A solid-phase radioimmunoassay for cyclic AMP.

A solid-phase radioimmunoassay for cAMP in tissues, body fluids, and cultured cells has been developed using 125I-2'-O-monosuccinyl adenosine-3':5'-cyclic monophosphate tyrosyl methyl ester and High Binding EIA microtiter strips coated overnight at 4 degrees C with a rabbit or sheep polyclonal anti-cAMP antibody. After washing and blocking of wells, samples or standards were added, followed by the addition of radiolabel. Bound 125I-cAMP was separated from free by washing with phosphate buffer containing Tween 20. Bound 125I-cAMP was inversely proportional to cAMP in samples or standards. Cyclic AMP content of unknowns was calculated from a standard curve run concurrently with each assay. Both antibodies showed sensitivity of approximately 1 fmol, an assay range between 15 and 1,000 fmol, a maximum displacement ratio of up to 11-12, and no cross-reactivity with other cyclic nucleotides. Recoveries were 86.5%-106.8%, intraassay coefficients of variation were 2.4%-6.0%, and interassay coefficients of variation were 7.4%-10.2% for both antibodies. The cAMP content of tissues (brain > heart > kidney, liver > muscle) from rat, rabbit, and guinea pig, cultured rat lymphocytes from three lymphoid tissues, and human serum and urine were tested. This solid-phase RIA is a reliable, sensitive, rapid, and relatively inexpensive method for determination of cAMP.