Concerns In The Development Of Homogeneous Enzyme Immunoassays: An Assay For Cyclic-amp.

The development of a homogeneous competitive binding assay for the determination of cyclic AMP in the 10('-8) M range is described. The basis for this assay is the antibody-induced inhibition of the catalytic activity of an enzyme label. O('2')-Monosuccinyladenosine 3',5'-cyclic monophosphate (SCAMP) was coupled to the carrier protein human serum albumin for use as an immunogen to prepare antibodies and to glucose-6-phosphate dehydrogenase as the analyte label in the assay. Both the mixed anhydride and the NHS ester methods were used to activate the carboxyl group of the cyclic AMP derivative for coupling purposes. The effect of various reaction conditions on the degree of conjugation to the proteins and on the residual activity of the enzyme were investigated. Enzyme conjugates were prepared that retained 49% of the activity of the native enzyme and were inhibited 85% by anti-cyclic AMP antibody. Chromatographic and immunological evidence is presented regarding the hydrolysis of the ester linkage of the O('2')-monosuccinyl cyclic AMP in neutral solutions. Loss of cyclic AMP from the immunogen (prior to or during immunization) leaves the succinyl bridging group bound to the protein. As a result, a population of anti-succinate-protein antibodies is formed. The effects of hydrolysis within the enzyme conjugate and the presence of the anti-succinate antibody population on the performance characteristics of the assay for cyclic AMP are discussed. The optimization of the assay protocol, the effect of sample acetylation on the detection limits of the assay, and recovery studies in which urine samples were spiked with various known amounts of cyclic AMP are described. The results of selectivity studies, the determination of cyclic AMP in the urine of normal human volunteers and a comparison study of values obtained by the new assay and those obtained by conventional radioimmunoassay in other laboratories are also presented.PhDAnalytical chemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/127847/2/8612489.pd