Construction of a single-chain variable-fragment antibody against the superantigen Staphylococcal enterotoxin B

Pawan Kumar Singh, Ranu Agrawal, Dev Vrat Kamboj, Garima Gupta, M Boopathi, Ajay Kumar Goel, Lokendra Singh
Applied and Environmental Microbiology 2010, 76 (24): 8184-91
Staphylococcal food poisoning (SFP) is one of the most prevalent causes of food-borne illness throughout the world. SFP is caused by 21 different types of staphylococcal enterotoxins produced by Staphylococcus aureus. Among these, staphylococcal enterotoxin B (SEB) is the most potent toxin and is a listed biological warfare (BW) agent. Therefore, development of immunological reagents for detection of SEB is of the utmost importance. High-affinity and specific monoclonal antibodies are being used for detection of SEB, but hybridoma clones tend to lose their antibody-secreting ability over time. This problem can be overcome by the use of recombinant antibodies produced in a bacterial system. In the present investigation, genes from a hybridoma clone encoding monoclonal antibody against SEB were immortalized using antibody phage display technology. A murine phage display library containing single-chain variable-fragment (ScFv) antibody genes was constructed in a pCANTAB 5E phagemid vector. Phage particles displaying ScFv were rescued by reinfection of helper phage followed by four rounds of biopanning for selection of SEB binding ScFv antibody fragments by using phage enzyme-linked immunosorbent assay (ELISA). Soluble SEB-ScFv antibodies were characterized from one of the clones showing high affinity for SEB. The anti-SEB ScFv antibody was highly specific, and its affinity constant was 3.16 nM as determined by surface plasmon resonance (SPR). These results demonstrate that the recombinant antibody constructed by immortalizing the antibody genes from a hybridoma clone is useful for immunodetection of SEB.

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