RESUMEN
BACKGROUND: Sperm acrosomal SLLP1 binding (SAS1B) protein is found in oocytes, which is necessary for sperm-oocyte interaction, and also in uterine and pancreatic cancers. Anti-SAS1B antibody-drug conjugates (ADCs) arrested growth in these cancers. However, SAS1B expression in cancers and normal tissues has not been characterized. We hypothesized that SAS1B is expressed on the surface of other common solid cancer cells, but not on normal tissue cells, and might be selectively targeted therapeutically. METHODS: SAS1B expression in human normal and cancer tissues was determined by immunohistochemistry, and complementary DNA (cDNA) libraries were employed to PCR amplify human SAS1B and its transcripts. Monoclonal antibodies (mAbs) to human SAS1B were generated using mouse hybridomas. SAS1B deletion constructs were developed to map SAS1B's epitope, enabling the creation of a blocking peptide. Indirect immunofluorescence (IIF) of human transfected normal and cancer cells was performed to assess SAS1B expression. SAS1B intracellular versus surface expression in normal and tumor tissues was evaluated by flow cytometry after staining with anti-SAS1B mAb, with specificity confirmed with the blocking peptide. Human cancer lines were treated with increasing mAb and ADC concentrations. ATP was quantitated as a measure of cell viability. RESULTS: SAS1B expression was identified in a subset of human cancers and the cytoplasm of pancreatic islet cells. Two new SAS1B splice variants were deduced. Monoclonal antibodies were generated to SAS1B splice variant A. The epitope for mAbs SB2 and SB5 is between SAS1B amino acids 32-39. IIF demonstrated intracellular SAS1B expression in transfected kidney cells and on the cell surface of squamous cell lung carcinoma. Flow cytometry demonstrated intracellular SAS1B expression in all tumors and some normal cells. However, surface expression of SAS1B was identified only on cancer cells. SB2 ADC mediated dose-dependent cytotoxic killing of multiple human cancer lines. CONCLUSION: SAS1B is a novel cancer-oocyte antigen with cell surface expression restricted to cancer cells. In vitro, it is an effective target for antibody-mediated cancer cell lysis. These findings support further exploration of SAS1B as a potential therapeutic cancer target in multiple human cancers, either with ADC or as a chimeric antigen receptor-T (CAR-T) cell target.
Asunto(s)
Inmunoconjugados , Neoplasias , Masculino , Humanos , Ratones , Animales , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Semen , Oocitos/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Epítopos , Péptidos/metabolismoRESUMEN
There has been a paradign shift in the status of immunoassays. There used to be a time where immunoassays had a very narrow role in clinical medicine, but that is not the case in today's world. Immunoassays have taken a central role in helping us better understand and treat human diseases. The literature around anti-ovarian antibodies (AOA) immunoassay testings have been conflicting. Researchers challenged the specificity of the reported assays, but a systematic study was never elucidated on what/who the trouble maker was in rendering these tests so nonspecific. Attempts were made by our group in Mumbai, India, to throw light on the culprit behind the nonspecificity casative factor in the immunoassays and a method to overcome this was reported and published. This review highlights the stories back five and a half decades to date, to demonstrate where the status of AOA testing was, is and will be.
Asunto(s)
Autoanticuerpos , Ovario , Femenino , Humanos , Inmunoensayo/métodos , IndiaRESUMEN
Successful therapeutic options remain elusive for pancreatic cancer. The exquisite sensitivity and specificity of humoral and cellular immunity may provide therapeutic approaches if antigens specific for pancreatic cancer cells can be identified. Here we characterize SAS1B (ovastacin, ASTL, astacin-like), a cancer-oocyte antigen, as an attractive immunotoxin target expressed at the surface of human pancreatic cancer cells, with limited expression among normal tissues. Immunohistochemistry shows that most pancreatic cancers are SAS1Bpos (68%), while normal pancreatic ductal epithelium is SAS1Bneg. Pancreatic cancer cell lines developed from patient-derived xenograft models display SAS1B cell surface localization, in addition to cytoplasmic expression, suggesting utility for SAS1B in multiple immunotherapeutic approaches. When pancreatic cancer cells were treated with an anti-SAS1B antibody-drug conjugate, significant cell death was observed at 0.01-0.1 µg/mL, while SAS1Bneg human keratinocytes were resistant. Cytotoxicity was correlated with SAS1B cell surface expression; substantial killing was observed for tumors with low steady state SAS1B expression, suggesting a substantial proportion of SAS1Bpos tumors can be targeted in this manner. These results demonstrate SAS1B is a surface target in pancreatic cancer cells capable of binding monoclonal antibodies, internalization, and delivering cytotoxic drug payloads, supporting further development of SAS1B as a novel target for pancreatic cancer.
RESUMEN
The heat shock proteins (HSPs) are a group of evolutionarily conserved proteins with important physiological functions, whose synthesis is enhanced by elevated temperature or other stresses. HSPs show high sequence homology between different species, from bacteria to humans. Despite the significant degree of evolutionary conservation, HSPs are highly immunogenic. Of the several HSPs, HSP90 is an abundant, constitutively expressed chaperone constituting around 1-2% of total cellular protein under non-stress conditions. This protein from even the most distantly related eukaryotes has 50% amino acid identity, and all have more than 40% identity with the Escherichia coli protein. They are immunodominant antigens for many common microbes, and thus their epitopes are recognized by the immune system. As HSPs are overexpressed at sites of acute and chronic inflammation, individuals are likely to be sensitized during the course of a microbial infection encountered during life. This chapter considers the evidence of a role for HSP90 in autoimmune ovarian failure, where autoantibodies to it have been observed in patients, and has been correlated to infertility.
Asunto(s)
Autoanticuerpos/inmunología , Proteínas HSP90 de Choque Térmico/inmunología , Ovario/inmunología , Ovario/patología , Animales , Formación de Anticuerpos/inmunología , Femenino , Humanos , Epítopos Inmunodominantes/inmunologíaRESUMEN
The metalloproteinase SAS1B [ovastacin, ASTL, astacin-like] was immunolocalized on the oolemma of ovulated human oocytes and in normal ovaries within the pool of growing oocytes where SAS1B protein was restricted to follicular stages spanning the primary-secondary follicle transition through ovulation. Gene-specific PCR and immunohistochemical studies revealed ASTL messages and SAS1B protein in both endometrioid [74%] and malignant mixed Mullerian tumors (MMMT) [87%] of the uterus. A MMMT-derived cell line, SNU539, expressed cell surface SAS1B that, after binding polyclonal antibodies, internalized into EEA1/LAMP1-positive early and late endosomes. Treatment of SNU539 cells with anti-SAS1B polyclonal antibodies caused growth arrest in the presence of active complement. A saporin-immunotoxin directed to SAS1B induced growth arrest and cell death. The oocyte restricted expression pattern of SAS1B among adult organs, cell-surface accessibility, internalization into the endocytic pathway, and tumor cell growth arrest induced by antibody-toxin conjugates suggest therapeutic approaches that would selectively target tumors while limiting adverse drug effects in healthy cells. The SAS1B metalloproteinase is proposed as a prototype cancer-oocyte tumor surface neoantigen for development of targeted immunotherapeutics with limited on-target/off tumor effects predicted to be restricted to the population of growing oocytes.
Asunto(s)
Anticuerpos/farmacología , Antígenos de Neoplasias , Inmunoconjugados/farmacología , Inmunoterapia/métodos , Metaloproteasas/antagonistas & inhibidores , Tumor Mulleriano Mixto/tratamiento farmacológico , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Neoplasias Uterinas/tratamiento farmacológico , Secuencia de Aminoácidos , Anticuerpos/metabolismo , Anticuerpos/toxicidad , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Endocitosis , Femenino , Humanos , Inmunoconjugados/metabolismo , Inmunoconjugados/toxicidad , Inmunoterapia/efectos adversos , Metaloproteasas/genética , Metaloproteasas/inmunología , Metaloproteasas/metabolismo , Tumor Mulleriano Mixto/enzimología , Tumor Mulleriano Mixto/genética , Tumor Mulleriano Mixto/inmunología , Tumor Mulleriano Mixto/patología , Datos de Secuencia Molecular , Terapia Molecular Dirigida , Oocitos/efectos de los fármacos , Oocitos/enzimología , Proteínas Inactivadoras de Ribosomas Tipo 1/metabolismo , Proteínas Inactivadoras de Ribosomas Tipo 1/toxicidad , Saporinas , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Neoplasias Uterinas/enzimología , Neoplasias Uterinas/genética , Neoplasias Uterinas/inmunología , Neoplasias Uterinas/patologíaRESUMEN
BACKGROUND: Sperm Acrosomal SLLP1 Binding (SAS1B) protein (ovastacin) is an oolemmal binding partner for the intra-acrosomal sperm protein SLLP1. RESULTS: Immunohistochemical localization revealed that SAS1B translation is restricted among adult tissues to the ovary and oocytes, SAS1B appearing first in follicles at the primary-secondary transition. Quiescent oocytes within primordial follicles and primary follicles did not stain for SAS1B. Examination of neonatal rat ovaries revealed SAS1B expression first as faint signals in postnatal day 3 oocytes, with SAS1B protein staining intensifying with oocyte growth. Irrespective of animal age or estrus stage, SAS1B was seen only in oocytes of follicles that initiated a second granulosa cell layer. The precise temporal and spatial onset of SAS1B expression was conserved in adult ovaries in seven eutherian species, including nonhuman primates. Immunoelectron micrographs localized SAS1B within cortical granules in MII oocytes. A population of SAS1B localized on the oolemma predominantly in the microvillar region anti-podal to the nucleus in ovulated MII rat oocytes and on the oolemma in macaque GV oocytes. CONCLUSIONS: The restricted expression of SAS1B protein in growing oocytes, absence in the ovarian reserve, and localization on the oolemma suggest this zinc metalloprotease deserves consideration as a candidate target for reversible female contraceptive strategies.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Mamíferos/metabolismo , Metaloproteasas/metabolismo , Oocitos/metabolismo , Folículo Ovárico/fisiología , Animales , Secuencia de Bases , Western Blotting , Clonación Molecular , Cricetinae , Cartilla de ADN/genética , Evolución Molecular , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Mamíferos/crecimiento & desarrollo , Ratones , Datos de Secuencia Molecular , Oocitos/crecimiento & desarrollo , Folículo Ovárico/metabolismo , Conejos , Ratas , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
Molecular mechanisms by which fertilization competent acrosome-reacted sperm bind to the oolemma remain uncharacterized. To identify oolemmal binding partner(s) for sperm acrosomal ligands, affinity panning was performed with mouse oocyte lysates using sperm acrosomal protein, SLLP1 as a target. An oocyte specific membrane metalloproteinase, SAS1B (Sperm Acrosomal SLLP1 Binding), was identified as a SLLP1 binding partner. cDNA cloning revealed six SAS1B splice variants, each containing a zinc binding active site and a putative transmembrane domain, with signal peptides in three variants. SAS1B transcripts were ovary specific. SAS1B protein was first detected in early secondary follicles in day 3 ovaries. Immunofluorescence localized SAS1B to the microvillar oolemma of M2 oocytes. After fertilization, SAS1B decreased on the oolemma and became virtually undetectable in blastocysts. In transfected CHO-K1 cells SAS1B localized to the surface of unpermeabilized cells. Recombinant and native SLLP1 co-localized with SAS1B to the microvillar domain of ovulated M2 oocytes. Molecular interactions between mouse SLLP1 and SAS1B were demonstrated by surface plasmon resonance, far-western, yeast two-hybrid, recombinant- and native-co-IP analyses. SAS1B bound to SLLP1 with high affinity. SAS1B had protease activity, and SAS1B protein or antibody significantly inhibited fertilization. SAS1B knockout female mice showed a 34% reduction in fertility. The study identified SAS1B-SLLP1 as a pair of novel sperm-egg binding partners involving the oolemma and intra-acrosomal compartment during fertilization.
Asunto(s)
Fertilización , Isoantígenos/metabolismo , Metaloproteasas/metabolismo , Oocitos/metabolismo , Proteínas de Plasma Seminal/metabolismo , Acrosoma/metabolismo , Empalme Alternativo , Animales , Unión Competitiva , Far-Western Blotting , Células CHO , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Femenino , Inmunoprecipitación , Isoantígenos/genética , Masculino , Metaloproteasas/genética , Ratones , Ratones Endogámicos ICR , Ratones Noqueados , Embarazo , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Plasma Seminal/genética , Interacciones Espermatozoide-Óvulo , Resonancia por Plasmón de Superficie , Técnicas del Sistema de Dos HíbridosRESUMEN
BACKGROUND: We earlier reported a simple specific test for detection of anti-ovarian antibodies in infertile women and identified number of specific molecular and cellular targets of which human heat shock protein 90-beta (HSP90 beta) was found to be the most immunodominant. The present study focuses on prediction and validation of the immunodominant epitope/s of this protein using sera from infertile women having anti-HSP90 autoantibodies. METHODS: Delineation of the immunodominant epitopes of HSP90 beta was done by using epitope prediction algorithms and 10 peptides (EP1-EP10) were custom synthesized. Their immunoreactivity was measured by ELISA using sera from patients and controls. To determine the most immunodominant epitope, the results were subjected to statistical analysis. The immunoreactivity of the immunodominant peptides were confirmed by dot blots using sera from patients. A rabbit polyclonal antibody against the immunodominant epitope was generated and its immunoreactivity to the parent protein in ovarian extracts as well in oocytes and embryos was investigated. RESULTS: Experimentally and statistically, peptide EP6 (380-389) seems to be the major antigenic epitope for the serum antibody binding followed by EP1 (1-12) and EP8 (488-498). Predicted 3D structures of these peptides demonstrated that they exist in the loop conformation which is the most mobile part of the protein. Also, analysis of the sequences of HSP90 beta across several species reveals that EP6 peptide forms a part of a well conserved motif. The polyclonal antibody generated to the immunodominant epitope- EP6 confirms similar biochemical and cellular immunoreactivity as seen with the patients' sera having anti-HSP90 autoantibodies. CONCLUSIONS: The decapeptide EP6 is a major immunogenic epitope of HSP90 followed by EP1 and EP8. Knowledge of binding epitopes on the autoantigen is necessary to understand the subsequent pathologic events. The study might generate new tools for the detection of disease-inducing epitopes and a possible therapeutic intervention.
Asunto(s)
Proteínas HSP90 de Choque Térmico/inmunología , Epítopos Inmunodominantes/inmunología , Infertilidad Femenina/inmunología , Animales , Autoanticuerpos/sangre , Autoantígenos/inmunología , Femenino , Humanos , Oligopéptidos/inmunología , Fragmentos de Péptidos/inmunología , ConejosRESUMEN
OBJECTIVE: To establish importance of anti-ovarian antibodies (AOA) testing in infertile women. DESIGN: A clinical reproductive outcome comparative study between two groups of women undergoing IVF-ET. Group 1 consists of women tested positive for AOA, put on corticosteroid therapy, reverted to AOA negative and then taken up for IVF-ET. Group 2 were seronegative for AOA. SETTING: Major urban infertility reference centre and National research institute. PATIENT(S): Five hundred seventy infertile women enrolled for IVF-ET. INTERVENTION(S): AOA testing, corticosteroid therapy and IVF-ET/ICSI. MAIN OUTCOME MEASURE(S): Comparable clinical outcome and significance of AOA testing established. RESULTS: AOA positive serum samples were sent periodically to re-investigate presence of AOA after corticosteroid therapy and women turned AOA negative were taken up for IVF-ET. Of the 70/138 women in group 1 who were treated with corticosteroids and turned seronegative for AOA, 22/70 were poor responders and needed donor oocyte-recipient cycles. Results demonstrated that fertilization and clinical pregnancy rates between both groups are comparable. Nevertheless, it is also observed that there is poor response to stimulation protocol, smaller number of oocytes retrieved and more spontaneous abortions in group 1 women. Hence not all outcomes following the treatment are comparable between the two groups. Usefulness of the test was established in two case studies. CONCLUSIONS: AOA testing could be included in the battery of tests investigating and treating infertility.
Asunto(s)
Anticuerpos Antiidiotipos/sangre , Autoanticuerpos/sangre , Infertilidad Femenina/inmunología , Ovario/inmunología , Corticoesteroides/uso terapéutico , Adulto , Anticuerpos Antiidiotipos/inmunología , Autoanticuerpos/inmunología , Femenino , Humanos , Infertilidad Femenina/tratamiento farmacológico , Oocitos/inmunología , Embarazo , Índice de Embarazo , Inyecciones de Esperma Intracitoplasmáticas/métodosRESUMEN
PURPOSE: To provide an update of putative auto-antigens identified and proposed to be involved in human ovarian autoimmunity. METHODS: Review of literature pertaining to ovarian auto-antigens / proteins identified with various immunological tools using sera of infertile women as a probe for investigation. RESULTS: An overview of autoimmune targets known till date in the study of human ovarian autoimmunity. CONCLUSIONS: Anti-ovarian antibodies (AOA) to multiple components and compartments of the ovary are present in the sera of infertile women. Researchers propose that these AOA may be responsible for ovarian failures and therefore render women to be infertile. Evaluation of AOA can be effective as a prognostic factor in the treatment of infertile patients and for the IVF-ET program.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Enfermedades del Ovario/inmunología , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Autoinmunidad/inmunología , Femenino , HumanosRESUMEN
OBJECTIVE: To report autoantibodies to human heat-shock protein 90-beta (HSP90 beta) in sera of women with infertility. DESIGN: Prospective, controlled observations. SETTING: Major urban infertility referral center and research institution. PATIENT(S): Fifty women with premature ovarian failure, 65 infertile women enrolled in the in vitro fertilization-embryo transfer program, and 60 normally menstruating fertile women as controls. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Identification and complete characterization of a 90-kd protein, the most immunodominant autoantigen. RESULT(S): Our previous studies employing a novel blocking demonstrated several cellular and molecular ovarian antigenic targets using patient's serum. Of all these antigens, the 90-kd protein designated as EP90 was found to be conserved across species, was serine-threonine phosphorylated, and was expressed from the primordial stage to the graafian-stage ooplasm of the oocytes during follicular development. Using high-throughput proteomic technologies like liquid chromatography/mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF), and tandem mass spectrometry analysis revealed the identity of this protein to be HSP90 beta. Commercially available recombinant protein immunoreacted with the sera from patients with antiovarian antibodies against the 90-kd antigen. In parallel, using monoclonal antibody to human HSP90, we found that it reacts with the eluted protein from a crude ovarian extract. CONCLUSION(S): This is the first report to show the presence of ovarian autoantibodies to human HSP90 in sera of women with infertility. This protein could be involved in human ovarian autoimmunity and thereby be a causative factor in early ovarian failure.
Asunto(s)
Autoanticuerpos/sangre , Fertilidad/inmunología , Proteínas HSP90 de Choque Térmico/inmunología , Infertilidad Femenina/etiología , Insuficiencia Ovárica Primaria/inmunología , Animales , Autoanticuerpos/aislamiento & purificación , Autoanticuerpos/metabolismo , Enfermedades Autoinmunes/complicaciones , Enfermedades Autoinmunes/inmunología , Autoinmunidad/inmunología , Autoinmunidad/fisiología , Estudios de Casos y Controles , Femenino , Humanos , Infertilidad Femenina/inmunología , Ratones , Ovario/inmunología , Ovario/metabolismo , Insuficiencia Ovárica Primaria/sangre , Insuficiencia Ovárica Primaria/complicaciones , Conejos , Ratas , Ratas Sprague-Dawley , PorcinosRESUMEN
Progesterone has been implicated in the process of spermatogenesis. This study aimed to investigate the association of progesterone receptor (PR) expression with spermatogenesis in the testis of infertile men. PR mRNA and protein were assessed by in-situ hybridization and immunohistochemistry in testicular biopsies obtained from 18 infertile men. The extent of spermatogenesis was assessed by Johnsen scoring. None of the patients included in the study had Yq microdeletions. PR expression was almost undetectable in all the testicular sections displaying Sertoli cell only (SCO) or arrest at spermatogonia. Weak cytoplasmic expression was observed in biopsies showing arrest at different stages of meiosis. In biopsies displaying spermatogenesis up to the round spermatid stages, PR expression was observed in both nucleus and cytoplasm of different cell types at intensity lower than that detected in normal biopsies. Normal PR expression was observed in biopsies demonstrating hypospermatogenesis. In biopsies showing mixed phenotypes, the tubules with SCO or spermatogonia arrest showed absence of PR expression; normal PR expression was observed in adjacent tubules showing complete spermatogenesis. Semi-quantitative assessment of PR expression and Johnsen scores in the testicular biopsies of infertile men demonstrating different phenotypes indicated a direct relationship between PR expression and extent of spermatogenesis.
Asunto(s)
Regulación hacia Abajo , Infertilidad Masculina/genética , Receptores de Progesterona/genética , Testículo/metabolismo , Adulto , Deleción Cromosómica , Cromosomas Humanos Y , Mutación de Línea Germinal , Humanos , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , Masculino , Persona de Mediana Edad , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Progesterona/metabolismo , Espermatogénesis/genética , Testículo/patología , Distribución TisularRESUMEN
Serum anti-ovarian antibodies (AOAs) have been shown in autoimmune premature ovarian failure and in vitro fertilization-embryo transfer (IVF-ET) cases. The specificity of assays detecting these antibodies has been questioned. Researchers have used several techniques (e.g., ELISA and indirect immunofluorescence). Few have reported on the non-specificity and the type of molecular and cellular targets. We reported earlier on the presence of naturally occurring anti-albumin antibodies as the likely factor for non-specificity. Having developed a novel blocking recipe, we show substantial elimination of this non-specificity. With these standardized tests, we hereby report multiple targets at protein and histological levels. In our study group, 15 of 50 (30%) patients with premature ovarian failure and 13 of 50 (26%) IVF-ET patients showed the presence of AOAs. Western blotting showed a large number of patients making AOAs to a 90-kDa protein, followed by 97- and 120-kDa proteins. Histochemically, it was evident that the sera of these patients predominantly react with the oocyte; other somatic cellular targets are also involved. The specific non-invasive test developed by us was found to be useful because it could carry out a reliable diagnosis of an autoimmune etiology that would be very helpful to select patients in whom immune-modulating therapy could be recommended, which in turn may restore ovarian function and fertility.
Asunto(s)
Autoanticuerpos/sangre , Infertilidad/inmunología , Ovario/inmunología , Insuficiencia Ovárica Primaria/inmunología , Adulto , Animales , Autoantígenos/sangre , Transferencia de Embrión , Femenino , Fertilización In Vitro , Humanos , Inmunoensayo/métodos , Epítopos Inmunodominantes/sangre , Inmunohistoquímica , Infertilidad/terapia , Ratas , Ratas Sprague-Dawley , Sensibilidad y EspecificidadRESUMEN
Autoimmunity is a well-established causative factor of premature ovarian failure (POF), and evidence for the same has been well reported in the literature. Detection of specific autoantibodies remains the most practical clinical research marker of any autoimmune disease. Variation in efficiency and specificity in the detection of ovarian autoantibodies has been reported. However, the frequency of false positivity and a solution to overcome this has not yet been reported. Herein, we report autoantibody to albumin as the likely responsible agent for false positivity. Our data indicate that presence of naturally existing autoalbumin antibodies in the circulation of normal women is responsible for the false signal seen in SDS-PAGE Western blot analysis and in immunohistochemistry (IHC). Having shown the presence of anti-albumin antibody in normal women as well as in the sera of POF patients, we have developed a novel blocking agent to overcome this problem. A high titer polyclonal antibody against human serum albumin was generated. This antibody showed immunoreactivity to albumin obtained from various sources. Preincubation of Western blots and IHC sections with this antibody drastically reduced background signals. The advantage of using this blocking was evident by identification of specific anti-ovarian antibodies in a group of POF patients. This blocking procedure made it possible to obtain a clear indication of the ovarian antibody status in women presenting with autoimmune POF.