Radioimmunoassay for the Measurement of Adenosine 3‧,5‧-CYCLIC Phosphate

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 AMP 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 AMP was prepared by synthesizing succinyl cyclic AMP tyrosine methyl ester (SCAMP-TME) and iodinating the phenolic hydroxyl group of the tyrosine moiety with ¹²⁵I. 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 ¹²⁵I-SCAMP-TME 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 ¹²⁵I-SCAMP-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.