Development Of Enzyme-Linked Immunosorbent Assay Test Kit For Detecting Anti-Classical Swine Fever Virus Antibodies

The recombinant plasmid pETE2 was transformed into E.coli strain BL21(DE3) plysS and the expression was induced by IPTG. Finally, the SDS-PAGE and Western blot detection analysis showed that E2 was expressed in the form of inclusion body at the level high up to 28% of total bacterial protein. After E.coli BL21(DE3)pLysS was fissioned by ultrasonication, The E2 inclusion body was washed by 1M NaCl, 0.5% TritonX-100, 2M urea, 3M urea, 4M urea, then subjected to solution and denaturation in 1%SDS and B-ME, In order to obt in higher purity the protein E2 were further purified by SDS-PAGEThe highly antigenic major structural protein E2 of CSF virus was used as detecting antigen in indirect enzyme linked immunosorbent assay (ELISA) to detect anti-CSFV serum. The optimal reaction conditions of ELISA were determined, The optimal concentration of recombinant E2 protein for coating microplate was 2Mg/ml, the coated microplate incubated for 36 hours at 4C, then 100ul of 1%BSA was added as blocking agent and incubated for 2 hours at 37C add 12 hours at 4C, The best serum dilution buffer was 0.05%Tween-20 / 1%BSA/ 2% supernatant of the normal abstracts of E.coli/PBS, the dilution of serum sample was 1:100. the working concentration of HRP-labeled rabbit anti-porcine IgG was 1:1100 and HRP-labeled rabbit anti-porcine IgG should be incubated at 37C for 20 min, the substrate for ELISA was incubated at room temperature for 30 min before terminated with the stopping solution.150 serum samples which were negative by the indirect fluorescent antibody test (IFA) and the vrus > neutralization test were used for the determination of threshold value of ELISA, If the OD value less than or equal to 1.5 times standard negative serum OD value, the test sample is negative. Another test results revealed the assay has good specificity for the detection of CSFV antibody in serum. The The variant coefficients of the OD value among wells in a plate and among plates for ELISA were both less than 10%, which showed the assay had a good Repetition.70 serum samples from swines were detected for the antibody to CSFV by using our developed ELISA and the IDEXX CSFV Antibody Test Kit simultaneously. It was found thatour developed ELISA have the highly sensitivity and correspondence. Though there are dignosic difference between several sera, the rabbit neutralization test and the indirect fluorescent antibody test prove our ELISA kit is right. The assay have met the standards of diagnosis method abroad. The storage conditions of the kit was explored, It was shown that the kit could be stored for 6 months at 4C. Moreover, the antigen of the indirect ELISA < as recombinant of CSFV, whose production cost is very low, and this makes it own a big advantage, compared with the IDEXX test kit.The result of clinical practice demonstrated our ELISA Test Kit is sensitive, specific and reliable. The kit was quick, simple and available, which is therefore suitable for large scale monitoring of classical swine fever, and will have greatly significant to CSF prevention and control.