DEVELOPMENT OF A STREPTAVIDIN-CONJUGATED SINGLE-CHAIN ANTIBODY THAT BINDS BACILLUS-CEREUS SPORES

Control of microorganisms such as Bacillus cereus spores is critical t o ensure the safety and a long shelf life of foods. A bifunctional sin gle chain antibody has been developed for detection and binding of B. cereus T spores. The genes that encode B, cereus T spore single-chain antibody and streptavidin were connected for use in immunoassays and i mmobilization of the recombinant antibodies. A truncated streptavidin, which is smaller than but has biotin binding ability similar to that of streptavidin, was used as the affinity domain because of its high a nd specific affinity with biotin, The fusion protein gene was expresse d in Escherichia coli BL21 (DE3) with the T7 RNA polymerase-T7 promote r expression system. Immunoblotting revealed an antigen specificity si milar to that of its parent native monoclonal antibody. The single-cha in antibody-streptavidin fusion protein can be used in an immunoassay of B, cereus spores by applying a biotinylated enzyme detection system , The recombinant antibodies were immobilized on biotinylated magnetic beads by taking advantage of the strong biotin-streptavidin affinity. Various liquids were artificially contaminated with 5 x 10(4) B, cere us spores per mi. Greater than 90% of the B. cereus spores in phosphat e buffer or 37% of the spores in whole milk were tightly bound and rem oved from the liquid phase by the immunomagnetic beads.