Bacterial Expression and Characterization of a Picloram-Specific Recombinant Fab for Residue Analysis

Abstract

Complete κ-light chain and VH-CH1 (Fd) genes were cloned by PCR from cDNA synthesized from RNA isolated from a picloram (4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid)-specific hybridoma cell line. Both genes were cloned into the phagemid vector pComb3 for expression of soluble Fab. Extracts from the periplasmic space of recombinant Escherichia coli expressing the Fab exhibited specificity to picloram with an IC50 of 50 ng/mL, as determined by competitive indirect ELISA. This value was comparable to that obtained when using the parent monoclonal antibody (IC50 = 20 ng/mL). Two different matrices, river water and soil extract, did not interfere with the sensitivity and specificity of the assay. Cross-reactivity was detected to the pyridine herbicide clopyralid (IC50 > 30 μg/mL) but not to the pyridine herbicides triclopyr and fluoroxypyr. A single-chain variable fragment was constructed with the same variable chain sequences, but no specific activity to picloram was detected. The soluble Fab was found to be a suitable recombinant antibody fragment for the purpose of quantifying picloram in environmental samples. Keywords: Competitive indirect ELISA; monoclonal antibody; recombinant Fab; herbicides; picloram