A Single-domain Antibody Cross-reacts with Lipopolysaccharide from Diverse Gram-negative Bacteria

Abstract

Gram-negative bacterial sepsis and endotoxemia are the leading causes of morbidity and mortality in animals and humans worldwide, and attempts have continued to design rational therapies. Endotoxin/lipopolysaccharide (LPS)-neutralizing polyclonal and monoclonal antibodies have been produced, but are not free from problems. Single-domain antibodies (dAbs), constructed from the camelid and the shark ‘heavy chain antibodies’ by phage display technology possess favorable features for in vivo applications. The objective of this study was to test cross-reactivity of a dAb clone with LPS from five genera of G-ve bacteria. The most suitable of several LPS-binding dAb clones selected from a previously constructed phage display library of the LPS-immunized Indian desert camel, viz., dAb clone 26 (dAb Cl26) was used in the present study. LPS was extracted and purified from E. coli, Salmonella Typhimurium, Pseudomonas aeruginosa, Klebsiella sp. and Pasteurella multocida B:2. The dAb Cl26 was expressed with C-terminal 6xHis tag in VHH-pET303/BL21(DE3) expression system under IPTG induction. The expression of 17 kDa dAb.6xHis Cl26 in whole cell lysate was confirmed by SDS-PAGE, and the His-tag-specific immunoblotting and direct ELISA. The dAb.6xHis Cl26 was purified by Ni-chelate chromatography under denaturing conditions. After renaturation, the clone bound to lipid A and LPS from five genera in indirect ELISA. In conclusion, dAb Cl26 specifically bound to lipid A and exhibited cross-reactive binding to LPS from diverse bacteria.

Keywords: Antigenic cross-reactivity, bacterial lipopolysaccharides, single-domain antibody, phage display library

Cite this Article

Banerjee S, Singh A. A Single-domain Antibody Cross-reacts with Lipopolysaccharide from Diverse Gram-negative Bacteria. Research & Reviews: A Journal of Immunology. 2016; 6(2): 29–37p.