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Highly Sensitive Assay for Anticholinesterase Compounds Using 96 Well Plate Format

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Abstract: The rapid and sensitive detection of organophosphate insecticides using a 96 well plate format is reported. Several features of this assay make it attractive for development as a laboratory-based or field screening assay. Acetylcholinesterase (AChE) was stabilized in a gelatin film. The remarkable properties of the dry immobilized AChE preparation include its stability to prolonged storage at room temperature as well as its stability to short term elevated temperatures (60 C). The enzyme could be maintained in dry gel form for 365 days at room temperature without substantial loss of activity. The absorbance assay used to measure enzyme activity was evaluated using several solvent systems including water, phosphate buffer, hexane, methanol and ethanol. The AChE activity in hexane was similar to that in the aqueous systems, however, the more polar organic solvents such as methanol and ethanol decreased the enzyme activity by about 90%. The microwell assay includes a procedure to oxidize less potent P=S organophosphate compounds to their more inhibitory oxon forms. Log-logit or four parameter equations were applied to the data to determine IC50 (molar concentration yielding 50% inhibition) values. The use of this assay to analyze field samples contaminated with mixtures of organophosphates is also reported.

The U.S. Environmental Protection Agency (EPA), through its Office of Research and Development (ORD), funded this research and approved this manuscript for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation of these products by the EPA.
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Citation:Mishra, N. N., J. A. Pedersen, and K. R. Rogers. Highly Sensitive Assay for Anticholinesterase Compounds Using 96 Well Plate Format.M. L. Phillips (ed.), Chemicals in the Environment: Fate, Impacts, and Remediation, Chapter16. American Chemical Society, Washington, DC, (2001).
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Contact: Liz Hope - (919) 541-2785 or hope.elizabeth@epa.gov
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Division: Human Exposure & Atmospheric Sciences Division
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Branch: Human Expsoure Research Branch
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Product Type: Book Chaptr
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Published: 12/31/2001
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Bullet Item Biosensors Research for Development of Innovative Monitoring Techniques That Support Exposure Assessment Related to the Superfund Program
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Last Updated on Monday, October 22, 2007
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