RADIOIMMUNOASSAYS FOR CYCLIC-AMP CROSS-REACT WITH PHOSPHODIESTERASE INHIBITORS AND BUFFER COMPONENTS

We addressed the issue of cross-reactivity of several commonly used ph osphodiesterase inhibitors with radioimmunoassays for cyclic AMP, afte r we had observed a considerably high cross-reactivity with a noncomme rcial antibody. Theophylline, pentoxifylline, penthydroxifylline (BL 1 94), albifylline (HWA 138), torbafylline (HWA 448), A 80 2715, isobuty l methylxanthine, and the nonmethylxanthines amrinone and rolipram wer e dissolved in supplemented and boiled cell culture medium (RPMI 1640) . These samples were assayed for apparent cyclic AMP in two different, commercially available radioimmunoassay kits (based on polyclonal ant ibodies), applying the nonacetylated protocol. Cross-reactivity was do se-dependent and nonlinear. Samples containing theophylline and amrino ne exhibited the strongest cross-reactivity in assay A (NEN/DuPont): 3 .0 +/- 0.5-nM and 2.4 +/- 1.1-nM apparent cyclic AMP +/- SD at 1-mM sp ike, respectively. With the more sensitive assay B (Amersham), higher concentrations of apparent cyclic AMP were detected: from 7.9 +/- 0.4 nM (for albifylline) to 3.5 +/- 0.1 nM (for rolipram). Values were cal culated from standard curves set up in the respective assay buffer, wh ere culture medium controls resulted in 1.8 +/- 0.3 nM and 3.1 +/- 0.1 nM for assay A and B, respectively. The culture medium interference i ncreased with rising cyclic AMP concentrations. Although comparatively low, this degree of cross-reactivity is relevant for in vitro experim ents. Phosphodiesterase inhibitors are commonly administered at millim olar concentrations, and resulting cyclic AMP levels are often in the nanomolar range. Neglecting these findings may lead to falsely high re adouts of cyclic AMP concentrations.