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 IC50 values of 1.6, 3.4, and 2.2 ng/ml, respectively. Cross-reactivity with other organophosphonis (OP) pesticides was below 0.1% except for parathion-methyl (<= 2.8%). The IC50 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 x 10(-10)M for ScFv-AF50, 1.42 x 10(-9) M for ScFv-AF93, and 2.66 x 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. (C) 2011 Elsevier Inc. All rights reserved.