A modification of a protein-binding method for rapid quantification of cAMP in cell-culture supernatants and body...

A modification of a protein-binding method (B. L. Brown et al., 1971, Biochem. J. 121, 561, 562) for measurement of adenosine 3',5'-cyclic monophosphate (cAMP) in cell-culture supernatants and urine is described. With filtration over glass-fiber filters and a semiautomatic cell harvester instead of charcoal precipitation as in the original method, free [3H]cAMP tracer was rapidly and uniformly separated from that which was protein-bound. Sensitivity was increased with a high ionic strength buffer and a tritiated cAMP tracer with high specific activity. There was good agreement between cAMP values obtained with the protein-binding method and commercially available radioimmunoassays that were used as reference methods. cAMP could be determined accurately over the range 0.15-8.0 pmol/sample. More than 500 samples could be assayed in duplicate or triplicate in less than 6 h.