Radioimmunoassay for the Measurement of Adenosine 3′,5′-Cyclic Phosphate

Abstract

Abstract.-A sensitive and specific radioimmunoassay for adenosine 3',5'-cyclic phosphate (cyclic AMP) has been developed which allows measurement of the nucleotide in extracts of 5-10 mg of tissue. The radioimmunoassay is sufficiently specific for cyclic AMP to eliminate the need for prior chromatographic separation of the cyclic nucleotide from other tissue nucleotides. The radioimmunoassay system is based upon competition of cyclic AMP with a labeled cyclic AMP derivative of high specific activity for binding sites on an antibody specific for the cyclic nucleotide. Antibody to cyclic Ai\IP was obtained by immunizing rabbits with an antigen prepared by conjugating succinyl cyclic AMP with human serum albumin. A high specific activity derivative of cyclic AAMP was prepared by synthesizing succinyl cyclic AMP tyrosine methyl ester (SCAMP-TME) and iodinating the phenolic hydroxyl group of the tyrosine moiety with "25I. Free and antibody-bound 125I-SCAMP-TME were separated by precipitation of the antibody-bound fraction with a second antibody (goat anti-rabbit gamma globulin). Displacement of 1251-SCAMP-T]NIE by unlabeled cyclic AMP when plotted as a semilogarithmic function was linear over a concentration range of 2-100 picomoles. The specificity of the antibody was tested against structurally related nucleotides, nucleosides, and purine bases. All had less than 0.005 per cent of the potency of cyclic AMP in inhibiting 125IJ SCAMIP-TME binding. The marked differences in affinity of the various cyclic nucleotides to cyclic AMP antibody would suggest that antibodies can be developed for each of the cyclic nucleotides by the principles used in this work. Adenosine 3',5'-cyclic phosphate (cyclic AMP) mediates a remarkable array of physiological phenomena' 2 and has been identified as a "secondary messenger" in the concept of hormone action proposed by Sutherland and his associates.' The methods available for the measurement of this nucleotide,3 -7 which is present in mammalian tissues in very low concentrations (0.2-1.5 m,4moles/ gm), involve a variety of complex enzymatic procedures36 and require the initial separation of cyclic AMP from other nucleotides by chromatographic techniques. We have developed a radioimmunoassay sensitive to 1-2 picomoles of cyclic AMP which permits rapid measurement of large numbers of samples using as little as 10-20 mg of tissue. The radioimmunoassay is highly specific for cyclic AMP, and therefore eliminates the need for prior chromatographic separation from other tissue nucleotides. This immunoassay is similar to the radioimmunoassay techniques developed for peptide hormones8 I and involves the competition of labeled and unlabeled cyclic AMP for antibody. In the assay to be described, free and antibody-bound cyclic AMP are then separated by precipitation of the latter with a second antibody.

The antitumour agent 5-(3,3-dimethyl-1-triazeno) imidazole-4-carboxamide (DTIC) inhibits rat liver cAMP phosphodiesterase and amplifies hormone effects in hepatocytes and hepatoma cells

“…cAMP was measured by a radioimmunoassay (Steiner et al, 1969) using acetylation of the samples (Harper & Brooker, 1975;Frandsen & Krishna, 1976) and acetylated [3H]cAMP as ligand (Skomedal et al, 1977).…”

Stimulation by Alcohols of Cyclic AMP Metabolism in Human Leukocytes

“…In most experiments the cyclic nucleotides were extracted from cells by boiling and sonicating and cAMP and cGMP were measured by radioimmunoassay as previously described (27)(28)(29). For comparative and confirmative pturposes, in selected experiments cyclic nucleotides were pturified before assay by perchloric acid protein precipitation, heating to 85°C for 3 min, and ion-exchange column chromatography (20).…”

Effects of prostaglandin on substrate uptake and cell division in human diploid fibroblasts

“…After neutralization with KOH, centrifugation, and chromatography on a Dowex 50 column (6mmx 35mm) with water as eluent, the supernatant containing the cyclic AMP fraction was assayed for the cyclic nucleotide content by radioimmunoassay (Steiner et al, 1969).…”