Quantitative kinetic assays for glutathione S-transferase and general esterase in individual mosquitoes using an EIA reader

Abstract

An enzyme immunoassay (EIA) reader capable of making kinetic determinations of change in absorbance in the visible and near u.v. spectrum was evaluated for monitoring enzyme activity in individual mosquitoes and compared to a research spectrophotometer. The instrument compared favorably, and appears especially suitable for the small tissue samples common in insect biochemistry. It is important for several assays that the instrument can reach the near u.v. Specifically, kinetic assays for alcohol dehydrogenase (ADH), glutathione S-transferase (GST) and general esterase (EST) enzyme activity were developed. The latter two assays were adapted for individual mosquitoes using 1 10 of a mosquito for each replicate. Extinction coefficients for GST and EST were linearly related to well volume. Both GST and EST, as well as protein concentration could be measured in a single individual mosquito. A nested analysis of variance of the GST and protein results for Aedes aegypti showed that most of the variability in the assay (≥72%) is due to variability among individuals. Other sources of error were caused by day to day variability in the assay technique. EST, GST and protein were measured in individuals from field and lab populations of Culex tarsalis. There was no significant difference in GST activity among field and laboratory individuals. EST activity however, was as much as 15 times greater in some field individuals compared to the laboratory population. The role of EIA readers in biochemistry laboratories in general, and in the study of resistance is discussed.