A Microassay Method for Neurotoxic Esterase Determinations

A microtiter plate reader with an associated computer to averagetriplicate samples and subtract blanks was used for readingand calculating neurotoxic esterase (NTE, also known as neuropathytarget esterase) activities in spinal cord regions of hens 4hr after administration of diisopropylphosphorofluoridate (DFP,0.5 mg/kg sc). Although NTE inhibition is an early indicatorof organophosphorus ester-induced delayed neuropathy, DFP-inducedinhibition was not greater in regions of the spinal cord wherepathological changes are most notable. Acetylcholinesterase(AChE) activities and protein determinations were also doneon these tissues using microassay methods. DFP-induced AChEinhibition was similar to NTE inhibition. In addition to thecapability to be used for small regional esterase activity measurements,the microassay was advantageous because the number of samplesincorporated into a single assay was increased and the timeneeded for the NTE assay was reduced by 50%. Total volume ofincubate in each well was 0.3 ml; the incubate contained 1/20quantities of sample and reagents necessary in more conventionalassays. Validation of the microassay was performed by comparisonwith more conventional assays when measuring inhibition of NTEand AChE in brains of control and experimental hens of two differentgenetic strains (B13B13 and B21B21 white leghorns). Experimentalbirds were given DFP, 0.5 mg/kg sc, 24 hr before samples werecollected. NTE activities in brains of control hens were similarusing both types of NTE analytical procedures. Percentage inhibitionof NTE caused by DFP was within 4% using both assay proceduresin both strains of hens. The microassay was sensitive enoughto detect NTE activity in 42 µg of hen brain after 15min of incubation. Hen lymphocytes could also be examined foreffects of organophosphorus esters on NTE activity, with 14.1±2.2 and 8.3 ±2.2 /xmol/15 min/mg protein in 1x10⁶cells measured in samples taken before and 4 hr after administrationof 0.5 mg/kg SC DFP.