ABSTRACT
Human chorionic gonadotropin (hCG) hormone is required for maintenance of early pregnancy and is a potential marker in the diagnosis and prognosis of both pregnancy and trophoblastic diseases. Murine hybridomas were generated against purified hCG. Seven hybrid clones secreting antibodies against hCG molecule with IgG1/kappa subclass were established. The indirect ELISA result demonstrated that six MAbs (BEL-1 to BEL-6) recognized hCG in both holo and free beta subunit form with negligible cross-reactivity against a closely related hormone, human luteinizing hormone (hLH). In this fusion, only one MAb (ALC-1) showed a cross-reaction with hLH, which designated an alpha subunit specific. The outcome of Western blot ascertained that ALC-1 recognized the conformational epitope on alpha subunit of hCG at Mr 23 kDa band in nonreducing condition (NR). In contrast, epitopes belonging to all MAbs recognized beta subunit of hCG were in linear peptide structure at Mr 34 kDa band (NR) and Mr 26 kDa band (R). These six MAbs were further characterized by using two beta subunit carboxy-terminal synthetic peptides (beta109-119 and beta109-145). Three of them (BEL-1, BEL-3, and BEL-4) recognized only epitope harboring in beta109-145 fragment, the others recognized both types of the synthetic peptide. In order to select the most suitable MAbs specific to beta subunit of hCG for exploiting with ALC-1 in the sandwich-type immunometric assay, competitive ELISA was employed. Six individual MAbs specific to beta subunit of hCG were used to compete with biotinylated ALC-1 to evaluate the proximity of their epitopes on the holo form of hCG molecule. Of all six MAbs, BEL-5 depicted the lowest percent inhibition result, which indicated the bottom-most steric hindrance effect. Consequently, MAb BEL-5 will be the most appropriate antibody to utilize in concert with ALC-1 in place of devising a sandwich-type immunometric assay for measuring holo-hCG level.
Subject(s)
Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Ascites/immunology , Biotinylation , Blotting, Western/methods , Chorionic Gonadotropin, beta Subunit, Human/genetics , Cross Reactions/immunology , Enzyme-Linked Immunosorbent Assay/methods , Epitopes/genetics , Female , Glycoprotein Hormones, alpha Subunit/genetics , Humans , Luteinizing Hormone/immunology , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Sequence HomologyABSTRACT
It has been proposed that automated systems for immunoenxymometric assay (IEMA) may substitute traditional radioimmunoassay (RIA) for measurement of luteinizing hormone (LH) and follicle stimulating hormone (FSH) in blood due to the advantage of being more rapid, higher sensitivity, lower cost and not requiring radioactive reagents. The study was designed to evaluate both systems using serum samples to determine luteinizing hormone (LH) and follicle stimulatin hormone (FSH) concnetrations. The automatic system (ES-300) for IEMA utilized two monoclonal antibodies, one of them on the solid phase was the specific extractant for the antigen, and the other was a peroxidase labeled antibody which recognizes a different epitope in the antigen molecule, specifically bound in linear proportion to the antigen concentration. Blood samples were obtained from patients who were treated at the hospital for various clinical problems ("problem group") as well as blood samples from patients in whom FSH and LH concentrations were already known ("high", "medium" and "low" levels) by previous RIA ("control group"). IEMA showed a higher sensitivity, 0.42 and 0.96 mIU/ml for FSH and LH, respectively, whereas RIA was 1.95 mIU/ml for both hormones. Intra and interassay coefficient of variation were below 10 percent within the range of 15-50 mIU/ml for FSH and 5-100 mIU for LH; however, the coeffcient of variation was 15 - 25 percent at lower concentrations of FSH and LH. Accuracy of IEMA was evaluated by recovery percentage, thus when high and medium concentrations of FSH and LH were analyzed the recovery was between 99 -104 percent. On the other hand, the recovery was 100 percent when low levels of FSH and LH were used. In coclusion, IEMA resulted reliale when FSH and LH concentrations are in the middle and high range; likewise, the detection limit of IEMA was lower than RIA, particularly for FSH. On the bases of these results, IEMA showed several advantages over RIA, but its reliability diminishes when serum samples contain low FSH and LH concentrations. It is important to extend theses studies to steroid assays and elaborate a database in each laboratory
Subject(s)
Humans , Female , Adult , Middle Aged , Follicle Stimulating Hormone/blood , Follicle Stimulating Hormone/immunology , Luteinizing Hormone/blood , Luteinizing Hormone/immunology , Radioimmunoassay , Reproducibility of Results , Sensitivity and Specificity , Immunoenzyme Techniques/instrumentationSubject(s)
Animals , Mice , Antibodies, Monoclonal/biosynthesis , Glycoproteins/immunology , Hormones/immunology , Antibodies, Monoclonal/immunology , Cell Fusion , Chorionic Gonadotropin/immunology , Fluoroimmunoassay , Follicle Stimulating Hormone/immunology , Glycoprotein Hormones, alpha Subunit/immunology , Chorionic Gonadotropin, beta Subunit, Human/immunology , Luteinizing Hormone/immunology , Mice, Inbred BALB C , Thyrotropin/immunologyABSTRACT
Activities of delta 5-3 beta- and 17 beta-hydroxysteroid dehydrogenase (delta 5-3 beta-HSD and 17 beta-HSD), Leydig cell nuclear area (LCNA) and spermatogenesis in the testis were observed after injection of lithium chloride in the 'antiserum to luteinizing hormone (LH)' treated toad. A significant decrease in the activities of steroidogenic enzymes, LCNA and spermatogenesis were noticed after the injections of 'antiserum to LH' to toads. Further decrease in the activities of the above parameters was observed in the lithium chloride--'antiserum to LH' treated toad. It is suggested that lithium chloride may inhibits testicular function without modulating the pituitary activity.