Enzyme-linked immunofiltration assay used in the screening of solid supports and immunoreagents for the development of an azinphos-methyl flow immunosensor

Abstract

Azinphos-methyl (AM), O,O-dimethyl S-[(4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl] phosphorodithioate, is a dithiophosphorous insecticide extensively used for the control of fruit culture pests. In this work the ELIFA system, initially developed and marketed to substitute conventional ELISA methods, was used for the screening of supports and immunoreagents in the development of a flow immunosensor to AM. The objective was to find the optimal antibody concentration, support quantity and enzymatic tracer concentration to develop a sensitive and reusable immunosensor. The influence of chitosan as protein stabilizing agent was also investigated. We observed that, on the basis of immunosorbent characterization, chitosan-modified silica with immobilized LIB-MFH14 monoclonal antibody (MAb) showed the best sensitivity, with a I50 value of 6 nM AM. All of the immobilized MAbs either in alkylaminated or chitosan-modified silica showed I50 values between 10 and 36 nM. Regarding the regeneration capability, the best desorption agent tested was 0.1 M glycine/HCl, pH 2.0, performing in most cases a 100% desorption after just one wash and maintaining the antibody activity even after 20 cycles of regeneration. The chitosan-modified silica seemed to be the best support for this purpose.