Lewis J G, Bason L M, Elder P A
Steroid and Immunobiochemistry Laboratory, Canterbury Health Laboratories, Christchurch, New Zealand.
Steroids. 1996 Dec;61(12):682-7. doi: 10.1016/s0039-128x(96)00189-4.
Mice were immunized with 5-androstene-3 beta-ol-7,17-dione-7-CMO:bovine serum albumin (DHEA-7-O-CMO-BSA) or 5-androstene-3 beta-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-lg 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.
用5-雄烯-3β-醇-7,17-二酮-7-羧甲基肟：牛血清白蛋白（DHEA-7-O-CMO-BSA）或5-雄烯-3β-醇-17-酮半琥珀酸酯-牛血清白蛋白（DHEA-3HS-BSA）结合物免疫小鼠，并制备、鉴定单克隆抗体，选择与硫酸脱氢表雄酮（DHEAS）结合能力最强的抗体。在这些杂交瘤中，来自用DHEA-3HS-BSA免疫的小鼠的四个克隆符合用于血浆中DHEAS竞争性酶联免疫吸附测定（ELISA）的开发标准。来自用DHEA-7-O-CMO-BSA免疫的小鼠的一种杂交瘤上清液对硫酸雄酮和硫酸表雄酮均表现出360%的交叉反应性。这使得在校正DHEAS的贡献后，有可能直接测定血浆中的硫酸雄酮和硫酸表雄酮。两种ELISA均使用吸附在标准96孔微量滴定板孔中的DHEA-3HS-甲状腺球蛋白结合物。标准品或稀释血浆样品中的DHEAS与固定化的DHEA-3HS-甲状腺球蛋白竞争抗体结合位点。通过进一步洗涤、加入邻苯二胺底物并读取492nm处的吸光度，用抗小鼠Ig过氧化物酶检测抗体。这些ELISA方法简单、可重复且可靠，据我们所知，它们是首批使用针对DHEAS的单克隆抗体的检测方法。
