Development Of An Indirect ELISA Method For Detection Of Bovine ETEC K99 Fimbrial Antibodies

Enterotoxigenic Escherichia coli(ETEC)is one of major pathogens which cause young stock diarrhea . The adhesive function of fimbrial adhesins is the primary condition of ETEC disease. Studies have shown adhesions to be immunogenic protein, and antibodies against adhesions can be produced by immunization using whole bacteria with adhesion or purified adhesion.In order to reduce influence of tag-protein to ELISA results.K99 gene was cloned from pET-32a(+)– K99 to pQE-30 Xa and transformed into the competent cells E. coli XL1-Blue. The protein induced by IPTG was detected by SDS-PAGE and Western blot. The results showed that the molecular weight of the recombinant protein was about 20.2Ku, and the expression level was 38% analyzed by Bandscan. The recombinant protein was highly purified by Ni–NTA agarose, and its concentration was 1.1mg/mL determined by GeneQuant. Western blot analysis using rabbit-anti-K99 polyclonal serum showed that the recombinant protein had good antigenicity and could be used as ELISA diagnostic antigen.The purified protein was used as coating antigen in indirect ELISA to detect anti-K99 antibody. The optimal reaction conditions of ELISA were determined. The optimal coating buffer was TBS (0.05M pH7.6), and the optimal concentration of protein for coating microplate was 0.7μg per well. 1% gelatin was added as the optimal blocking agent and incubated for 1 hour at 37℃. The best serum dilution buffer was 5% nonfat dry milk, and the dilution of serum sample was 1:800. The working concentration of HRP-labeled rabbit-anti-bovine IgG was 1:6000, coloration time was 10min. The cutoff value for ELISA was defined as absorbance values among negative serum samples(n=166) from field. If the OD value is more than or equal to 0.336, the test sample is positive, otherwise negative. By comparising with PCR, it was found that the specificity and sensitivity of the developed ELISA were 96.4% and 94.8%, respectively.