DEVELOPMENT OF A SCREENING METHOD FOR ANTI-6-BETA-HYDROXYCORTISOL ANTIBODY USING AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY (ELISA) AND ITS APPLICATIONS

An indirect enzyme-linked immunosorbent assay (ELISA) for the detectio n anti-6-beta-6-beta-hydroxycortisol (6 beta-OHC) antibody has been de veloped. After immunization of 6 beta-OHC-protein conjugates in New Ze aland White rabbits, specific polyclonal antibody to 6 beta-OHC was de tected by ELISA in which the wells of microtiter plates were coated wi th 6 beta-OHC conjugated to protein. The rabbit anti-6 beta-OHC antibo dy titer was above 1:500,000 dilution. Cross-reactivity with other str ucturally related steroids such as cortisol hydrocortisone was less th an 5%. The sensitivity of the polyclonal the polyclonal antibody was c omparable to previous studies reported, and was within the accepted de tection limit for 6 beta-OHC in man and in laboratory animals. The ass ay has a low detection limit of 1 ng/ml, an intraassay variation of 3. 1% and an interassay variation of 5.2%. The application of the anti-6 beta-OHC-based-ELISA to detect urinary 6 beta-OHC was tested by measur ing the concentration of 6 beta-OHC in man before and after enzyme ind uction by rifampicin treatment. The mean 24 h urine output of 6 beta-O HC in man subjects was 370 +/- 105 mu g and 1350 +/- 201 mu g before a nd after rifampicin administration, respectively This polyclonal anti- 6 beta-OHC antibody-based ELISA can be modified to detect mouse anti-6 beta-OHC IgG with equally good precision and specificity which should be useful in screening positive clones of a 6 beta-OHC IgG-secreting mouse hybridoma currently being developed for detecting enzyme inducti on of CYP3A4 in man and laboratory animals.