Synthesis of hapten for immunoassay of chlorpyrifos pesticide

Abstract

The production of chlorpyrifos specific antibodies requires presence of a conjugated form of the pesticide to develop an immune response in animals. Thus in turn demands the design of an appropriate chemical structure (a hapten) which can be covalently coupled to a carrier protein. Chlorpyrifos being a low molecular weight compound (M.W.350.4 gm) which is below the threshold for a molecule to be antigenic so, unable to elicit an effective immune response to generate anti chlorpyrifos antibodies. Hence to make it antigenic it needs covalently linkage with suitable carrier molecules. This was carried by replacing chlorine atom at 6th position of pyridyl ring in the chlorpyrifos molecule with thiopropanate group. Characterization of prepared hapten by thin layer chromatography (TLC) showed the appearance of two bands one for standard chlorpyrifos having 0.67 Rf value and an another (unknown) band for chlorpyrifos antigen showing the attachment of thiopropanate group with 0.41Rf value. It was further analyzed by nuclear magnetic resonance (NMR) and spectral data of hapten was recorded in terms of peaks at 400.13 Mhz. NMR study indicated the highest peak with 7.94Mhz representing the aliphatic chain with sulphur group and showing the presence of thiopropanate group thereby confirming the formation of hapten. While the other small peaks with 4.39, 4.14, 3.36, 1.41 and 1.39 Mhz values represent benzene groups in the NMR spectrum. After that hapten containing active carboxyl group was then coupled with carrier protein i.e. BSA for its further usage as antigen.