hybridizes to SEQ ID NO:12;

(b) a polynucleotide encoding a polypeptide of a translation of SEQ ID NO: 12 or a polypeptide encoded by a cDNA sequence which hybridizes to SEQ ID NO: 12; (c) a polynucleotide encoding a polypeptide with similar immunological specificity to the polypeptide encoded by one or more of SEQ ID NO: 12;

(d) a polynucleotide which is a variant of SEQ ID NO: 12.

RECTIFIED SHEET (RULE 91)