Abstract
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°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 125IcAMP 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.
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