BACTERIAL EXPRESSION AND CHARACTERIZATION OF A PICLORAM-SPECIFIC RECOMBINANT FAB FOR RESIDUE ANALYSIS

Complete kappa-light chain and V-H-C-H1 (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. Bo th genes were cloned into the phagemid vector pComb3 for expression of soluble Fab. Extracts from the periplasmic space of recombinant Esche richia 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 monoclona l antibody (IC50 = 20 ng/mL). Two different matrices, river water and soil extract, did not interfere with the sensitivity and specificity o f the assay. Cross-reactivity was detected to the pyridine herbicide c lopyralid (IC50 > 30 mu g/mL) but not to the pyridine herbicides tricl opyr and fluoroxypyr. A single-chain variable fragment was constructed with the same variable chain sequences, but no specific activity to p icloram was detected. The soluble Fab was found to be a suitable recom binant antibody fragment for the purpose of quantifying picloram in en vironmental samples.