Immunoassays for avian butyrylcholinesterase: Implications for ecotoxicological testing and clinical biomonitoring

Limitations of using the cholinesterases as biomarkers for assessing organophosphate (OP) pesticides impact on avian wildlife are a matter of concern for both toxicologists and environmentalists. One limitation appears to be the assumption of proportionality between enzyme activity and enzyme protein content. However, this proportionality does not hold during exposure to OP pesticides which can cause changes in both factors. Therefore, RIA and ELISA were developed to investigate the relationship between BChE enzyme mass and activity during exposure to OP pesticides in birds. The sensitivity of RIA was 0.60 ng protein/ml and of the competitive ELISA was 0.15 ng protein/ml. A third enzyme-antigen immunoassay (EAIA) for measuring BChE catalytic activity was also developed to increase the specificity of cholinesterase assays by immunoprecipitation. Results also showed that although exposure of birds to malathion produced 47% (±6%) decrease in serum BChE activity, this inhibition was associated with a statistically significant (p < 0.0001) increase in immunoreactive concentration of BChE in all exposed birds. The increased BChE concentration observed in the present study while its activity continued to be inhibited will diminish the apparent overall inhibition of the enzyme. Hence, the degree of exposure to OP pesticides will be underestimated if enzyme activity is the only biomarker used. There is now a case for measuring the “actual” specific activities of serum esterases as a rational diagnostic tool. This approach requires the simultaneous measurement of enzyme mass and enzyme activity.