A microassay-based procedure for measuring low levels of toxic organophosphorus compounds through acetylcholinesterase inhibition

Using a microtiter plate spectrophotometric system, an assay procedure was developed for the following toxic organophosphorus compounds: 1,2,2-trimethylpropyl ester of methylphosphonofluoridic acid (1, soman); ethyl N,N-dimethylphosphoramidocyanidate (3, tabun); O-ethyl S-2-bis(1-methylethyl)amino]ethyl]- methylphosphonothiolate (4, VX); the diethyl 4-nitrophenyl ester of phosphoric acid (5, paraoxon); and bis(1-methylethyl) phosphorofluoridate (6, DFP). The procedure, based on the Ellman assay method, uses inhibition of eel acetylcholinesterase (0.01 unit per well) to carry out the determination of inhibitor concentrations for both a standard curve and the unknown samples on a single 96-well microtiter plate. On a typical plate, samples of both unknowns and standards (a minimum of six concentrations were used per standard curve) were assayed five times per sample, with three control (uninhibited) enzyme activity points included for each sample. The time required for carrying out a single plate was approx 30 min. Sensitivity for the most potent acetylcholinesterase inhibitor tested was 0.4 nM under the conditions used for a typical assay. It should be noted, however, that no attempt was made to optimize the assay procedure for sensitivity.