Radioimmunoassay for Cyclic Nucleotides: I. PREPARATION OF ANTIBODIES AND IODINATED CYCLIC NUCLEOTIDES

Abstract

Abstract Sensitive and specific radioimmunoassays have been developed for cyclic adenosine 3',5'-monophosphate (cAMP), cyclic guanosine 3',5'-monophosphate (cGMP), cyclic inosine 3',5'-monophosphate (cIMP), and cyclic uridine 3',5'-monophosphate (cUMP). These assays are based upon competition of the cyclic nucleotide with isotopically labeled cyclic nucleotide derivatives for binding sites on specific antibody. Antibodies to the cyclic nucleotides were obtained by immunizing rabbits with an antigen prepared by conjugating the 2'-O-succinyl derivative of the cyclic nucleotide with protein (human serum albumin or keyhole limpet hemocyanin). High specific activity derivatives of the cyclic nucleotides (g150 Ci per mmole) were prepared by iodinating (125I) the tyrosine methyl ester derivative of the succinylated cyclic nucleotide. Free and bound 125I-labeled cyclic nucleotides were separated by either the second antibody precipitation technique or (NH4)2SO4 fractionation. Binding equilibrium was reached in 24 hours, but sensitive and reproducible assays were obtained after 4 to 6 hours of incubation. The sensitivity and assay range for the various radioimmunoassays are (picomoles per tube): cAMP, 0.01 to 2; cGMP, 0.01 to 1.0; cIMP, 0.12 to 10.0; and cUMP, 0.1 to 10.0. Structurally related purine and pyrimidine nucleotides and nucleosides exhibited ≤0.005% competitive binding in all immunoassays except cIMP which showed 0.35% competitive binding in the cGMP assay, cGMP 1% in the cIMP assay, and cIMP 1% in the cUMP assay.