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

Immunotherapy with antibodies (Abs) against the lipopolysaccharide (LPS) of Pseudomonas aeruginosa remains an alternative to serotype-specific LPS-based vaccines due to their limited use and to antibiotics due to the intrinsic resistance to antimicrobials observed in P. aeruginosa. We have chosen a monoclonal Ab (MAb), MF23-1, that binds to the O antigen of the most clinically relevant serotype, IATS O6, for producing a recombinant antibody. Heavy (H) and light (L) chain genes were isolated from MF23-1 to form a functional Fab molecule in the periplasm of Escherichia coli and on the surface of phage by using phagemid vector pComb3. The entire L chain gene was used, but the H chain gene was amplified to 2 amino acids past cysteine 128 which is involved in interchain disulfide bond formation with the L chain. The truncated H chain associated with the L chain in the periplasm of E. coli to form a functional Fab molecule that bound in both enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay to O6 LPS. Therefore, the remainder of the C H 1 past cysteine 128 is not essential for stable formation of the Fab portion of MF23-1. This recombinant Fab (r-Fab) was shown to be specific for the LPS of the most predominant clinical isolate, serotype O6, while no cross-reactivity was detected to the LPS of the other 19 remaining serotypes. This r-Fab was also expressed on the surface of filamentous phage upon addition of helper phage to recombinant E. coli containing phagemid. Recombinant phage from clones MT13 and MT24 bound specifically to O6 LPS in ELISA. These results represent an important step toward the design of therapeutic Abs to be used against P. aeruginosa infections.

The molecular nature of the murine Ab response to Pseudomonas aeruginosa LPS was examined using a panel of 10 well-defined anti-LPS mAbs. Abs to P. aeruginosa LPS are encoded by diverse V-genes, with at least five VH and four V kappa gene families represented in these Abs. The Abs that bind to hydrophilic O-polysaccharide side chains of B-band LPS and A-band LPS are encoded by VH J558, SM7, and J606 gene families, while Abs to hydrophobic core and lipid A regions are encoded by X24, SM7, and Q52 gene families. All active JH and only two J kappa (J kappa 2 and J kappa 5) germ-line genes are utilized in the anti-LPS Abs examined. Four of six anti-P. aeruginosa mAbs used diversity genes of the DSP2 gene family. Interestingly, JH1 and JH2 use was observed in three mAbs that reacted with hydrophilic LPS epitopes (O-polysaccharide, A-band LPS), whereas JH3 and JH4 use was observed in three mAbs that bound to the more hydrophobic regions of LPS (core, lipid A). Point mutations were observed in framework and complementarity-determining regions (CDRs) of VH and VL genes, suggesting an Ag-driven maturation process in response to P. aeruginosa LPS. Mutations occurred in all heavy chain CDRs, as well as in CDR1 and CDR3 of the light chain, indicating an important role of these regions in binding to LPS. These data suggest that diverse VH and V kappa genes encode Abs to LPS from P. aeruginosa.