Development of an IgG/IgY Sandwich-ELISA for the Detection of Cholera Toxin Subunit B

Introduction: Cholera is a lethal diarrheal disease caused by Vibrio cholerae. Cholera toxin (CTX) is one of the major virulence factors in V. cholerae pathogenesis. One of the major strategies in dealing with the poisoning of the bacteria is their rapid detection. The aim of this study was to design and set up a double-sandwich ELISA diagnostic method for the direct detection of cholera toxin B (CtxB) based on chicken immunoglobulin Y (IgY) and rabbit immunoglobulin G (IgG).Materials and Methods: Recombinant CtxB protein was expressed in E. coli BL۲۱ (DE۳) cells by addition of IPTG and was purified using an NiNTA column. Chickens and rabbits were immunized subcutaneously and the generated antibodies were purified from egg yolks by polyethyleneglycol (PEG) precipitation and from the rabbits' sera by protein G column. These antibodies were used to set up the ELISA method. The sensitivity of the designed ELISA method was evaluated using serial dilutions of the protein and the specificity of this method was evaluated.Results: Recombinant protein expression analysis showed an appropriate expression of the protein (۳۰۰ µg/ml). ELISA assay results showed an increased serum antibody levels against the protein in rabbits’ and chickens’ sera after each injection. The yield of the purified IgY and IgG was ۱۰ and ۲.۵ mg/ml, respectively. The sensitivity of the ELISA method was about ۳۹ ng for recombinant CtxB. The results showed the high specificity of this technique.Conclusions: Results suggest that IgY/IgG-based sandwich ELISA in this study provides a convenient preparation for the development of an immune-based method for the detection of CtxB.