DEVELOPMENT OF A MONOCLONAL-ANTIBODY TO 6-BETA-HYDROXYCORTISOL AND ITS APPLICATION IN AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY (ELISA) FOR 6-BETA-HYDROXYCORTISOL IN URINE

A novel monoclonal antibody to 6 beta-hydroxycortisol (6 beta-OHC) was generated and incorporated into an antigen-coated indirect enzyme-lin ked immunosorbent assay (ELISA) using 6 beta-OHC-protein conjugate as the steroid-coating antigen. The monoclonal antibody is specific to 6 beta-OHC and 6 beta-OHC-3-carboxymethyloxime. Cross-reactivity with ot her structurally related steroids such as cortisol, cortisone, and 6 b eta-hydroxycortisone was less than 10%. Two different clones (clone 5C 1 and 19F) of the monoclonal anti-6 beta-OHC antibody have been develo ped, each with slightly different sensitivity and specificity. The sen sitivity of the MAb clones was not significantly improved when compare d to the rabbit polyclonal antibodies in this study, but still within the accepted detection limit for 6 beta-OHC in both human and laborato ry animals. The assay had a detection limit of 200 ng/ml, an intraassa y variation of 6.4% and an interassay variation of 7.3%. The applicati on of the anti-6 beta-OHC-MAb-based-ELISA was tested by measuring the urinary output of 6 beta-OHC in human before and after enzyme inductio n by rifampicin treatment. The mean 24-h urine output of 6 beta-OHC in human subjects was 485 +/- 100 mu g and 1478 +/- 281 mu g before and after rifampicin administration, respectively. In conclusion, the mono clonal anti-6 beta-OHC antibody developed in this study has the requir ed specificity and sensitivity as an alternative method for measuring urinary 6 beta-OHC in the detection of enzyme induction or enzyme inhi bition of CYP3A in humans and laboratory animals. (C) 1997 Elsevier Sc ience Inc.