Development of a screening method for anti-6 beta-hydroxycortisol antibody using an enzyme-linked immunosorbent assay...

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of anti-6 beta-hydroxycortisol (6 beta-OHC) antibody has been developed. After immunization of 6 beta-OHC protein conjugates in New Zealand White rabbits, specific polyclonal antibody to 6 beta-OHC was detected by ELISA in which the wells of microtiter plates were coated with 6 beta-OHC conjugated to protein. The rabbit anti-6 beta-OHC antibody titer was above 1:500,000 dilution. Cross-reactivity with other structurally related steroids such as cortisol hydrocortisone was less than 5%. The sensitivity of the polyclonal antibody was comparable to previous studies reported, and was within the accepted detection limit for 6 beta-OHC in man and in laboratory animals. The assay 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 measuring the concentration of 6 beta-OHC in man before and after enzyme induction by rifampicin treatment. The mean 24 h urine output of 6 beta-OHC in man subjects was 370 +/- 105 micrograms and 1350 +/- 201 micrograms before and 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 induction of CYP3A4 in man and laboratory animals.