A SOLID-PHASE RADIOIMMUNOASSAY FOR CYCLIC-AMP

A solid-phase radioimmunoassay for cAMP in tissues, body fluids, and c ultured cells has been developed using I-125-2'-O-monosuccinyl adenosi ne-3':5'-cyclic monophosphate tyrosyl methyl ester and High Binding EI A microtiter strips coated overnight at 4 degrees C with a rabbit or s heep polyclonal anti-cAMP antibody. After washing and blocking of well s, samples or standards were added, followed by the addition of radiol abel. Bound I-125-cAMP was separated from free by washing with phospha te buffer containing Tween 20. Bound I-125- cAMP was inversely proport ional 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. Recover ies 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 bot h antibodies. The cAMP content of tissues (brain > heart > kidney, liv er > muscle) from rat, rabbit, and guinea pig, cultured rat lymphocyte s from three lymphoid tissues, and human serum and urine were tested. This solid-phase RIA is a reliable, sensitive, rapid, and relatively i nexpensive method for determination of cAMP.