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

• Published in: Analytical biochemistry Vol. 421; no. 1; pp. 130 - 137
• Main Authors: Luo, Yihui, Xia, Yuxian
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.02.2012
• Subjects: Amino Acid Sequence; Animals; Antibody Affinity; Antibody Specificity; Base Sequence; Biosensing Techniques - methods; clones; complementary DNA; cross reaction; Cross Reactions; cucumbers; dissociation; DNA, Complementary - genetics; enzyme-linked immunosorbent assay; Enzyme-Linked Immunosorbent Assay - methods; Escherichia coli; Female; Fenitrothion; Fenitrothion - analysis; Fenitrothion - immunology; Food Contamination - analysis; genes; Haptens - immunology; Humans; Immunoassay; inhibitory concentration 50; Insecticides - analysis; Insecticides - immunology; Mice; Mice, Inbred BALB C; Molecular Sequence Data; monoclonal antibodies; Organophosphorus pesticide; Oryza sativa; parathion-methyl; Peptide Library; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Ribosome display; ribosomes; Ribosomes - immunology; rice; Sequence Homology, Amino Acid; Single-Chain Antibodies - genetics; Single-chain variable fragment; Single-chain variable fragment; Immunoassay; Organophosphorus pesticide; Fenitrothion; Ribosome display
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2011.10.044

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.