Development And Evaluation Of A Blocking ELISA For The Detection Of JEV Antibody

Japanese encephalitis is a mosquito borne zoonosis caused by Japanese encephalitis virus(JEV),which threats both human and several kinds of livestock and has important impact in public health in china.Many animals can become infected,the pigs are most susceptible,commercial swine were inapparent infection,infection in pregnancy is there sows miscarriage,mummy and stillbirth,infection in boar orchitis,to the pig industry caused great economic loss.Currently,there is no popular Japanese encephalitis in the treatment of the effects of drugs,prevention mainly rely on vaccination and mosquito control,therefore,the evaluation of antibody levels after vaccination in susceptible population is of great significance.In this study,JEV Cap-ED3 recombinant protein was used to immune BALB/c female rats,and 4 monoclonal antibodies that could reactive to JEV envelope protein were produced by using of cell fusion technology.Indirect ELISA,Western-blotting reaction,indirect immunofluorescence test,plaque reduction neutralization test and the additive ELISA analysis of these antibodies titer,antigenicity,neutralizing activity,recognition differences,cross reactivity and other characteristics.On this basis,the peroxidase labeled JEV monoclonal antibody 5F9 and JEV recombinant protein ED3 were used to establish a blocking ELISA for the detection of JEV antibody in animal serum.The sensitivity and specificity of the method were tested by parallel experiments with indirect ELISA detecting JEV antibody.Serum samples of cattle,goat,chicken and duck collected from different regions of Jiangsu Province for the laboratory 2014 collection were detected by the blocking ELISA method and the detection results were confirmed by serum neutralization test.The results showed that the blocking ELISA for the detection of JEV antibody has good specificity and sensitivity.Specific as follows:1?Production and Characterization of monoclonal antibodies reactive to envelope protein of Japanese Encephalitis VirusIn the present study,panel of monoclonal antibodies(MAbs)reactive to JEV envelope protein were produced by using of cell fusion technology.The purified cap and ED3 fusion protein which form viral like particle was used to immunize BALB/c female mice.For the screening of envelope specific MAbs,we expressed a soluble recombinant ED3 protein in BL21 E.coli cells.Analyzed by immunogical methods,MAbs 1B10 and 3F5,4hl and 5F9 were selected on the basis of their reactivity.All of the four MAbs can recognize naive JEV NJ2008 strain in the western-blotting and IFA tests.The titers of antibodies secreted into the culture supernatant and ascitic fluid were measured by indirect ELISA and the highest antibody titer of ascitic fluid was 409 600 for 5F9.However,none of the four MAbs identified to have neutralizing activity against JEV NJ2008 strain in the plaque reduction neutralization test.The result of additional ELISA indicates that 3F5 and 4H1 recognize the same antigen epitope while 1B10 and 5F9 react to other antigen sites.In general,four JEV envelope protein reactive MAbs produced in the present pave a way for the developing a JEV antigen or antibody detecting ELISA.2.Development and evaluation of a blocking ELISA for the detection of JEV antibodyTo provide a serological method for detecting antibodies against JEV,a blocking ELISA was developed based on a monoclonal antibody(mAb)to JEV enverlope protein.The conditions for each step were optimized.The optimal concentration of the coating antigen was 0.05?g/ml;the dilution ratio of the sera samples is 1:2500;and it is 1:2500 for the HRP labeled mAb strain 5F9.The ELISA results of 58 JEV negative sero samples were statistically analyzed,and the cutoff of blocking ELISA was determined that the sample presenting a%IN ?40 value is considered positive;while samples with a calculated%IN<40 will be rated negative.There was 98.17%coincidence rate between the blocking ELISA and the indirect ELISA based on the detection of 219 clinical cases of swine serum samples.Preliminary application of the method was carried out to detect about 500 serum samples collected in 2014 from Jiangsu Province by our laboratory.The positive rates of JEV antibody were 12.67%,4.67%,10.34%and 44.44%for the cattle,sheep,chicken and duck sample,respectively.In the PRNT,pig,sheep and cattle original ELISA positive samples showed significant inhibition effects on JEV multiplication,while no neutralizing activity was observed for the chicken and duck serum samples with positive ELISA values.However,inhibition effect on DTMUV was identified for duck serum samples with positive ELISA values.Furthermore we found that these MAbs have no cross reactivity with duck tembus virus(DTMUV)except 5F9,DTMUV is a newly emerging flavivirus widely spread in china with higher gene homology to JEV.The results show that this established blocking ELISA for the evaluation of JEV antibodies in swines,cattle,goats,and other large animals of strong specificity,high sensitivity,but the evaluation of JEV antibodies in poultry may have cross-reaction with DTMUV antibodies.