Development Of Monoclonal Antibody Of Japanese Encephalitis Virus

Japanese encephalitis virus which is an arthropod-borne virus,can lead to epidemic encephalitis type B,and seriously harm to human and animal.It spreads main by the intermediary of Culex tritaeniorhynchuso.The definition of McAbs is a kind high purity antibody,and it can only aim directly at one specified epitope structure.Because of its high purity and strong specificity,people do much investigations about it from the diagnoise,therapy and biopreparate.To parapare the McAbs of Japanese encephalitis virus can lead a important significance.This essay had done a research about the development of monoclonal antibody of Japanese encephalitis vires.1.Preparation and concentration of Japanese encephalitis virus antigenIn this experiment,the Japanese B encephalitis virus were proliferated with BHK-21 cell,we can see the CPE when the BHK-21cells were inoculated with JEV for about 24 h. And during 48-72 h we froze the cell when the CPE reach to 80%,after freeze thawing for three times,we use the PEG6000 and Nacl to condense and extrat the sample.After centrifugate,The crude extract sample was took a single step purification for 45000 r/3 h. At the same time,normal untreated BHK-21 cell was set as the negative control group.We do PCR and SDS-PAGE with the sample,and determine the antigen protein level,the results was that we can gain the purpose strap from the purity sample;when do SDS-PAGE with the sample,we can see more strips,it comes to say that the purified sample have the immunoreactivity.The antigen protein level was 19.78 mg/mL.With this result,we had successly prepared the Japanese encephalitis virus antigen,it can establish the basis of preparing the McAbs.2.Preparation of monoclonal antibody JEVThe purified JEV were used to immune the BALB/c mice with routine immunization. The spleen cells of the Balb/c mouse which immunized with JEV SA14-14-2 strain were fused with mouse myeloma cells(SP2/0).The own indirect ELISA method was used for preliminary screening,the negative and over one clone cell holes were abandoned,and the kit was used to check the masccline hole again.At last,we chose the best one to subclone for four times by the limited dilution.The result was:the fusion rate of routine immunization was about 34%,the masccline rate was about 1%,three strains were selected and named as JEV.1,JEV.2,JEV.3.3.Identification of monoclonal antibody JEVThe subclass of JEV.1,JEV.2,JEV.3 antibodies were all IgG₁,its light strand wasκby using kit to detect.The indirect ELISA result indicated that these three strains all have a favourable passage and thermostabile stability.The titres of three McAbs cell supernatants were 10³,the ascitic fluids were 10⁵,and in the serum were 10⁴.The specificity of McAbs by addition test showed that they can recognize the same antigenic determinant on the surface of the virion.The McAbs also showed favourable specificity in IFA and specificity experiment.The relative affinity of the McAbs are as follows:JEV.1 5 mol/L,JEV.2 4 mol/L,JEV.3 5 mol/L,based on the elution method with MSCN,the Mcabs of JEV do have a good affinity.