An IgG/IgY sandwich-ELISA for the detection of heat-labile enterotoxin B subunit of enterotoxigenic Escherichia coli

Abstract

ObjectiveEnterotoxinogenic Escherichia coli is a health-threatening pathogen, especially in children. LT toxin this bacterium causes diarrhea and loss of body fluids. The development of rapid and cost-effective methods for diagnosis will help the patient survive and prevent the spread of this bacterium. The aim of this study was to design and set up the double sandwich ELISA diagnostic method for the detection of LT toxin using egg yolk (IgY) and rabbit antibody (IgG). MethodsRecombinant LTB protein was expressed in E coli BL21 (DE3) and purified using a Ni-NTA column. Chickens and rabbits were immunized subcutaneously and the generated antibodies were purified from egg yolks by polyethylene glycol (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. ResultsExpression of recombinant protein led to the production of LTB with molecular weight of 15 kDa. Yield of protein was 6 mg/L. ELISA assay results showed, increased serum antibody levels against the protein in chickens and rabbits sera after each injection. The yield of purified IgG was 2/5 mg/mL. The yield of purified IgY was 27 mg per egg. The sensitivity of the ELISA method was about 39 ng for recombinant LTB. The results showed the high specificity of this technique. ConclusionResults suggest that IgY/IgG-based sandwich ELISA in this study provides a convenient preparation for the development of immune-based method for the detection of LTB.