Group-specific enzyme-linked immunosorbent assay for fenitrooxon and 3-methyl-4-nitrophenol in water samples based on a polyclonal antibody

Abstract

Fenitrooxon [ O,O-dimethyl- O-(4-nitro- m-tolyl)phosphate] is the major metabolite of the organophosphorus insecticide fenitrothion, and 3-methyl-4-nitrophenol is its major degradation product. In the present study, we describe the development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of these compounds in water samples based on a group-specific polyclonal antiserum generated with a “bifunctional hapten”, which has two functions: the conventional function of producing an antibody against an antigen and a unique function of promoting the production of the antibodies in rabbit. For application to water samples, the influence of several factors such as organic solvent, pH, and detergent was studied. Under optimized conditions, the quantitative working range of the fenitrooxon ELISA was 0.71–27 ng ml −1 with a limit of detection (LOD) of 0.32 ng ml −1, and the fenitrooxon concentration giving 50% reduction of the maximum signal (IC 50) was 4.2 ng ml −1. The quantitative working range of the 3-methyl-4-nitrophenol ELISA was 0.67–27 ng ml −1 with a LOD of 0.38 ng ml −1 and an IC 50 of 3.7 ng ml −1. No significant matrix effect originating from the water sample (river water, tap water, purified water, and bottled water) was shown by addition of Tween 20 to the assay buffer. Water samples spiked with each of these compounds at 1, 5, 10, and 20 ng ml −1 were directly analyzed without extraction and clean-up by the proposed ELISA. The mean recovery was 100.9%, and the mean coefficient of variation (CV) was 7.7% for the fenitrooxon ELISA and for the 3-methyl-4-nitrophenol ELISA, the mean recovery was 97.6%, and the mean CV was 7.2%. The proposed ELISA allows precise and accurate determination of these compounds in water at such low levels.