A simplified procedure for cyclic nucleotide radioimmunoassay and its application to human blood leukocytes.
Rapid treatment of leukocyte suspensions was found to be an effective alternative to acid treatment in the preparation of these cells for radioimmunoassay of cyclic GMP and cyclic AMP. A 2-sec heating to boiling temperature, followed by sonication and micropore filtration, was employed. This procedure adequately inactivated or removed enzymes and binding proteins that can alter cyclic nucleotide concentrations or otherwise interfere with the radioimmunoassay. Moreover, heating in this manner did not appear to affect the stability of cyclic nucleotides or cause significant formation of cyclic GMP from endogenous GTP. Recovery of cyclic nucleotides after heating and filtration was high (89-96%), making possible the measurement of both cyclic GMP and cyclic AMP in small cellular samples. Variation in cyclic nucleotide recovery was small (+/- 2-4% SD), therefore individual recovery determinations were unnecessary.
D001798:Blood Proteins; D055598:Chemical Phenomena; D002621:Chemistry; D005374:Filtration; D006160:Guanosine Triphosphate / Q000097:blood; D006358:Hot Temperature; D006801:Humans; D009504:Neutrophils / Q000032:analysis*; D009712:Nucleotides, Cyclic / Q000097:blood*; D011489:Protein Denaturation; D011863:Radioimmunoassay / Q000379:methods
D001798:Blood Proteins; D009712:Nucleotides, Cyclic; D006160:Guanosine Triphosphate