| Japanese encephalitis is a mosquito-borne zoonotic disease caused by the Japanese encephalitis virus (JEV) which belongs to Flaviviridae. And a variety of animals are susceptible, pigs are very susceptible, but the incidence rate is not high, so that pigs become the amplification reservoir of JEV, which have a significant impact on the public health safety. There are currently no methods for JE treatment, therefore timely and effective detection and prevention of JEV is very important. JEV envelope (E) protein domain III (ED III) is one of the major structural proteins, which it can induce virus specific humoral immunity in the host anti-JEV infection. Hemagglutination inhibition test (HI) and plaque reduction neutralization test (PRNT) are the classic method for detecting JEV antibodies. But due to test material, the operator’s proficiency and special device needed of these assays, wide clinical application of these methods are limited. Enzyme-linked immunosorbent assay (ELISA) test is a widely used antibody detection method, which is simple operation, rapid detection, high sensitivity, high specificity and highthroughput. However, the clinical significance of ELISA test results evaluation requires a lot of correlation between epidemiologically supported datas.Therefore, based on the previous studies of JEV E protein domain III expression and an indirect ELISA method established for JEV antibody detection which using recombinant E protein as coating antigen in our laboratory. In the present, the indirect ELISA method for detecting JEV antibody was futher improved and its correlation-ship with HI and PRNT were analyzed by contrast detecting clinical swine serum samples. The aim of this study is to provide a basis for clinical signigficance interpretation of the ELISA results and paved the way to the development of JEV antibody detecting ELISA kit. Specific studies are as follows. 1. The establishment of indirect ELISA for JEV antibody detection based on prokaryotically expressed JEV EDIII antigenBased on previous study in our laboratory, JEV EDIII protein expression vector pET-28a-EDⅢ and BL21host bacterial were choosed in the present study to produce coating antigen. After induced expression by IPTG, JEV EDⅢ protein was soluble expressed in E. coli when cultivated in low temperature, but the protein expression level was low; while induced expression in37℃produced large amount of target protein,but as form of inclusion bodies. Then relative pure recombinant JEV EDIII protein was obtained by Ni+column affinity chromatography purification. After the protein expression and purification for three times, the average protein concentration was measured as2.84mg/mL, and the average availability yield was9.4mg per liter. The result of Westen blot analysis for the expressed protein shows the positive reaction of the EDIII protein with JEV antibody and His tag monoclonal antibody which indicates that the inclusion body proteins of the expressed EDIII have good immunogenicity and specificity, which can be used as coating antigen. The optimal reaction condition of the JEV EDIII antigen as coating antigen, the reaction conditions were optimized, established to detect JEV antibody by indirect ELISA. The optimal reaction conditions are as follows, the concentration of coating antigen is0.7μg/mL,4℃overnight;1%BSA in PBST, blocked overnight at4℃; tested serum dilution is1:40, incubation for1h at37℃; the second antibody as1:5000dilution (concentration of working solution is0.2μg/mL),37℃for1h. The value of the OD450nm≥0.436is judged as positive, OD450nm≤0.363was negative, for the median suspicious, if rechecked still as suspicious, then judged as negative. The OD450value of standand positive serum of Porcine reproductive and respitory syndrome, Classic swine fever and pseudorabies were lower than criteria value in the ELISA test which indicates that the established ELISA is specific. Inter-and intra-batch test proved to be reproducible. In summary, the JEV antibody detection ELISA based on recombinant EDIII antigen has good specificity, reproducibility and sensitivity.2. The test on the correlation ship of JEV EDⅢ antigen based ELISA and indirect hemagglutination inhibition and plaque reduction neutralization test in porcine JEV antibody detectionTo provide a basis for clinical signigficance interpretation of the ELISA results, the correlation-ship of JEV antibody level of serum sample detected by ELISA with HI and PRNT were analyzed by contrast detecting clinical swine serum samples in the present. JEV antibody level of127swine serum samples were tested by indirect ELISA, and then their HI titer were determination and then21samples were randomly selected and tested by PRNT. The paired chi-square test showed that compare to HI, ELISA sensitivity is72.5%and a specificity of52%, the compliance rate of the two methods was68.5%, difference between the two detection methods was not significant (X=5.535, P=0.17>0.05). Compare to PRNT the HI test sensitivity of72.2%and a specificity of33.3%, the compliance rate was66.7%, the difference was not significant (X2=0.039, P=0.453>0.05); Compare to PRNT, the sensitivity of ELISA80.0%and a specificity of50.0%, and the compliance rate is71.4%, the difference was not significant (X2=1.890, P=1>0.05). The result indicates that these three detection methods can effectively detect JE antibody from porcine serum, and relatively the ELISA test is more sensitive.3Serum epidemiological investigate of the swine.JE based on indirect ELISAIn order to further verify the validity of the indirect ELISA established based on JEV EDⅢ antigen in the present study for porcine JEV antibody detection in Clinical. Relatively large amount of clinical porcine serum samples were collected, which include339swine serum samples collected in the JE vaccine evaluation test and samples of the different immunization backgrounds from different size swine farms in Anhui, Jiangsu.Then the JEV antibody prevalence of the different regions, different sizes and different immunization programs were analyzed. The results showed that JEV antibody level was in accordance with the serum background characteristics which depend on different ages,different areas, different farm sizes and the different immunization program.These results also shows that the established JEV EDIII antigen indirect ELISA is reliable for the swine JEV serum antibody detection. As a conclusion, the results of present study provide a more detailed and reliable basis for the development of porcine Japanese encephalitis antibody detection kit. |
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