PRODUCTION OF A MONOCLONAL-ANTIBODY TO CORTISOL - APPLICATION TO A DIRECT ENZYME-LINKED-IMMUNOSORBENT-ASSAY OF PLASMA

Cortisol mouse monoclonal antibodies were produced and characterized. Of the four clones studied, supernatant from one clone (A2), compared with other cortisol monoclonal antibodies, showed minimal cross-reactivity to other C21 steroids and was suitable for the direct determination of cortisol in plasma by enzyme-linked immunosorbent assay using a standard 96-well microtiter plate. The enzyme-linked immunosorbent assay uses the immobilized antigen approach, in which cortisol in plasma samples or standards competes with immobilized steroid for antibody-binding sites. After washing, the cortisol antibody bound to the wells of the microtiter plate is detected with antimouse immunoglobulin conjugated to horseradish peroxidase. Following further washing, o-phenylenediamine substrate is added. The enzyme-linked immunosorbent assay is robust and semiautomated. The mean +/- SD recovery from plasma was 97% +/- 6%. Precision studies on three different plasma pools showed mean coefficients of variation of 7.6% and 8.6% for within- and between-assay variation, respectively. The satisfactory performance criteria allow, its use in the routine laboratory.