Original Articles

Radioimmunoassay for guanosine 3’,5’-cyclic phosphate (cGMP)

Jing-sheng Liu, Yue-ming Li, Zhen-gang Wang, Xue-bin Wang, Pei Qi, Ci-hui Jiang, Gui-fang Cheng, Xing-guo Chen


Since the content of cGMP in tissue fluids is less than 1/10 of that of cAMP, it is difficult to measure cGMP with general protein binding competitive method. A radioimmunoassay was established by us. This method required no purification of the sample, thus making the cGMP measurement as easy as that of cAMP.
cGMP was synthetized by us. Purified cGMP solution was mixed with succinyl anhydrideto form 2’-O-ScGMP. By passing through QAE-Sephadex A-50 columns, nearly 95% of it was recovered. Then ethyldimethyl aminopropyl carbodiiide-HCl was used to conjugate with hemocyanin to produce an immunogen. Each molecule of hemocyanin was able to conjugate about 80 molecules of cGMP.
Antiserum to cGMP was obtained by multiple subcutaneous injections on the back of rabbits (a total of 1 mg cGMP/rabbit at each immunization). Booster dosage was given every other month. Blood for antiserum was collected on 10-14 day after the last booster dosage. Finally the titre of the antibody in RIA was 1:1400. The specific activity of [3H]cGMP was 22 Ci/mmol.
A typical standard curve was plotted from 0.25-4 pmol/tube, and the sensitivity could reach 0.15 pmol/tube. The cross reactivity to cAMP with cGMP antiserum was not significant.
This method has been applied in several biological researches.

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