Homogeneous colorimetric enzyme inhibition immunoassay for cortisol in human serum with Fab anti-glucose 6-phosphate dehydrogenase as a label modulator

Abstract

The Fab fragment of rabbit IgG antibody to bacterial glucose 6-phosphate dehydrogenase covalently linked to the cortisol retained the capacity to inhibit the enzyme completely. In optimal conditions the antibody to cortisol effectively bound the cortisol residues of the cortisol-Fab conjugate, making it incapable of inhibiting the enzyme. The enzyme modulatory properties of the cortisol-Fab conjugate were exploited to set up a direct competitive homogeneous enzyme immunoassay for cortisol in human serum. The procedure involved the use of the auxiliary enzyme diaphorase, specific for NADH, which converts the nitro blue tetrazolium salt to a colored formazan. The procedure detects modulated glucose 6-phosphate dehydrogenase activity by a single-point measurement without serum interference. The assay working range was between 20 and 640 μg/1 of cortisol and used 50 μ1 of sample.