4 new type of radioimmunoassay of cyclic AMP is described; it consists in taking into account the remarkable affinity of anticyclic AMP antibodies for 2'-0-succinyl derivatives of cyclic AMP, loo-fold higher than that for cyclic AMP itself. The samples to assay are submitted to a simple and rapid treatment which converts cyclic AMP into 2'-O-succinyl cyclic AMP with a 109% yield. Then, the radioimmunoassay is performed by equilibrium dialysis. The sensitivity is lOO-fold increased, as compared to that of the usual radioimmunoassay.
The specificity and the reproducibility are also improved. lo-l5 Mole cyclic AMP is routinely assayed, the minimum detectable being under 10." mole.
-4mong the various methods used for the assay of cyclic AMP (l-8) the most popular ones are those based upon kinase inhibitors such as Gilman protein (6) and antibodies which were first developed by Steiner et al. (8,9). These two methods are limited by the dissociation constant with cyclic AMP, which lies around 10ms M in both cases. The antibody produced against cyclic AMP-succinylalbumin as an immunogen, shows a greatly enhanced affinity for 2'0-succinyl cyclic AMP. The dissociation constant now reaches 8 X lo-l1 M (10). Therefore, the succinylation of cyclic AMP on its 2'0 position will theoretically be sufficient to increase the sensitivity of the radioimmunoassay by a factor of at least 100. In the present work, we describe the succinylation reaction and its application to tissue extracts.
Methods
Material. The anti-3',5' cyclic AMP antibody used for the radioimmunoassay consist,ed of a dilution of a rabbit immune serum previously studied (11)) produced in the laboratory of Pr R. Depieds, CHU-Nord, Marseille. 2'-0-succinyl cyclic AMP (SCAMP) and 2'-0-succinyl cyclic AMP tyrosine methyl ester iodinated by lz51 (lz51SCAMPTME) were prepared in our laboratory as described before (12).
Uniformly labeled ['"Cl 3',5' cyclic AMP (117 mCi/mmole) was 394