Selection of single-chain variable fragment antibodies against fenitrothion by ribosome display.

A single-chain variable fragment (ScFv) complementary DNA (cDNA) library against fenitrothion was constructed, and ScFvs specific for fenitrothion were selected by ribosome display from the library. After three rounds of ribosome display, the ScFv genes were cloned into Escherichia coli for expression. The expressed ScFvs of 160 clones were analyzed by indirect enzyme-linked immunosorbent assay (ELISA). Of these, 40 clones produced antibodies with relatively high activity against fenitrothion, and 3 were selected for Biacore and ELISA analysis. These 3 antibodies-ScFv-AF50, ScFv-AF93, and ScFv-AF132-had IC(50) values of 1.6, 3.4, and 2.2 ng/ml, respectively. Cross-reactivity with other organophosphorus (OP) pesticides was below 0.1% except for parathion-methyl (≤2.8%). The IC(50) values and cross-reactivity were lower than achieved previously with polyclonal or monoclonal antibodies against fenitrothion. The equilibrium dissociation constant (K(D)) values determined by Biacore analysis were 4.56×10(-10)M for ScFv-AF50, 1.42×10(-9)M for ScFv-AF93, and 2.66×10(-10)M for ScFv-AF132. These results demonstrate that the ribosome display has great potential in selection of ScFvs against pesticides. Recoveries of fenitrothion from fortified rice and cucumber were in the range 80.6 to 108%, indicating that the ELISAs with the isolated ScFvs can accurately determine fenitrothion in food samples after the simple and rapid extraction procedure.

Enzyme-linked immunosorbent assays for the insecticide fenitrothion. Influence of hapten conformation and sample matrix on assay performance.

This study aimed at developing competitive enzyme-linked immunosorbent assays (ELISAs) for the organophosphorus (OP) insecticide fenitrothion using a monoclonal antibody. The hapten used to obtain the antibody had an ideal structural feature that allowed minimal functional group sacrifice. By using the antibody and a coating antigen, a competitive indirect ELISA was developed, which showed an IC50 of 14 ng mL(-1) with a detection limit of 3.0 ng mL(-1). A competitive direct ELISA using an enzyme tracer was also developed, which showed an IC50 of 17 ng mL(-1) with a detection limit of 1.6 ng mL(-1). The antibodies in both assays showed negligible cross-reactivity with the metabolites of fenitrothion and other OP pesticides except with the insecticides parathion-methyl and parathion-ethyl. Recoveries of fenitrothion from fortified rice and lettuce samples were determined and the bias in the recovery values was rationalized by using the standard curves obtained in the matrix extract.