Enzyme immunoassay for cytidine 3′,5′-cyclic monophosphate (cyclic CMP)

An enzyme immunoassay for cytidine 3′,5′-cyclic monophosphate (cyclic CMP) is presented. This assay is based upon the principles of competitive reaction and the double antibody solid phase method. Succinyl cyclic CMP-human serum albumin conjugate was injected into rabbits. Specific anti-cyclic CMP antibodies were incubated with a mixture of succinyl cyclic CMP labelled with β-D-galactosidase and standard or sample cyclic CMP that had been succinylated prior to assay. The antibody-bound β-D-galactosidase-cyclic CMP conjugate was separated from that of free with a second antibody, anti-rabbit immunoglobulin G, that was immobilized to a polystyrene ball. Then, activity of the enzyme on the solid phase was fluorometrically determined. When cyclic CMP contents in biological materials were estimated, acid extracts were partially purified by Dowex 1 × 8 formate column chromatography. The present immunoassay allows the detection of as little as 0.5 fmol of cyclic CMP with practically no interference from other cyclic nucleotides and cytidine analogs. By use of the enzyme immunoassay technique, we determined the amounts of cyclic CMP in various tissues of rats. They were found to be as little as 0.24∼0.51 pmol/g wet weight, which was roughly 3000 to 20,000 and 100 to 500 times less than those of cyclic AMP and cyclic GMP, respectively.