An enzyme immunoassay for a metabolite of a new sleep inducer (450191-S), a ring-opened derivative of 1,4-benzodiazepine, in human serum

A competitive enzyme immunoassay was developed for metabolites of 450191-S, a ring-opened derivative of triazolobenzodiazepines, in human serum. Three kinds of labelled antigens, beta-D-galactosidase conjugates, and antisera from three kinds of immunogens, bovine serum albumin conjugates, were tested and the hapten heterologous assay was selected. B/F separation was performed using immobilized second antibody and the enzyme activity was measured using fluorescent substrate. One of the serum metabolites, the carboxyl form, was extracted from acidified serum and measured by the assay. The minimum detectable concentration was 1.5 ng/ml in serum, 15 pg/tube. The intra-assay and inter-assay variances were 5.5% and 7.7% at 6 ng/ml of serum, respectively. Standard curves of other related metabolites were assessed with various combinations of labelled antigens and antibodies.