WO1994028021A1 - Endometrial proteins, antigenic compositions and methods for detecting endometriosis - Google Patents

Abstract

A purified antigen having a molecular weight of about 64 to 66 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Purified endometrial proteins, free of albumin and having molecular weights of about 64 kDa and isoelectric focusing points of 3.5, 4.0, 6.0, 6.5 and 8.0 are provided. A purified endometrial protein, free of albumin and having molecular weight of 94-97 kDa and iseolectric focusing point of 3.5 is also provided. The presence of antigen composition or endometriosis-associated endometrial protein is a marker for endometriosis in a subject. A purified monoclonal antibody specifically reactive with the antigen is also provided. A method of diagnosing endometriosis in a subject is also provided. The method comprises the steps of: (a) contacting an antibody-containing sample from the subject with the antigen composition or purified endometrial proteins of the invention; and (b) detecting the reaction of the antigen with an antibody from the sample, the reaction indicating endometriosis in the subject. Having provided an endometriosis associated antigen and monoclonal antibody, a method of diagnosing endometriosis in a subject, using the MAb is also provided.

Description

ENDOMETRIAL PROTEINS, ANTIGENIC COMPOSITIONS AND METHODS

FOR DETECTING ENDOMETRIOSIS

This is a continuation of patent application Serial No. 08/069,171 filed May 28, 1993.

BACKGROUND OF THE INVENTION

This invention was made with government support under National Institutes of Health Research Grant HD 27256. The government has certain rights in the invention.

Field of the Invention

The invention relates to purified endometriosis- associated proteins (free of albumin), purified endometriosis-associated antigenic compositions, and methods of diagnosing endometriosis.

Background Art

Endometriosis is a complex disease with possibly multiple causes frequently seen in association with infertility. Dysmenorrhea (menstrual abnormalities) is often seen in patients with endometriosis. Currently, the only way to diagnose endometriosis is by painful laparoscopic procedures. Thus, there exists a need for a noninvasive method for diagnosing endometriosis. Several obstacles to the development of such methods are presented in the literature. The presence of elevated titers of immunoglobulin G (IgG) auto-antibodies to endometrium in the serum of women with endometriosis have been reported (Mathur et al., 1982, 1988, 1990; Saiffudin et al., 1983; Badawy et al. , 1984, 1990; Wild and Shivers, 1985; Chihal et al. , 1986; Meek et al., 1988; Kennedy et al., 1990; Garza et al., 1991; Wild et al. , 1991). Serum endometrial antibody titers and the specific endometrial antigens to which these antibodies bind (Badawy et al. , 1984; Meek et al., 1988; Mathur et al., 1988; Garza et al. , 1991; Mathur et al. , 1990) do not correlate or vary with the stage or the degree of infiltration of the disease. 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.

However, further knowledge regarding the identities and specificity of endometrial auto-antigens that elicit these auto-antibody responses is essential for the development of an endometrial antibody assay for diagnosis, monitoring and therapy for endometriosis. Among the factors making such an assay difficult is that Genetic Gm and Am factors on human immunoglobulins (de Lange, 1989) and human leukocyte antigens (Ferrone and Solhei , 1982) show variations according to the race and geographical location of the various individuals. Furthermore, women with endometriosis develop autoimmune responses to ovarian antigens (Mathur et al., 1982) as well as to nuclear and phospholipid antigens (Confino et al., 1990) further complicating the use of antibodies for specific diagnosis. Contamination with white blood cell (WBC) and histone antigens may also confound the issue.

Eluting 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.

SUMMARY OF THE INVENTION

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 is also provided. The method 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. Having provided an endometriosis associated antigen and monoclonal antibody, a method of diagnosing endometriosis in a subject, using the MAb is also provided. 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. An example of passive hemagglutination, immunofluorescence, ELISA and Western blot analyses are provided in the Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

DETAILED DESCRIPTION OF THE INVENTION

Antigen

Purified antigenic proteins

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.

As used herein, "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. The term "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. 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.

Having purified the proteins provided above, well known protein sequencing methods can be applied to elucidate the amino acid sequence of each protein (Schleisinger, 1988). 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.

Thus, 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.

Antigenic fragments

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.

Once 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.

Antigenic compositions

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 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.

Further provided is 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, 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.

Other endometriosis-associated antigens provided 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.

Having provided antigenic polypeptide fragments of the present proteins, a composition comprising two or more of the antigenic polypeptide fragments of the present antigenic proteins is also provided. The 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 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.

Antigen Bound to Substrate 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. For example, the antigen can be fixed to the well of an ELISA plate simply by drying the antigen on to the surface of the plate. Alternatively, 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. Such 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). Likewise, the ligand can be a purified polyclonal antibody or a fragment thereof that specifically binds the antigen.

Purified Antibodies

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. Those positive clones can then be sequenced as described in the Examples or by other methods (see, for example, Kelly et al., Bio/Technoloσv 10:163-167, 1992 and Bebbington et al., Bio/Technoloσv 10:169-175, 1992).

The antibody can be bound to a solid support or labeled with a detectable moiety or both bound and labeled. The 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.

Nucleic Acids

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). By "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. By "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.

As used herein to describe nucleic acids, 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. Thus, 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. Depending on the length of the probe or primer, it can range between 70% complementary bases and full complementarity and still hybridize under high stringency conditions. For example, for the purpose of detecting the presence of the antigen- encoding gene, 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. Thus, 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. In general, 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. (Molecular Cloning: A laboratory manual, pages. Second edition, ed.; Cold Spring Harbor Lab., Cold Spring Harbor, NY., 1987, chapter 11). The 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.

One skilled in the art can readily obtain the nucleic acids of the present invention using routine methods to synthesize a full gene as well as shorter nucleotide fragments. For example, techniques for obtaining nucleic acids such as those provided in the Sequence Listing are specifically provided in the application. Furthermore, 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)) and Wosnick et al. ( Gene 76:153-160 (1989)) show routine methods to synthesize a gene of known sequence. More specifically, Ferretti et al. teach the synthesis of a 1057 base pair synthetic bovine rhodopsin gene from synthetic oligonucleotides. The synthesized gene was faithful to the known sequence (first sentence, page 603), demonstrating the reliability of this method of gene synthesis. Additionally, Wosnick et al. teach the synthesis of a maize glutathione-transferase (GST) gene using an efficient, one-step annealing/ligation protocol. This technique also produced a complete synthetic gene with 100% fidelity, which demonstrates the routine nature of this protocol.

Modifications to the 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. Likewise, 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).

Vectors and Hosts

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.

There are numerous 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. In these 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). In addition, 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

Southern analysis and production of an RNA corresponding to the antigen coding sequence can be confirmed by Northern analysis. A number of other 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.

Serological Detection (Diagnosis) Methods Detecting Antibody with Antigen

A method of diagnosing endometriosis in a subject is also provided. The method 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.

In a further method of diagnosing endometriosis in a subject by detecting endometriosis associated antibodies comprising the steps described above, 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.

contacted by a fluid sample such as serum, peritoneal fluid, urine, saliva or cervical mucous. Alternatively, 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. An example of passive hemagglutination, immunofluorescence, ELISA and Western blot analyses are provided in the Examples.

In addition, as in a typical sandwich assay, 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.

In the diagnostic methods taught herein, the antigen can be bound to a substrate and contacted by 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. In this manner, antibodies specific for the antigen (the primary antibody) will specifically react with the bound antigen. Thereafter, a secondary antibody bound to, or labeled with, a detectable moiety can be added to enhance the detection of the primary antibody. Generally, 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. Thus, for example, several molecules of the secondary antibody can react with each primary antibody, making the primary antibody more detectable.

Detectable Moieties

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. Examples of 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).

Nucleic Acid Detection (Diagnosis) Methods

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. Alternatively, the nucleic acid is detected utilizing direct hybridization or by utilizing a restriction fragment length polymorphism. For example, the present invention provides a method of diagnosing endometriosis, comprising ascertaining the presence of a nucleotide sequence associated with a restriction endonuclease cleavage site. In addition, 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. 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.

Depending on the intended mode of administration, 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. By "pharmaceutically acceptable" is meant 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. Antibody-Detecting Kit

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. Alternatively, 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. Such 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.

Antigen-Detecting Kit

An antigen detecting kit, comprising a detectable amount of the monoclonal or purified polyclonal antibody of the invention bound to a solid support is provided. Particularly, 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. Such 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.

The following examples are intended to illustrate, but not limit, the invention. While they are typical of those that might be used, other procedures known to those skilled in the art may be alternatively employed.

EXAMPLE 1

Endometriosis-Specific Antigenic Compositions

Study subjects and samples.

24 fertile women (20 white, 3 black and 1 Asian), free of endometriosis, aged 25 to 35 yr., gravida 2 to 5 and undergoing laparoscopic tubal ligation (n=23) or hysterectomy (n=1), served as the controls. Seventy-six infertile women (mostly with primary infertility) in the same age group as the controls from Charleston, Chicago, Dallas and Boston, and with laparoscopically proven endometriosis, served as the study group. None of the women had infectious endometrial pathology or were under exogenous steroid hormone therapy in the month preceding surgery. Twenty-four of the infertile women were in Stage II, 20 were in Stage I, 19 were in Stage III and 13 were in Stage IV of the disease. The clinics in Charleston, Chicago, Dallas and Boston, from which the subjects were selected, cater to a mixed racial population.

Sera from 20 patients with endometriosis, 7 with chronic pelvic inflammatory disease (PID), 4 with adenomyosis, 6 with uterine leiomyomata, 30 anovulatory women and 321 women with ovarian cancer were tested for IgG to endometrial and ovarian antigens.

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.

Western blot analysis of serum and P.F. for endometrial antibodies. Antigenic extracts were prepared (Mathur et al., 1988, 1991; Garza et al., 1991) from intrauterine endometrium and implants. Briefly, fifty μl each extract (2 mg per ml) was mixed 1:1 with the extraction buffer for 5 minutes at 100°C and centrifuged at 1000 X g for 10 to 15 seconds. The extracts were subjected to electrophoresis in a 7.5% to 15% gradient polyacrylamide gel (PAGE) containing 0.1% sodium dodecyl sulfate (Laemmli, 1970). The Western blot analysis was performed using serum and PF (Burnette, 1981; Mathur et al., 1988, 1991). Peroxidase labeled F(ab)₂ anti-human IgG was used as the secondary antibody. Blots without serum or P.F. layered over them (blanks) were processed at each run to rule out the bands caused by endogenous antibody or peroxidase activity. There was no endogenous peroxidase activity in the blanks. The mobility indices for the MW standards run simultaneously were semilogarithmically calibrated against their MW. The MW of unknown antigenic bands were determined and were reproducible (± 4 kDa).

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. After centrifugation for 4 min. at 400 X g, the plates were read at a 45° angle in a microtiter plate reader. The end point was the dilution at which the button formation stopped and the streaking began. Positive ovarian or endometrial antibody titers of ≥32 (reciprocal of end-point dilution; log₂ ≥5) are > mean + 2 S.D. of titers in normal fertile controls (Mathur et al., 1982). Sera from 28 of 30 normal healthy men (n=30), used as negative controls, had endometrial antibodies at titers < 8 (log₂ 3). Two men with chronic gastrointestinal problems had a titer of 64. All sera from men were negative for ovarian antibodies.

Immunofluorescent antibody assay for ovarian and endometrial antibodies.

Frozen 5 μm 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. After washing, the cells were mounted in PBS-glycerol mixture and scored using an IMT 35 Zeiss fluorescent microscope (Mathur et al., 1982). All sera were heat inactivated and absorbed with pooled and washed human RBCs and lyophilized human liver powder. For the determination of specificity, the sera were absorbed with endometrium from controls or patients.

Testing Serum for anti—nuclear antibodies.

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. Of the patients in Chicago, 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.

Five human sera (positive controls) with strong anti-nuclear activity (The Binding Site, London, England) and monoclonal antibodies to 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 log₂ 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. Of these, 3 had IgA auto-antibodies to cardiolipin and total histone, IgM auto-antibodies to histone fraction H2B or IgA auto-antibodies to phosphatidyl-ethanolamine. Four patients (1 treated with prednisone; 2 with danazol) were negative for endometrial antibodies (titers < 16). Of these, 1 was positive for IgA antibodies to histone fraction H2B and ssDNA, while another was positive for IgA auto-antibodies to histone fraction H2A.

The Western blot analysis of 5 sera positive for nuclear antibodies against endometrial and implant antigens was unremarkable. 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

Electro—elution of endometrial/implant proteins.

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.

Immunization of rabbits with the native and eluted endometrial proteins.

Eight to 10 week old, young adult female NZW rabbits were intradermally immunized with 0.15 mg per ml protein emulsified with 0.05% complete Freund's adjuvant in the following groups (n = 2 per group): (1) Sterile saline (control); (2) Endometrial extracts from patients with endometriosis; (3) Implant extracts; (4) Eluted endometrial and implant proteins with MW 29 to 68 kDa; and (5) Eluted endometrial and implant proteins with MW >68 kDa. The antisera were absorbed with pooled normal endometrium and human liver powder to remove non-specific antibody binding. Absorption with implants was also done. Passive hemagglutination, immunofluorescence and Western blot analyses, as described above, were used to screen the antisera for endometrial antibodies. Cross-reactivity of the polyclonal antibodies with human ovary, spermatozoa, lymphocytes, macrophages, kidney and gastric duct was assessed by immunofluorescence assay.

Western blot analysis of patients' PF and serum for IgG against the eluted proteins confirmed that all the proteins have been eluted from the gels. Serum or PF from normal fertile controls failed to have IgG binding with the eluted implant proteins.

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 intensities were the highest in patients' sera and rabbit antisera to eluted endometrial and implant proteins (Table 4). The rabbit antisera to eluted endometrial or implant proteins and native endometrium from patients failed to react against ovary (Table 4), sperm, WBCs and brain. The MW of the specific endometrial and implant antigens that elicited immune responses from rabbits were the same as those in women with endometriosis (Tables 1 and 2; 4). This confirms the immunogenicity of endometrial or implant antigens from patients with MW of 34, 48, 64/66, 84, 94/97 and 120 kDa. What is more important, the endometrial glands, and not the stroma, bear the target antigenic epitopes for both patient serum and rabbit antiserum IgG.

Absorption of the rabbit antisera with controls' endometrium did not abolish immunoblot and immunofluorescent reactivities against patients' endometrium and implants. Absorption with patients' implants, on the other hand, abolished the antibody activity.

The immunization studies show that endometrial and implant antigens are highly immunogenic and, what is more important, that they may be antigenically and biochemically related. Rabbits immunized with 29 to 68 kDa endometrial proteins from patients had serum IgG binding to the same set of endometrial and implant antigens as those immunized with >68 kDa proteins (Table 4) and exhibited similar immunofluorescent patterns. This suggests a partial sequence homology of all these proteins. It is also possible that the large MW antigens are aggregates of smaller MW antigen (34 kDa).

Geographical differences in the endometrial and implant antigens.

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. (Tables 1 and 2). Of these, the antigen(s) with MW ranging from 64-66 kDa showed binding with IgG in serum and P.F. from all patients with endometriosis, irrespective of the city (Table 2). The with MW of 140 kDa was more reactive against PF than serum IgG. Antigens with MW 84 and 140 kDa were found mostly in the implants, while the other specific antigens were present in both endometrium and implants of the patients. All antigens were undetectable in normal endometrium. Also, the concentration of all antigens, as denoted by antibody activity of serum or PF of patients with endometriosis, was much higher in implants than in the endometrium of similar protein content. The serum or PF from fertile controls rarely had IgG to these antigens. The stage of endometriosis did not affect the antigenicity.

Incidence of endometrial and ovarian autoimmunity in endometriosis and other gynecological disorders. Positive titers (> 1:32) of circulating endometrial auto-antibodies (by hemagglutination and immunofluorescence assays) were observed primarily in patients with endometriosis and in a few with adenomyosis and certain gynecological disorders that may co-exist with endometriosis, such as PID, uterine leiomyomata and anovulation (Table 3). Since any of the above conditions can coexist with endometriosis, this finding does not challenge the specific association of endometrial antibodies with endometriosis.

The incidence of ovarian and endometrial auto-antibodies was independent of each other (P not significant by Chi-square analysis). Patients with endometriosis often also demonstrate ovarian antibodies (Mathur et al., 1982). Ovarian intrusion by endometriotic implants may release an excess of ovarian cell component antigens, eliciting an autoimmune response. The data (Table 3) strongly suggest that ovarian autoimmunity is independent of endometrial autoimmunity in these patients. The failure of rabbit antisera against endometrial antigens to bind to ovarian antigens (Table 4) further proves this point.

All but 15 of 321 cancer patients had negligible titers of endometrial antibodies. Endometriosis could not be ruled out in the 15 patients with significant endometrial antibody titers.

Summary of Results:

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. There was no correlation between endometrial and ovarian antibodies in sera from 321 patients with ovarian cancer, 7 with pelvic inflammatory disease (PID), 4 with adenomyosis, 6 with uterine leiomyomata, 20 with endometriosis and 30 anovulatory women. The same was true of nuclear and endometrial antibodies in 20 other patients with endometriosis. Immunogenicity: Serum from rabbits immunized with the native and eluted (MW 29 to 68 kDa and > 68 kDa) endometrial proteins had IgG binding to the same glandular epithelial antigens as those bound by the patients' serum, and did not bind to WBC, ovarian or nuclear antigens. 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.

MW (kDa) of Percentage of patients with antigens in: antigens in:

Charleston and Boston Dallas

Endometrium (Number of samples tested 45 _61

34' 36 100 42 100 83

46/48 77 100 64/66 100 83 84^b 4 0 94/97 27 100 120 49 83 140° 4 0 150 11 100 170 0 100 Implants:

(Number of samples tested 45 _6±

34 100 100

42 100 100

46/48 100 100

64/66 100 100

84^b 77 100

94/97 67 100

120 77 100

140^b 40 100

150 10 100

170 0 100

^•Highlighted antigens found in > 25% of patients from all cities; "These antigens were found predominantly in the_ implants rather than the endometrium Table 2: The percentage of endometriosis patients with IgG antibodies in their serum and peritoneal fluid (reactivities in both fluids being similar, except for antigens with MW 140 kDa) binding to the antigens on patient endometrium and implants. None of the controls had reactivities to these antigens.

MW (kDa) of Percentage of patients with antigens: antibodies in:

Charleston Boston Chicago⁰ and Dallas

(Number of samples tested 45 IJ 20)

34

42

46/48

64/66¹

84^b

94/97

120

140^b

150

170

^•Serum and PF IgG to highlighted antigens found in > 25% of patients from at least 2 of 3 cities;

•These antigens were found predominantly in the implants rather than the endometrium. Antigen with MW of 140 kDa reacted with PF IgG of 38 to 56 (79%) of patients from Charleston, Dallas and Boston. ^cSerum samples only and tested against endometrium and implants from Charleston subjects. Table 3: Number of patients with ovarian and/or endometrial auto-antibodies (AB).

No. with No. with endometrial No. with Disorder Total ovarian AB AB both AB

Endometriosis 20 7 19 7

Others:

0

0

0

7

Table 4: Immunofluorescent IgG antibody (IFA) reactions (all antisera tested at 1:32 dilution), and hemagglutinating antibody (Hemagg) titers (reciprocal end point dilutions) of rabbit antisera to native {groups (Gp) 2 and 3} and eluted (groups 4 and 5) endometrial and implant proteins. MW (kDa) of endometrial and implant antigens (10 Western blot runs) binding with rabbit IgG in each group.

64,94 64,94

4. ++++ ++++ 64 256 34,48,58, 34,48,48, 64,84,94, 64,84,94, 120,140 120,140

64 64 34,48,58, 34,48,48, 64,84,94, 64,84,94, 120,140 120,140

EXAMPLE 2

Fast Protein liquid chromatography.

FPLC was conducted on endometrium and endometrial implants was performed. Fast protein liquid chromatography (FPLC) equipment along with fraction collector is from the Pharmacia and gel running conditions

10 have been standardized. 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

15 together and concentrated by using Centricon concentrators. The protein yield was approximately 0.5 to 1.0 mg/ml. Gel filtration technique was used. The running conditions are specified in the Tables 8 and 9. Phosphate buffered saline was used as the eluent.

5 The FPLC data are presented in TABLES 5 to 11 ) . In addition to the data in the tables data on 12 implant runs and 8 endometrial extract runs produced similar data.

FPLC separation of endometrial and implant proteins 10 from patients with endometriosis results in four significant fractions. Fraction numbers 2 and 3 contain the 34, 48, 64-66 and 72 kDa bands (See the FPLC and the Western blot analysis).

15 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.

20

Table 5: FPLC Molecular Weight Standards

TABLE 6 : FPLC Data on Endometrial Antigens

Implant antigens Endometrial antigens Peak No . Retention MW Peak No . Retention MW Time (RT ) (kDa ) Time (RT ) (kDa)

1 2 3 4 5

TABLE 7. Endometrial Antigens from Patients with Endometriosis (pooled from patients 1-4):

TABLE 8. Endometrial Antigens from Patients with Endometriosis (pooled from the same four patients in Table 7): 200 μl sample; 0.1 v range, 0.3 Flow rate, 0.2 AUFS

TABLE 9. Endometrial Antigens from Patients with Endometriosis (pooled from the same four patients in Tables 7 and 8 (summary of 8 runs): 200 μl sample; 0.1 v range, 0.3 flow rate, 0.2 AUFS (Duplicate Run)

TABLE 10. Implant Antigens from a fifth patient (Representative data of 4 runs):

TABLE 11. Implant Antigens from patients 6-8 (Representative data of 8 runs); also from patients 9 , 3 and 8 (4 runs):

Electrophoresis and Western Blot Analyses.

When the FPLC fractions were subjected to polyacrylamide gel electrophoresis followed by Western blot analysis, two proteins were apparent in the 64-66 kDa region.

PAGE Coomasie blue-stained gels of FPLC fractions from endometrium (MW marker. Void fraction. Factions 1- 11), whole endometrial extracts, normal endometrium and pooled endometrial extracts from patients with endometriosis were performed. The unique antigen with MW 64 kDa elutes in fractions 3 and 4, whereas the albumin band of about 66 kDa extends throughout the gel.

Western blot analysis of serum from a fertile control without endometriosis against endometrial antigens from patients with endometriosis, endometrial antigens from controls and implant antigens was performed. The MW of reactive bands are 15, 25/27, 30 and 52/54 kDa. Of these, 25/27 and 52/54 kDa are light and heavy chains of endogenous immunoglobulins that appear in the blank runs without serum and are ignored. There was no endogenous peroxidase activity in the blanks.

Western blot analysis of P.F. from a patient with endometriosis against endometrial antigens from the patients, endometrial antigens from controls, implant antigens from patients and eluted implant proteins with MW > 68 kDa and 29 to 68 kDa was performed. Endometrial and implant antigens are the same as used in the control serum Western Blot above. 27 and 52 kDa bands are the light and heavy chain respectively of endogenous IgG. Patients' endometrium and implants show the antigens with MW of 34, 42, 42, «64, 94/97, 120 and 140 kDa.

Data obtained from the Western blot studies is presented in Table 12. TABLE 12. Western Blot Analysis Data of FPLC Fractions from Endometrium (endo) or Implants from Patients with Endometriosis Tested Against Patients' (patient numbers given) Serum or P.F. vs Negative Controls or Blank Runs (specified as such) .

Glycan Detection.

Using a glycan detection kit, it was ascertained that 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. In contrast the protein band at 64 kDa did not include glycoproteins. As shown above, the 64 and 66 kDa bands are present in both endometrium and implants of patients with endometriosis.

Passive Hemagglutination. To show that patients with endometriosis do not have antibodies to albumin, passive hemagglutination was performed to document that patients with endometriosis do not have antibodies to human albumin in their serum or peritoneal fluid.

The procedure was as follows: Prepare gelatinized saline: 0.250 g gelatin to 250 ml of saline. Heat with stirring to boil. Cool the solution before using. Prepare the solution daily. Wash red blood cells (RBCs): Place 2 drops of 0, Rh positive RBCs in a tube and suspend in 5 ml of saline. Spin in Serofuge for 30 sec. Aspirate supernatant. Repeat two more times. Remove the frozen samples and thaw. To separate tubes of packed washed RBCs, add 6 drops each of endometrial extract from the patient and endometrial extract from the control (RBC, antigen ratio:1:3). Mix well, cover with parafilm and incubate in 37°C water bath for 2 hrs. Prepare 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. Place one drop of the respective samples in the first well of each row. Dilute samples with automatic diluter or hand-held mini diluter. Add 5 ml gelatinized saline to appropriate number of 5 ml tubes (1 tube per microtiter plate). From each tube of washed RBCs, add 2 or 3 drops to a tube of gelatinized saline and mix well. The color of the RBC suspension should be very light peach. Add a drop of this diluted (0.005%) suspension to all the wells of the microtiter plates with samples. Incubate at room 5 temperature for 2 hrs. Then centrifuge for 5 mins at 1000 rpm. Place the plates on the plate reader at 45° angle. Wait for 5 to 10 min and record the results. Dots are positive reactions, while streaks are the negative reactions. Read the plates when the cell control has 10 streaks. The end point is where the button formation stops and streaking begins. Antibody titers of > 32 are positive.

This experiment ruled out the possibility that the 15 unique protein identified along with albumin in the 64-66 kDa band by PAGE and Western blot analysis is an altered form of albumin (Table 13).

TABLE 13. Hemagglutinating Antibodies to Human Albumin in Patients' Serum.

Sample Titer

Pooled Serum from patients with:

Endometriosis 2

Fibroids 2

Control 2

Enzyme Linked Immunosorbent Assay.

To show that patients with endometriosis do not have antibodies to albumin, an ELISA was performed to document that patients with endometriosis do not have antibodies to 5 human albumin in their serum or peritoneal fluid.

Briefly, coat the ELISA plate with purified Ag in coating buffer (1.05 g Na₂C0₃, 3.36 g NaHC0₃, 0.1 M, pH 9.6 in 500 ml distilled water) 50-100 μl/well at 50-100 μl/ml/well. Place the plates overnight in the refrigerator; Wash 5X in Plate washer with 15 - 30 sec delay and 350μl volume. Proceed with 100 to 200 μl of Blocking solution (1% BSA in 1 x PBS) in each well; incubate at 37°C for 1 hr; Wash 5X in Plate washer with 15 - 30 sec delay and 350 μl volume of wash buffer (0.32 g. NaH₂P0₄, 1.10 g. Na₂HP0₄, 8.58 g. NaCl, 2 ml TWEEN-20 (0.2%), Distilled water 1 L, use in auto plate washer); Add patient serum at desired dilution, except in the blanks; incubate at 370°C for 1- hr; Wash as above; Add goat anti human IgG-peroxidase, 1:1000, incubate 37°C for 1 hr; Wash as above; Add ABTS substrate (ABTS Substrate Buffer from 10X Stock - 1 ml in 9 ml dH₂0, 2 ABTS 5 mg Tablets (Calbiochem)/10 ml (1 mg/ml), 6.7 μl 30% H^0₂ (Prepare fresh just before development) and develop for desired time recommended 2 hrs; Stop solution by addition of 50 μl 10% H₂S0₄ or 50 μl 10% SDS; Read the absorbance in ELISA reader after blanking out the assigned blank wells.

The data rule out the possibility that the novel protein identified in the 64-66 kDa band along with albumin by PAGE and Western blot analysis is an altered form of albumin (Table 14).

TABLE 1 4. Antibodies to Human Albumin and IgG in Serum and Peritoneal Fluid from Patients with Endometriosis and Fibroids and Serum from Normal Controls by ELISA (ELISA done at 1 : 32 dilution of the samples )

Positive ( + ) or negative (-) for antibodies to: Sample Albumin IgG

Patients with endometriosis:

17 Serum - +

18 Serum

18 P.F.

19 Serum

19 P.F. 16 Serum 16 P.F.

20 Serum

20 P.F.

21 P.F.

22 Serum

22 P.F.

Patients with fibroids:

23 Serum - +

24 Serum Control Serum Control Serum

Absorption of Serum With Anti-Albumin and Anti—IgG.

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.

5 grams of CNBr-activated Sepharose 4 B (Pharmacia) (25 ml) was soaked in 100 ml of 1 mM HC1 for 20 mins. It was then washed several aliquots and then dried. Goat 10 Anti-human Albumin (Cappel: 40 mg in 20 of coupling buffer) was dissolved in 20 ml of coupling buffer (0.1 M NaHC0₃ (pH 8.3) containing 0.5 M NaCl) and measure the protein concentration by A280(1 :11=0.315) . The solution was then mixed with the gel in a stoppered vessel for 2 hr at room temperature rotating end to end. Centrifuge the gel and save the supernatant to measure the protein concentration by A280 (=0.045) and calculate the coupling efficiency.

0.045x20 X 100 = 99% 0.315x11x20

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.

In Coomasie blue-stained PAGE gels of MW standards; Unabsorbed endometrial extracts; Endometrial extracts absorbed with protein A (to remove endogenous IgG and anti-albumin antibodies) show that the endometrial protein of the 64-66 kDa region is still intact even after the absorption.

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.

Western Blot with Absorbed and unabsorbed Serum.

In Western blot analysis, the unabsorbed and absorbed endometrial antigens are reacted against serum and peritoneal fluid (P.F.) of patients with endometriosis.In Western blot analysis of blank run, control man's serum and control woman's serum against unabsorbed endometrial antigens and endometrial antigens absorbed with protein A and anti-albumin, as expected, there is minimal reactivity in all the three blots.

In Western blot analysis of serum and peritoneal fluid (P.F.) of patients 17 and 25 with endometriosis against unabsorbed endometrial antigens and endometrial antigens absorbed with protein A and anti-albumin both the serum and P.F. of patients with endometriosis react against the « 64 kDa antigen irrespective of the absorption.

In Western blot analysis of serum and peritoneal fluid (P.F.) of a patient (26) with endometriosis against unabsorbed endometrial antigens and endometrial antigens absorbed with protein A and anti-albumin, both the serum and P.F. of the patient with endometriosis react against an antigen at the « 64 kDa position irrespective of the absorption. The intensity of 64-66 kDa band is unaffected by the absorption and is unique to the patients with endometriosis. In a blank run (no serum), and against control male serum and female serum, there was no reactivity against the 64-66 kDa antigen bands. In fact, there was no reactivity at all except for a single band in the 25-29 kDa area. These data clearly demonstrate that the serum and P.F. from patients with endometriosis bound with an antigen other than albumin in this area.

Hemagglutination and ELISA With Preabsorbed Serum.

Hemagglutination and ELISA studies are repeated as described above using the absorbed antigens. The antibody titers remained unaltered in pooled serum and P.F. samples from patients with endometriosis.

Generation of an Endometrial Implant cDNA Library. 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.

EXAMPLE 3

Two Dimensional Gel Electrophoresis

2-Dimensional Electrophoresis of Preabsorbed Proteins.

Pooled endometriosis implant extracts were passed first through a column containing cyanogen bromide- activated sepharose B coupled with anti-human albumin antiserum (to remove human albumin). The eluate was again passed through cyanogen bromide-activated sepharose B coupled with protein A (to remove IgG). The final eluate was concentrated using Centri-Prep concentrator 10 according to the manufacturers instructions (Amicon).

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.

In the second dimension. Excel Gel SDS gradient 8-18 was used, according to the manufacturer's protocol (Gόrg et al. 1988, Gorg 1991, Gorge 1993, Hanash et al. 1987, Dameral et al. 1988; Gorge et al. 1987, Gorge et al. 1985, Heukeshoven et al., Merril et al. 1981, Dunn, M.J. Vol. 1).

Molecular weight standards were run in one gel and the absorbed implant material in the other at the same run so as to compare the unknowns with the known standards (Tables 15 and 16; and Fig. 2).

Two dimensional electrophoresis shows that there are at least five proteins in addition to albumin in the 64-66 kDa range.

TABLE 15. The pi Values and Molecular Weights (MW) of the standards .

No. Standard pi Value MW (kDa)

1 Hen egg white conalbumin 2 Bovine serum albumin 3 Bovine muscle actin 4 Rabbit muscle GAPDH 5 Bovine carbonic anhydrase 6 Soybean trypsin inhibitor 7 Equine myoglobin

TABLE 16. The pi Values and MW (kDa) of the absorbed implant proteins .

Implant protein No. pi Value MW (kDa)

1 6.6 120

2 4.5 120

3 3.5 97

4 4.5 72

5 6.5 64

6 6.0 64

7 4.0 64

8 8.0 64

9 3.5 64

10 6.6 34

11 4.5 34

12 6.6 22

13 6.0 18

14 6.6 18

15 4.5 16

In the absorbed implant extracts there are no spots corresponding to albumin at the 64 kDa MW. There are proteins at 64 kDa with pi values of 3.5, 4.0-4.2, 6.0, 6.5 and 8.0. 2-Dimensional Electrophoresis of Nonabsorbed Proteins.

Pooled endometriosis implant extracts which were not absorbed with anti-albumin or protein A were subjected to two dimensional gel electrophoresis using the equipment from Pharmacia. Isoelectric focusing was performed in the first dimension, separating the proteins according to their pi values. The pH gradient that was used in the immobiline dry strips was pH 3 to 10. In the second dimension. Excel Gel SDS, gradient 0-18 was used as described above.

Molecular weight standards were run in one gel and the implant in the other at the same run so as to compare the unknowns with the known standards (Tables 15 and 17; and Fig. 2, which schematically represents the position of unabsorbed endometrial and implant proteins from patients with endometriosis in relation to the standards in two dimensional gel electrophoresis run.

TABLE 17. The pi Values and MW (kDa) of the endometrial and endometriotic implant proteins from patients with endometriosis.

Implant protein No. pi Value MW (kDa)

1 4.75 150

2 5.0 150

3 5.0 120

4 8.3 120

5 3.5 97

6 5.0 72

7 6.3 72 3.5 64 5.5 66

10 6.5 64

11 6.0 64

12 4.0 64

13 8.0 64

14 4.0 52 5 4.5 48 6 4.5 42 7 4.6 40 8 4.5 38 9 4.5 34 0 6.6 34 1 4.5 34 2 4.0 27 3 4.5 26 4 4.0 26 5 5.0 26 6 4.5 16 7 4.6 16 8 5.0 16 9 4.5 14 0 4.0 14 1 5.0 14 In the unabsorbed implant extract there are 6 proteins in the 64/66 kDa MW range, with p.I.s of approximately 3.5, 4.0, 5.5 (albumin), 6.0, 6.5 and 8.0. Hence, there are 5 proteins besides albumin in this region.

Amino Acid Sequencing.

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 NH₄HC0₃, 0.05% trifluoroacetic acid (TFA), or 50% CH₃CN/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.

The sequencing data revealed that one of the proteins migrating in the 64-66 kDa electrophoretic band is human serum albumin (66 kDa) and another has a unique sequence (SEQ ID N0:1 ) .

Western Blot of 2—Dimensional Gel.

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. Thus, these 5 purified proteins are markers for endometriosis.

Furthermore, because 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.

Throughout this application various publications are referenced within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully 5 describe the state of the art to which this invention pertains. Full citations for these publications are as follows:

REFERENCES

Abramovitz, A.S., Randolph, V., Mehra, A. and Christakos, S. Isolation of proteins from preparative gels. Prepar. Biochem. 14:205 -221, 1984.

Badawy, S.Z.A., Cuenca, V., Stitzel, A.B., Jacobs, R.D.B. and Tomar, R.H. Autoimmune phenomena in infertile patients with endometriosis. Obst . Gynecol . 63:271 - 276, 1984.

Badawy, S.Z.A., Cuenca, V., Freliech, H. and Stefanu, C.

Endometrial antibodies in serum and peritoneal fluid of infertile patients with ajid without endometriosis. Fertil . Steril . 53:930 - 932, 1990.

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 .

Biochem. 12:195-201, 1981. Chihal, H.J., Mathur, S., Holtz, G.L. and Williamson, H.O.

Endometrial antibodies in the detection and evaluation of therapy of endometriosis. Fertil . Steril . 46:408-411,

1986. Confino, E, Harlow, L. and Gleicher, N. Peritoneal fluid and serum auto-antibody levels in patients with endometriosis.

Fertil . Steril . 53:242-251, 1990. De Lange, G.G. Polymorphisms of human immunoglobulins: Gm, Am,

Em and Km allotypes. Exp. Clin . Immunogenet . 6:7-17,

1989. Fedele, L., Marchini, M., Bianchi, S., Dorta, M., Arcaini, L. and Fontana, P.E. Structural and ultra structural defects in preovulatory endometrium of normo-ovulating infertile women with minimal or mild endometriosis. Fertil . Steril .

53:989-993, 1990. Ferrone, S. and Solheim, B.G. HLA Typing: Methodology and

Clinical Aspects. CRC Press Inc, Boca Raton, pp. 189,

1982. Garza, D., Mathur, S., Dowd, M. , Smith, L.F. and Williamson,

H.O. Antigenic differences between endometrium from patients with and without endometriosis. J. Reprod. Med.

36:177-182, 1991. Kamat, B.R. and Isaacson P.G. The immunocytochemical distribution of leukocyte sub populations in human endometrium. Am . J . Pathol . 27:66-73, 1987. Kennedy, S.H., Sargent, I.L., Starkey, P.M., Hicks, B.R. and

Barlow, D.H. Localization of anti-endometrial antibody binding in women with endometriosis using a double-labeling immunohistochemical method. Br. J.

Obstet . Gynaecol . 97:671-674, 1990. Kreiner, D., Fromowitz, F.B., Richardson, D.A. and Koenigsborg,

D. Endometrial immunofluorescence associated with endometriosis and pelvic inflammatory disease. Fertil .

Steril . 46:243-246, 1986. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature

227:680-685, 1970. Mathur, S., Chihal, H.J., Homm, R.J. , Garza, D.E., Rust, P.F. and Williamson, H.O. Endometrial antigens involved in the autoimmunity of endometriosis. Fertil . Steril .

50:860-866, 1988. Mathur, S., Garza, D.E., Smith, L.F. and Williamson, H.O.

Endometrial auto antigens eliciting immunoglobulin (Ig) G,

IgA and IgM responses in endometriosis. Fertil . Steril .

54:56-63, 1990. Mathur, S., Peress, M.R. , Williamson, H.O., Youmans, CD.,

Maney, S.A. and Garvin, A.J. Autoimmunity to endometrium and ovary in endometriosis. Clin . Exp. Immunol .

50:259-266, 1982. Meek, S.C, Hodge, D.D. and Musich, J.R. Autoimmunity in infertile patients with endometriosis. Am. J. Obstet .

Gynec. 158:365-373, 1988. Saiffudin, A., Buckley, CH. and Fox, H. Immunoglobulin content of the endometrium in women with endometriosis. Int . J.

Gynecol . Pa thol . 2:255-261, 1983. Schleisinger, David H. High sensitivity microsequencing of the peptides and proteins. Macromolecular Sequencing

Synthesis: Selected Methods and Applications.

Schleisinger (Ed.) Alan R. Liss pp. 35-44, 1988. Wild, R.A., Medders, D. and Zhang, R. F(ab')₂ segment is the active component of immunoglobulin G auto-antibody generation with endometriosis. Fertil . Steril . 56, 900 -

903, 1991. Wild, R.A. and Shivers, CA. Endometrial antibodies in patients with endometriosis. Am. J. Reprod. Immunol . Microbiol .

8:84-90, 1985. Gδrg A., Postel W. and Gunther S. Electrophoresis 9:531-546,

1988 Gδrg A. Nature 349:545-546, 1991 Gδrg A. Biochem. Soc. Trans . 21:130-132, 1993 Hanash S.M., Strahler J.R., Somerlot L., Postel W. and Gorg A.

Electrophoresis 8:229-234, 1987 Dunn M.J. Advances in Electrophoresis VCH Publishers, Germany,

Ed. Chrambach A., Dunn M.J., Radola B.J., Vol. 1 Dameral C, Zivy M., Granier F. and de Vienne D. In Advances in

Electrophoresis VCH Publishers, Germany 2:265-340, 1988 Gόrg A., Postel W., Wester J., Gunther S., Strahler J. , Hanash

S. and Somerlot L. Electrophoresis 8:122-124, 1987 Gόrg A., Postel W., Gunther S. and Weser J. Electrophoresis

6:599-604, 1985 Heukeshoven J. and Dernick Electrophoresis Forum 186:22-27 Merril C.R., Goldmann D., Sedman S.A. and Ebert M.H. Science

211:1437-1438, 1981

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: The Government of the United States of America, as represented by the Secretary

(B) STREET: 6011 Executive Blvd, Suite 325

(C) CITY: Rockville

(D) STATE: Maryland

(E) COUNTRY: United States of America

(F) POSTAL CODE (ZIP): 20852

(G) TELEPHONE: 301/496-7056 (H) TELEFAX: 301/402-0220 (I) TELEX: NONE

(ii) TITLE OF INVENTION: ENDOMETRIAL PROTEINS, ANTIGENIC COMPOSITIONS AND METHODS FOR DETECTING ENDOMETRIOSIS

(iii) NUMBER OF SEQUENCES: 1

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(v) CURRENT APPLICATION DATA:

APPLICATION NUMBER: NOT YET ASSIGNED

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/069,171 (B) FILING DATE: 28-95-1993

(2) INFORMATION FOR SEQ ID N0:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME/KEY: Protein

(B) LOCATION: 3

(D) OTHER INFORMATION: /label= Xaa

/note= "Xaa can be Asn or Met" (ix) FEATURE:

(A) NAME/KEY: Protein

(B) LOCATION: 4

(D) OTHER INFORMATION: /label= Xaa

/note= "Xaa can be Ala, Tyr, He or Pro"

(ix) FEATURE:

(A) NAME/KEY: Protein

(B) LOCATION: 5

(D) OTHER INFORMATION: /label= Xaa

/note= "Xaa can be Ser or Leu"

(ix) FEATURE:

(A) NAME/KEY: Protei n

(B) LOCATION: 6

(D) OTHER INFORMATION: /label= Xaa

/note= "Xaa can be Val or Leu"

(ix) FEATURE:

(A) NAME/KEY: Protein

(B) LOCATION: 9

(D) OTHER INFORMATION: /label= Xaa

/note= "Xaa can be Phe or Met"

(ix) FEATURE:

(A) NAME/KEY: Protein

(B) LOCATION: 10

(D) OTHER INFORMATION: /label= Xaa

/note= "Xaa can be any amino acid"

(ix) FEATURE:

(A) NAME/KEY: Protein

(B) LOCATION: 16

(D) OTHER INFORMATION: /label■ Xaa

/note= "Xaa can be any amino acid"

(ix) FEATURE:

(A) NAME/KEY: Protein

(B) LOCATION: 17

(D) OTHER INFORMATION: /label= Xaa

/note= "Xaa can be Ser, Glu or Lys"

(ix) FEATURE:

(A) NAME/KEY: Protein

(B) LOCATION: 18

(D) OTHER INFORMATION: /label= Xaa

/note= "Xaa can be Asp, Glu or Lys"

(ix) FEATURE:

(A) NAME/KEY: Protein

(B) LOCATION: 1..18

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1: Glu Pro Xaa Xaa Xaa Xaa Val Thr Xaa Xaa Ala Lys He Ser Thr Xaa 1 5 10 15

Xaa Xaa

Claims

What is claimed is:

1. 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.

2. A purified antigen composition having a molecular weight of about 64 to 66 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis, wherein the presence of antigen composition is a marker for endometriosis in a subject.

3. The antigen of claim 2, bound to a solid support.

4. A method of diagnosing endometriosis in a subject comprising the steps of: a. contacting an antibody-containing sample from the subject with an endometriosis specific antigen composition having a molecular weight of about 64 to 66 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis; and b. detecting the reaction of the antigen with an antibody from the sample, the reaction indicating endometriosis in the subject.

5. A method of diagnosing endometriosis in a subject comprising the steps of: a. contacting an antibody-containing sample from the subject with an endometriosis specific antigen having a molecular weight or about 46 to 48 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis or an endometriosis specific antigen fragment; and b. detecting the reaction of the antigen with an antibody from the sample, the reaction indicating endometriosis in the subject.

6. A monoclonal antibody specifically reactive with the antigen of claim 2.

7. A monoclonal antibody specifically reactive with a purified antigen 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.

8. A method of diagnosing endometriosis in a subject comprising the steps of: a. contacting a sample from the subject with the monoclonal antibody specifically reactive with an endometriosis specific antigen; and b. detecting the reaction of the antibody with the antigen in the sample, the reaction indicating endometriosis in the subject.

9. The method of claim 8, wherein the monoclonal antibody is the antibody of claim 6.

10. The method of claim 8, wherein the monoclonal antibody is the antibody of claim 7.

11. The method of claim 8, wherein the sample is peritoneal fluid.

12. The method of claim 8, wherein the sample is serum.

13. A purified polyclonal antibody specifically reactive with the antigen of claim 2.

14. An antigen detecting kit, comprising a detectable amount of the monoclonal antibody of claim 6 bound to a solid support.

15. An antibody detecting kit, comprising a detectable amount of the antigen of claim 2 bound to a solid support,

16. 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.

17. 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.

18. 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.

19. 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.

20. A purified antigenic endometrial protein, separated from albumin and having a molecular weight of 94 to 97 kilodaltons as determined by sodium dodecyl sulphate gel electrophoresis under reducing conditions and an isoelectric focusing point of about 3.5.

21. An isolated nucleic acid encoding the protein of claim 1.

22. An isolated nucleic acid encoding the protein of claim 16.

23. An isolated nucleic acid encoding the protein of claim 17.

24. An isolated nucleic acid encoding the protein of claim 18.

25. An isolated nucleic acid encoding the protein of claim 19.

26. An isolated nucleic acid encoding the protein of claim 21.

27. An endometriosis-specific antigenic polypeptide fragment encoded by the nucleic acid of claim 21.

28. An endometriosis-specific antigenic polypeptide fragment encoded by the nucleic acid of claim 22.

29. An endometriosis-specific antigenic polypeptide fragment encoded by the nucleic acid of claim 23.

30. An endometriosis-specific antigenic polypeptide fragment encoded by the nucleic acid of claim 24.

31. An endometriosis-specific antigenic polypeptide fragment encoded by the nucleic acid of claim 25.

32. An endometriosis-specific antigenic polypeptide fragment encoded by the nucleic acid of claim 26.

33. A purified antigenic endometrial protein having the amino-terminal amino acid sequence defined in the Sequence Listing as SEQ ID NO:1.

34. An isolated nucleic acid encoding the amino acid sequence of SEQ ID NO:1.

35. An isolated nucleic acid that selectively hybridizes with the nucleic acid of claim 21 , under conditions of high stringency.

36. 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 to 97 kilodaltons as determined by sodium dodecyl sulphate gel electrophoresis under reducing conditions and an isoelectric focusing point of about 3.5.

37. 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 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.

38. 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.

39. 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, 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 5.0.

40. A method of diagnosing endometriosis in a subject comprising the steps of: a. contacting an antibody-containing sample from the subject with the purified antigenic endometrial protein of claim 1 ; and b. detecting the reaction of the antigenic protein with an antibody from the sample, the reaction indicating endometriosis in the subject.

41. A method of diagnosing endometriosis in a subject comprising the steps of: a. contacting an antibody-containing sample from the subject with the purified antigenic endometrial protein of claim 20; and b. detecting the reaction of the antigenic protein with an antibody from the sample, the reaction indicating endometriosis in the subject.

42. A purified antibody that specifically binds the protein of Claim 1.

43. A purified antibody that specifically binds the protein of Claim 20.