Development And Application Of An Indirect ELISA For Detecting Antibodies Against Swine Hepatitis E Virus

Hepatitis E is a kind of serious zoonosis caused by hepatitis E virus?HEV?,and swine is its natural reservoir host.Therefore,monitoring HEV infection in swinery is of significant importance to the prevention and control of this disease as well as the public health.So far,the detection technologies for swine HEV infection mainly include immune electron microscopy,nested RT-PCR and ELISA.Among them,ELISA has been widely used owing to its advantages of easy operation and fast detection.At present,the commercialized ELISA kits for swine HEV antibody detection is mainly based on the ELISA method usingHEV genotype 1 ORF2 or ORF3 protein as the envelope antigen.However,on the one hand,the predominant swine HEV circulating in China is genotype 4;on the other hand,there are significant differences in the swine sera antibody level when the samples are detected with ORF2 protein of different genotype,thereby resulting in low coincidence rate among test results of different ELISA kits.Therefore,it is urgently required to establish an ELISA method with higher accuracy and sensitivity,and good specificity,so as to provide a technical support for monitoring swine HEV infection.In this study,to improve the specificity and sensitivity of the ELISA method,the epidemic genotype 4 swine HEV ORF2 protein in China is used as the envelope antigen.Consequently,the swine HEV antibody indirect ELISA detection method is established through the optimization of reaction conditions.The sensitivity,specificity and repeatability of this method are analyzed,as well as the coincidence rates of commercialized kits,toevaluate its clinical application.SDS-PAGE results show that ORF2 protein is successfully expressed in Escherichia coli in the form of inclusion body,with the size of about 40 ku.The high-purity target protein is obtained after protein renaturation and size exclusion chromatography.With the positive serum of swine HEV used as primary antibody,Western blot results show that the antigenicity of purified sHEV-ORF2-C protein is good,and the protein can be used as the envelope antigen.After the optimization of ELISA conditions,it is finally determined that,the optimal enveloping amount of antigen is 200 ng/hole,the best dilution degree of serum is1:200,the envelop buffer solution is carbonate buffer?pH 9.6?,the optimal blocking condition is incubation with 2.5%of skimmed milk for 2 h at 37?,the optimal second antibody incubation condition is incubation with the IgG-HRP 2 diluted to 5,000-fold for 1h,and the final detection condition is incubation with TMB developing reagent for 10 min.This method is employed to detect 91 HEV negative serum specimens,and the critical value is determined to be 0.296.Further,the OD₄₅₀50 value of positive serum of other important swine epidemics and swine HEV negative serum is less than 0.296,indicating that this indirect ELISA method has a good specificity.The positive serum is diluted using multiple-proportion method,and the OD₄₅₀ value is less than 0.296 only when it is diluted to 12,800-fold,showingthat the sensitivity of this method is good.The variable coefficients of intra-plate and inter-plate duplicate tests are 5%and 10%respectively,representing that the established indirect ELISA method maintains good repeatability and stability.182 clinical swine serum specimens were detected with the ELISA method established in this study and commercialized kits from Beijing Wantai simultaneously.The ELISA method established in this study detected 93positive specimens,and the other 89 specimens were negative;while the commercialized kits from Beijing Wantai detected 77 positive specimens,and the other 105 specimens were negative,thus,the coincidence rate of the two detection methods was 91.2%.In addition,477clinical swine serum specimens submitted by the pig farms in different cities were detected.Among all the specimens,210 specimens were positive,giving the positive rate of 44.03%?210/477?,indicating that this method could be used for detecting swine HEV antibody in clinical swine serum.Hence,the swine HEV antibody indirect ELISA detection method established in this research is of good sensitivity and specificity,and high accuracy,making it suitable for the detection of clinical samples and serum epidemiology investigation.