Production and characterization of monoclonal antibodies to dehydroepiandrosterone sulfate: Application to direct enzyme-linked immunosorbent assays of dehydroepiandrosterone sulfate and androsterone/epiandrosterone sulfates in plasma

• Published in: Steroids 1996-12, Vol.61 (12), p.682-687
• Main Authors: Lewis, John G., Bason, Lois M., Elder, Peter A.
• Format: Article
• Language: English
• Subjects: Adult; Aged; Androsterone - analogs & derivatives; Androsterone - blood; Androsterone - immunology; androsterone sulfate; Animals; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - metabolism; Biological and medical sciences; Chromatography, High Pressure Liquid - methods; Cross Reactions; dehydroepiandrosterone sulfate; Dehydroepiandrosterone Sulfate - blood; Dehydroepiandrosterone Sulfate - immunology; DHEAS ELISA; Enzyme-Linked Immunosorbent Assay - methods; epiandrosterone sulfate; Fundamental and applied biological sciences. Psychology; Humans; Mice; Mice, Inbred Strains; Middle Aged; monoclonal antibody; Reference Values; Steroid hormones. Cholecalciferol derivatives; Vertebrates: endocrinology
• ISSN: 0039-128X; 1878-5867
• DOI: 10.1016/S0039-128X(96)00189-4

Mice were immunized with 5-androstene-3β-ol-7,17-dione-7-CMO:bovine serum albumin (DHEA-7-O-CMO-BSA) or 5-androstene-3β-ol-17-one hemisuccinate-bovine serum albumin (DHEA-3HS-BSA) conjugates and monoclonal antibodies were produced, characterized, and selected for maximum DHEAS binding. Of these hybridomas, four clones from DHEA-3HS-BSA-immunized mice had acceptable criteria for the development of a competitive enzyme-linked immunosorbent assay (ELISA) for DHEAS in plasma. One hybridoma supernatant from DHEA-7-O-CMO-BSA-immunized mice showed 360% cross-reactivity to both androsterone sulfate and epiandrosterone sulfate. This allows the possibility of the direct determination of androsterone sulfate and epiandrosterone sulfate in plasma after correction for the DHEAS contribution. Both ELISAs employ a DHEA-3HS-thyroglobulin conjugate adsorbed to the wells of a standard 96-well microtiter plate. DHEAS in the standards or diluted plasma sample competes with immobilized DHEA-3HS-thyroglobulin for antibody-binding sites. Antibody is detected with anti-mouse-Ig peroxidase by further washing, adding o-phenylenediamine substrate, and reading the absorbance at 492 nm. The ELISAs are simple, reproducible, and reliable and, to our knowledge, they are the first tests employing monoclonal antibodies to DHEAS.