Enzyme immunoassay of cyclic adenosine 5', 5' - monophosphate (AMP) using beta-D-galactosidase as label

Succinyl cyclic adenosine monophosphate (AMP) was synthesized and coupled to beta-D-galactosidase using 1-ethyl-3-(3-dimethylaminopropyl)carbodimide. An antiserum to cyclic AMP was raised in rabbits by immunization with a succinyl cyclic AMP-human serum albumin conjugate. An enzyme immunoassay of cyclic AMP was successfully performed via the competitive binding procedure using the succinyl cyclic AMP-enzyme conjugate and the antiserum to cyclic AMP. The sensitivity of this assay has been increased several hundredfold by prior 2'-O-succinylation of cyclic AMP as already described by Cailla et al. in the radioimmunoassay of cyclic nucleotides. The assay system makes it possible to ascertain values as low as 6 fmol of cyclic AMP/tube. Human plasma cyclic AMP could be accurately determined by this method without requiring a deproteinizing reagent as the first step of assay. The concentration of TCA-extracted cyclic AMP from various tissues of mice was determined by both enzyme immunoassay and radioimmunoassay. There was a good correlation between the values for cyclic AMP determined by the two methods (Y = 1.01X - 0.15, r = 0.996, n = 39). Succinylated nucleotides and nucleosides such as cyclic GMP, ATP, ADP, and AMP, adenosine, adenine, GTP, GDP, GMP, guanosine, and guanine had no effect on the immunoassay of cyclic AMP.