Preparation Of Monoclonal Antibody Against Japanese Encephalitis Virus And Establishment Of Rapid Detection Methods

Epidemic encephalitis B is caused by Japanese encephalitis virus(JEV),which is a zoonotic natural epidemic disease transmitted by mosquitoes and is one of the major infectious diseases that threaten the health of humans,especially children.Pigs are considered to be the"amplifying host"in the epidemic of JE.Japanese encephalitis virus can multiply in pigs,causing significant viremia.In addition,the virus can be further transmitted between pigs through mosquitoes,The pig industry has caused significant economic losses.Prevention and control of Japanese encephalitis requires effective vaccine immunization on the one hand,and simple,fast,and accurate diagnostic methods on the other.Monoclonal antibodies(Mc Ab)have the advantages of high specificity,so the diagnostic method of Japanese encephalitis antigen based on monoclonal antibodies has great application prospects.In this study,the preparation of Japanese encephalitis virus monoclonal antibody and the establishment and preliminary application of its antigen-antibody detection method were carried out.In this study,a dual-chromatographic method for the gentle purification of JEV was established.Compared with the virus stock solution,the virus recovery and total protein removal rate of JEV after dual-gel chromatography purification were 61.04%and 99.71%,respectively.The removal rates of HCP and BSA reached 98.72%and 99.72%,respectively,indicating that Capto ^TMCore700 chromatography after ultrafiltration membrane package concentration and agarose gel molecular sieve chromatography(TFF-SEC)can obtain higher purity and integrity Well,virus particles with good immunogenicity and biological activity.BALB/c mice were immunized with JEV virons purified by TFF-SEC-Capto ^TMCore700.A total of 5 strains of monoclonal antibodies were screened in the study:29E3,29F8,4B4,2F2,and 36A6.The hybridoma cell culture supernatant titer was between 1:800and 1:1600.After inoculating the mice,the ascites titer was prepared.Between 1:5.12×10⁵and 1:2.56×10⁶.Class and subtype identification showed that 4B4 and 36A6 are Ig G1.29E3,29F8,and 2F2 are Ig G2?.29E3,29F8,and 2F2 light chains are?chains,and 4B4 and 36A6light chains are?chains.Stability testing showed that all five hybridoma cells were able to stably and continuously secrete specific antibodies.Western blot analysis showed that 29F8and 2F2 monoclonal antibodies could specifically recognize the E protein of JEV virus.The purified JEV E protein monoclonal antibody was used to optimize colloidal gold test paper for the detection of JEV virus.The test strip has strong specificity,and the detection limit can reach 10³?10⁴TCID₅₀/100?L,which has good repeatability and stability,and can be widely used in production and clinical detection of a variety of samples.The study further optimized various detection conditions,determined the thresholds of negative and positive serum for clinical tests,and established a JEV monoclonal antibody blocking ELISA detection method.The method has good specificity,and the lowest detectable sex serum is 1:1600 times.The coefficient of variation of the repeatability test within and between batches is less than 5%.The clinical sample tests show that JEV is still active in the pig farms and even the entire Henan Province for inspection,and vaccine immunization prevention must be strengthened.In conclusion,we have successfully established an in vitro screening method for Japanese encephalitis virus monoclonal antibody and obtained 5 hybridoma cells that can stably and continuously secrete specific antibodies;A blocking ELISA for the detection of Japanese encephalitis virus(JEV)was successfully established.