Direct solid-phase fluoroenzymeimmunoassay of 5 beta-pregnane-3 alpha, 20 alpha-diol-3 alpha-glucuronide in urine

A competitive solid-phase fluoroenzymeimmunoassay has been developed for rapidly measuring 5 beta-pregnane-3 alpha, 20 alpha-diol-3 alpha-glucuronide (Pd-3G) directly in diluted specimens of pre-breakfast urine. The assay involves use of an antiserum to Pd-3G and enzyme-labeled antigen prepared by chemically linking glucose-6-phosphate dehydrogenase (EC 1.1.1.49) to Pd-3G. Antibody-bound and free antigen are separated by use of a solid-phase double antibody: sheep anti-rabbit gamma-globulin coupled to cellulose particles. The solid phase, isolated by centrifugation, is washed free of labeled antigen and endogenous enzyme interferences. Enzyme activity in the bound fraction is then measured fluorometrically with glucose 6-phosphate as the substrate and generated NADPH as a fluorogenic indicator of enzyme activity. The assay is sufficiently sensitive (30-35 pg per assay tube), specific, and reliable for routine use, and results correlate well (r = 0.98) with those by an established specific radioimmunoassay. Because it is suited to routine use, this assay may be applied to detecting ovulation, assessing the function of corpus luteum, and monitoring early pregnancy.