Immunoassay for parathion without its prior removal from solution in hexane

Abstract

Immunoaffinity methods are presently finding limited applications in environmental analysis because many target compounds are best handled in non-polar solvents such as hexane. These solvents can be detrimental to protein molecules, particularly when the solvating water which is necessary for bioactivity is lost. Consequently, a generic immunoassay principle which would allow the provision of samples dissolved in hexane was sought. A competitive enzyme-linked immunosorbent assay (ELISA) method for the measurement of aqueous parathion was developed, optimized and then adapted to the measurement of the analyte without its prior removal from hexane. The parathion-specific antiserum was encapsulated in reverse micelles composed of Aerosol OT and having aqueous centres, and a solution of parathion in hexane was then allowed to compete for antibody binding sites with the antigen-coated surface of a microtitre plate. The calibration graph for the hexane-compatible ELISA method is displaced from its aqueous counterpart in correspondence with the partition coefficient of parathion between water and hexane, this method being about 10⁴-fold more sensitive. It is probable that the thermodynamic barrier to the transfer of parathion from an organic bulk phase to the aqueous interior of a reverse micelle balances the antigen–antibody binding strength and thus reduces the potency of parathion as an antigenic competitor. Means are suggested for tuning the calibration graph of the hexane-compatible ELISA method to the analyte concentration range that is of interest.