ME-5, ME-2, and EPP2: human protein antigens reactive with autoantibodies present in the serum of women suffering from endometriosis

Abstract

Three human endometrial antigens, ME-5, ME-2 and EPP2, and combination thereof have been discovered that are the targets of antibodies present in the serum of women suffering from endometriosis. These protein products and the human antibodies reactive with them are used as tools for diagnostic assays that monitor patients with the disease of endometriosis.

Description

BACKGROUND OF THE INVENTION

[0001] Endometriosis is a female reproductive disorder characterized by the presence of endometrial tissue outside of the normal uterine location. Most frequently the endometriosis tissue is present in the peritoneal cavity, attaching to various tissues and organs in this location. Endometriosis is a benign disease affecting approximately 5 million women in the United States annually with a prevalence of 10 to 15 percent in women of childbearing age. The incidence increases to 60 to 80 percent of women who are infertile or present with pelvic pain (D. Gosselin et al.

[1999] Curr. Opin. Onco. Endo. & Metabol. Invest. Drugs 1:31). The conditions that predispose an individual to endometriosis are still unknown. Several authoritative reports suggest that retrograde menstruation may be a key-contributing factor, but this process is thought to be common in most women. This theory has also been questioned recently (D. B. Redwine

[2002] Fert. Steril. 78:686) due ...

Examples

example 1

Identification and Cloning of the ME-5, ME-2, AND EPP2 cDNAS

[0099] The endometriosis tissue cDNA library was generated using poly A+ RNA isolated from a deep embedded endometriosis tissue specimen donated by Professor Philip Koninckx at the Catholic University of Leuven. Total RNA was isolated from the tissue using Trizol reagent (Biorad Laboratories; Hercules, Calif.), and poly A+ RNA was prepared by hybridization to oligo poly T coupled magnetic particles using a commercial kit (PolyATract; Promega; Madison, Wis.). Library construction was carried out using the Lambda ZAP® II vector system following instructions obtained from the supplier (Stratagene; San Diego, Calif.). The initial ME-5 and ME-2 cDNA clones were identified by immunoscreening using, as primary antibody, a single endometriosis patient serum specimen obtained from a woman diagnosed with mild disease. This serum was adsorbed of nonspecific anti-E. coli / lambda phage antibodies by diluting the sera 1:50 in a commercia...

example 2

Characterization of ME-5, ME-2, and EPP2 cDNA and Protein

[0100] Sequence analysis of both strands of each of the original isolated ME-5, ME-2, and EPP2 clones was performed upon an ABI Biosystems 373 DNA Sequencer (PE Applied Biosystems; Foster City, Calif.). The nucleic acid sequences so generated were analyzed using Bionet software to identify nucleic acid and protein characteristics and for homology comparisons with nucleic acid and protein sequences present in the database.

[0101] The ME-5 cDNA sequence is presented in FIG. 1A (SEQ ID NO:1) and it is 1,279 base pairs in size excluding the poly dA track. A 5′ noncoding sequence of 112 base pairs was identified just upstream of the suspected ATG start codon. There is a 3′ non coding sequence of 254 base pairs down stream of the TGA stop codon and this is followed by a stretch of dA residues that would correspond to the poly A tail at the 3′ end of the mRNA. Both the start and stop codon are highlighted in bold type in FIGS. 1A an...

example 3

Northern Blotting with Radiolabeled ME-5, ME-2, and EPP2 Probes: mRNA Character and Expression Pattern

[0110] Gene expression profile of ME-5 from normal human tissues was done by performing Northern blot analysis with a commercial Multiple Tissue Northern Blot (BD Biosciences; San Diego, Calif.), the results of which are presented in FIG. 4. The commercial Northern blot contained RNA from the following tissues: spleen, thymus, prostate, testis, uterus, small intestine, colon (no mucosa), and peripheral blood leukocyte. The entire 912 base pair coding sequence was isolated by electrophoresis in a low melting agarose gel, and labeled with 32P by random priming. The 32P-labeled ME-5 probe was used for hybridization to the Northern blot using the procedure supplied by the manufacturer. After washing the blot was exposed to X-ray film. Upon development of the film a band at about 1.4 kb on the Northern blot corresponds to the ME-5 transcript of the expected size (FIG. 4). The transcript...

BACKGROUND OF THE INVENTION

[0001] Endometriosis is a female reproductive disorder characterized by the presence of endometrial tissue outside of the normal uterine location. Most frequently the endometriosis tissue is present in the peritoneal cavity, attaching to various tissues and organs in this location. Endometriosis is a benign disease affecting approximately 5 million women in the United States annually with a prevalence of 10 to 15 percent in women of childbearing age. The incidence increases to 60 to 80 percent of women who are infertile or present with pelvic pain (D. Gosselin et al.

[1999] Curr. Opin. Onco. Endo. & Metabol. Invest. Drugs 1:31). The conditions that predispose an individual to endometriosis are still unknown. Several authoritative reports suggest that retrograde menstruation may be a key-contributing factor, but this process is thought to be common in most women. This theory has also been questioned recently (D. B. Redwine

[2002] Fert. Steril. 78:686) due ...

Examples

example 1

Identification and Cloning of the ME-5, ME-2, AND EPP2 cDNAS

[0099] The endometriosis tissue cDNA library was generated using poly A+ RNA isolated from a deep embedded endometriosis tissue specimen donated by Professor Philip Koninckx at the Catholic University of Leuven. Total RNA was isolated from the tissue using Trizol reagent (Biorad Laboratories; Hercules, Calif.), and poly A+ RNA was prepared by hybridization to oligo poly T coupled magnetic particles using a commercial kit (PolyATract; Promega; Madison, Wis.). Library construction was carried out using the Lambda ZAP® II vector system following instructions obtained from the supplier (Stratagene; San Diego, Calif.). The initial ME-5 and ME-2 cDNA clones were identified by immunoscreening using, as primary antibody, a single endometriosis patient serum specimen obtained from a woman diagnosed with mild disease. This serum was adsorbed of nonspecific anti-E. coli / lambda phage antibodies by diluting the sera 1:50 in a commercia...

example 2

Characterization of ME-5, ME-2, and EPP2 cDNA and Protein

[0100] Sequence analysis of both strands of each of the original isolated ME-5, ME-2, and EPP2 clones was performed upon an ABI Biosystems 373 DNA Sequencer (PE Applied Biosystems; Foster City, Calif.). The nucleic acid sequences so generated were analyzed using Bionet software to identify nucleic acid and protein characteristics and for homology comparisons with nucleic acid and protein sequences present in the database.

[0101] The ME-5 cDNA sequence is presented in FIG. 1A (SEQ ID NO:1) and it is 1,279 base pairs in size excluding the poly dA track. A 5′ noncoding sequence of 112 base pairs was identified just upstream of the suspected ATG start codon. There is a 3′ non coding sequence of 254 base pairs down stream of the TGA stop codon and this is followed by a stretch of dA residues that would correspond to the poly A tail at the 3′ end of the mRNA. Both the start and stop codon are highlighted in bold type in FIGS. 1A an...

example 3

Northern Blotting with Radiolabeled ME-5, ME-2, and EPP2 Probes: mRNA Character and Expression Pattern

[0110] Gene expression profile of ME-5 from normal human tissues was done by performing Northern blot analysis with a commercial Multiple Tissue Northern Blot (BD Biosciences; San Diego, Calif.), the results of which are presented in FIG. 4. The commercial Northern blot contained RNA from the following tissues: spleen, thymus, prostate, testis, uterus, small intestine, colon (no mucosa), and peripheral blood leukocyte. The entire 912 base pair coding sequence was isolated by electrophoresis in a low melting agarose gel, and labeled with 32P by random priming. The 32P-labeled ME-5 probe was used for hybridization to the Northern blot using the procedure supplied by the manufacturer. After washing the blot was exposed to X-ray film. Upon development of the film a band at about 1.4 kb on the Northern blot corresponds to the ME-5 transcript of the expected size (FIG. 4). The transcript...

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