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HIGHLY SENSITIVE ASSAY FOR ANTICHOLINESTERASE COMPOUNDS USING 96 WELL PLATE FORMAT
Citation:
Mishra, N N., J. A. Pedersen, AND K R. Rogers. HIGHLY SENSITIVE ASSAY FOR ANTICHOLINESTERASE COMPOUNDS USING 96 WELL PLATE FORMAT. Chapter 16, M. L. Phillips (ed.), Chemicals in the Environment: Fate, Impacts, and Remediation. American Chemical Society, Washington, DC, , 289-305, (2001).
Impact/Purpose:
The overall objective of this task is to develop scientifically sound sampling and bioanalytical approaches for screening and monitoring of hazardous wastes. These techniques are expected to provide the Agency with improved screening and field portable methods to characterize, reduce, and control risk to human health and the environment. Specific objectives will include development and characterization of the following concepts:
SPMDs for passive accumulation of TICs
Bioassays for toxic and genotoxic compounds
MIPs for volatile and semivolatile toxic organics
Rapid screening assays using the previously listed components.
Description:
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.