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Expression of an anti-Pseudomonas aeruginosa lipopolysaccharide core recombinant antibody in Escherichia coli

Department of Microbiology, College of Biological Sciences, University of Guelph, Guelph, ON NIG 2W1, Canada

S.R.C. Malburg

Department of Microbiology, College of Biological Sciences, University of Guelph, Guelph, ON NIG 2W1, Canada

J.S. Lam

Department of Microbiology, College of Biological Sciences, University of Guelph, Guelph, ON NIG 2W1, Canada

Antibodies against Pseudomonas aeruginosa LPS are generally protective, however, this protection is usually serotype-specific. Thus the generation of antibodies against the more conserved core-lipid A epitopes has a potential for being more broadly cross reactive. Our laboratory has previously produced several monoclonal antibodies (mAb) against the core region and lipid A region of P. aeruginosa LPS. In this study, we cloned the immunoglobulin genes from mAb 7-4, an antibody with specificity for the inner core region of P. aeruginosa LPS. VH and VL genes of 7-4 were cloned into both of the M13-derived phagemid vectors, pComb3 and pComb8. 14 pComb3/VH/VL and 6 pComb8/V_H /V_L recombinant clones were isolated. The presence of recombinant F(ab) molecules in the periplasmic extracts of Escherichia coli was confirmed by Western immunoblots of these extracts with goat anti-mouse F(ab')2 and goat anti-mouse kappa light chain antibodies under non-reduced conditions. Recombinant 7-4 antibody also interacted with LPS prepared from a rough mutant P. aeruginosa strain AK43 in Western immunoblotting, and ELISA as well as with whole cells of AK43 in immunofluorescence. Thus, recombinant 7-4 antibodies expressed in E. coli are structurally and functionally correct.

Journal of Endotoxin Research, Vol. 2, No. 1, 53-61 (1995)
DOI: 10.1177/096805199500200107


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