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/

Mice were immunized with 5-androstene-3 beta-ol-7, 17-dione-7-CMO:bovi ne serum albumin (DHEA-7-O-CMO-BSA) or 5-androstene-3 beta-ol-17-one h emisuccinate-bovine serum albumin (DHEA-3HS-BSA) conjugates and monocl onal antibodies were produced, characterized, and selected for maximum DHEAS binding. Of these hybridomas, four clones from DHEA-3HS-BSA-imm unized mice had acceptable criteria for the development of a competiti ve 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 sul fate. This allows the possibility of the direct determination of andro sterone sulfate and epiandrosterone sulfate in plasma after correction for the DHEAS contribution. Both ELISAs employ a DHEA-3HS-thyroglobul in conjugate adsorbed to the wells of a standard 96-well microtiter pl ate. DHEAS in the standards or diluted plasma sample competes with imm obilized DHEA-3HS-thyroglobulin for antibody-binding sites. Antibody i s 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, th ey are the first tests employing monoclonal antibodies to DHEAS. (C) 1 996 by Elsevier Science Inc.