Use of Affinity Elution Chromatography for the Microassay of Adenosine 3′: 5′‐Monophosphate

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Abstract
Using the principle of affinity elution chromatography, a microassay was developed for the determination of picomolar and even smaller quantities of cyclic[c]AMP in biological samples. In principle, cAMP along with the other anions is adsorbed onto an anion exchange cellulose and subsequently, the cyclic nucleotide is specifically eluted with cAMP-dependent protein kinase. In the actual assay cAMP is determined by competitive displacement of radiolabeled cAMP by the non-labeled cyclic nucleotide. The described procedure results in a linear dependence for the displacement of the radioactive nucleotide on cAMP concentration and obviates the usual procedure of sample enrichment by lyophilization. The degree of interference of different nucleotides including cGMP was minimal, and in this respect the use of affinity elution chromatography was comparable to radioimmunoassay. [Bovine heart 3'':5''-nucleotide phosphodiesterase was used.].