Background: Regarding the importance of rapid detection of cholera toxin in environmental samples, an immunoassay method was developed for direct detection of cytotoxin B.
Materials and Methods:
The gene sequence of ctxB was cloned to pET28a vector and the recombinant protein was expressed in BL21 (DE3). The recombinant CtxB was purified by affinity chromatography and then used for immunization of chickens. The polyclonal anti-CtxB antibodies were purified from egg yolks by polyethylene glycol. The IgY antibodies were used to develop a sandwich ELISA. Analytical sensitivity of the assay was determined.
Results:
38 mg of recombinant CtxB, more than 95% purity, was purified from 1 liter of bacterial culture. The titer of anti-CtxB antibodies in the serum sample of immunized chicken was 1:32,000. The yield of purified IgY was 50.9 mg per egg yolk. The detection limit of the developed ELISA is about 33 pg/ml of recombinant CtxB.
Conclusion:
Management and control of Vibrio cholerae infection requires the availability of rapid diagnostic tools. The newly developed immunoassay provides a rapid and sensitive method for detection of cytotoxin B of V. cholerae. We plan to evaluate the performance of the developed assay in detection of cholera toxin in environmental and food samples.