Enzyme immunoassays of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate using acetylcholinesterase

Abstract

The pure tetrameric form of acetylcholinesterase (EC 3.1.1.7) from the electric eel Electrophorus electricus has been covalently coupled to 2'-O-succinyl-cAMP tyrosine methyl ester and 2'-O-succinyl-cGMP. Both enzymatic conjugates have been used as tracers in a classical heterogeneous competitive enzyme immunoassay allowing the determination of cAMP and cGMP, respectively. The test was performed in 96-well microtiter plates coated with a mouse monoclonal anti-rabbit Immunoglobulin antibody in order to ensure separation between bound and free moieties of the tracer. Acetylcholinesterase activity bound to the solid phase was measured by colorimetric assay. When standards or samples were first acetylated by treatment with acetic anhydride, the sensitivity of both assays appeared very good since minimum detectable concentration close to 0.04 pmol/mL (2 fmol/well) could be calculated for each assay. Precision was also very satisfying since the coefficient of variation was less than 5% in the 0.2-10 pmol/mL range. Good correlation was noted between enzymoimmunological and radioimmunological measurements of cAMP performed for different biological samples (urine, serum, or tissue extracts).