WESTERN BLOT ANALYSIS OF ORGANOPHOSPHATE EXPOSURE

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Organophosphate pesticides are neurotoxins with a high potential for human exposure due to their widespread use on fruit, vegetable and cotton crops. The acute health effects result from inhibition of acetylcholinesterase (AChE). Workers exposed to organophosphates are routinely monitored for AChE activities. This project will develop a Western blotting assay to detect erythrocyte AChE inhibition. This assay will simultaneously measure inhibited and native enzyme (IN assay) eliminating the problems encountered in those that measure only enzyme activity. AChE levels differ both between people and in successive measurement taken from the same individual. These variabilities necessitate the use of pre-exposure measurements and limits the ability to detect low levels of inhibition. The internal reference incorporated into the IN assay will permit direct measurements of inhibition and eliminate the need for comparison with earlier measurements. The IN assay will separate native and inhibited AChEs by electrophoresis and transfer the enzymes to a membrane where they will be detected with an immunoenzyme procedure using murine anti-human AChE monoclonal antibodies. The goal of optimization is to produce an assay able to detect inhibition levels of 2% or less. Once optimized, the impact of different organophosphates on the measured inhibition will be evaluated by comparing the results obtained with the IN assay with those from the Ellman's assay, which is the standard method for measuring AChE activities. The effect of the IN assay's internal control will be assessed by measuring the intraindividual, interindividual and analytical variabilities found in the assay and comparing them with the variabilities found in the Ellman's assay. Blood taken from a cohort of five people drawn weekly for five weeks and inhibited in vitro with diisopropylfluorophosphate will be used. Finally, the inhibition measured by the two assays will be compared using 12 samples from organophosphate exposed workers obtained from a clinical laboratory in Fresno, CA. The IN assay will help NIOSH fulfill its goals in two ways. 1) It will facilitate study of neurotoxic disorders in workers exposed to low levels of organophosphates. 2) The internal control will permit accurate measurements of AChE monitoring a more comprehensive early warning technique.