The Generation And Identcfication Of Monoclonal Antibodies Against Csfv-Gxwz02 Strain

In this study the CSFV-GXWZ02 strain was purified as antigen immunize mice to generate hybridomas which could excret monoclonal antibodies against classical swine fever virus (CSFV wuzhou strain). The CSFV-GXWZ02 strain is isolated with PK-15 cell, this strain could adapt to the cell well after going down to posterity in 5 generations, and it's infective tirer was 106TCID50/ml . Purified GXWZ02 as antigen immunize mice, the result is very well after 3 times immunity, the immune serum titre of little mouse of BALB/C is up to 1:32000. Using monoclonal antibody technique with the immune mouse's spleen cell and cell's routine method of SP2/0 bone myeloma to merge, it is carried on for two times, the result is 660 in all merges wells. Screened by indirect ELISA, positive well that merged have 468 well in all, selected 145 positive well to carry on clone 3 times by diluted, and attaining 19 strains that excreted McAb hybridization tumour cell against CSFV-GXWZ02 strain. They can still stably excreted McAb after being stored at -80℃ for 3months .Choosing 10 hybridization tumour cell to produce ascitic fluid , the antibody of ascitic fluid from 10 hybridomas were detected by indirect ELISA.The titers respectively ranges 1:400-1:6400, the titres of 1B3 and 2C6 were 1:6400, the titres of 2B5, 2A6, 1F4, 1F8 and 1B7 were 1:3200, 1C7 and 2B3 were 1:800, and 1A9 was 1:200. And also the antibody of ascitic fluid from 10 hybridomas were detected by indirect immnofluorescent assay(IFA), they reacted with CSFV-GXWZ02 strain prepared in PK-15 cell in 24 microplates and has specific fluorescent reaction, the dilution of 2C6 was 1:400, and 1F4, 1F8, 2A6, 2B5, 1B3 and 1B7 were 1:200, 1C7 and 2B3 were 1:100, and 1A9 was 1:50. The result of ELISA and IFA indicated these monoclonal antibody against CSFV-GXWZ02. Inoculating respectively CSFV-SM and CSFV-HCLV in PK-15 cell, the antibody of ascitic fluid from 10 hybridomas were detected by indirect immunofluorescent assay(IFA) and presented positive reaction, this result indicated these McAb against CSFV-GXWZ02 strain can discern CSFV-SM and CSFV-HCLV strain. This study offers the base on accumulated more technique of Monoclonal antibody and produced diagnosis box of CSFV.