Preparation Of Monoclonal Antibody Against Saccharomyces Cerevisiae Larvirus And Establishment Of Agar Diffusion Assay

ObjectiveIn this experiment,purified CSBV was used as an antigen to immunize BALB/c mice to prepare anti-CSBV monoclonal antibody,and its potency,specificity and virus-neutralizing ability were tested.On this basis,using the prepared anti-CSBV monoclonal antibody as a diagnostic antibody,a agar diffusion method for detecting CSBV was established.MethodIn this experiment,we used purified CSBV as antigen to immunize Balb/c mice.After three immunizations,the mice were colected blood by the cutting tail and measure their serum titer,the mice with higher potency were selected and enhanced the immune 3-4 days prior to cell fusion.The splenic lymphocytes from the immunized mice were fused with SP2/0 myeloma cells by hybridoma technique.Positive cells were screened by indirect ELISA and subcloned three times by limited dilution method to prepare ascites monoclonal antibody.Monoclonal antibodies that can stably secrete anti-CSBV were obtained,and the antibody titers and their specificity were measured by indirect ELISA.After purified ascites monoclonal antibody interacts with CSBV respectively,the monoclonal antibody are inoculated to 2 to 3 days old Chinese bee larvae,and the neutralization effect of the antibody is identified by observing larval mortality.On this basis,the prepared anti-CSBV monoclonal antibody was used as a diagnostic antibody,the agar plate configuration conditions,observation time of agar diffusion,optimal dilution concentration of CSBV and dilution of anti-CSBV monoclonal antibody are optimized to establish a Agar diffusion method for detecting CSBV,and its specificity,sensitivity and stability were verified.ResultsTen positive hybridoma cells were obtained by hybridoma technology and three times subclone and labeled as 10C3,10A1,10C4,9A5,11D7,5B10,5A5,5H2,1D3,3C10 respectively.And it was injected into the peritoneal cavity to prepare ascites,and 9 strains of ascites-type monoclonal antibodies that can stably secrete anti-CSBV were obtained,which were 10A1,10C4,9A5,11D7,5B10,5A5,5H2,1D3,3C10 respectively Indirect Elisa was used to detect the titer,which were all more than 1:16000.On this basis,the anti-CSBV monoclonal antibody subtype,stability and specificity were identified,the monoclonal antibody subtype results showed that monoclonal antibodies 5A5,1D3,3C10 is IgG1 subtype;10A1,5B10 is IgG2a;9A5,10C4 is IgG2 b subtype;11D7,5H2 is IgM subtype;stability test results show that monoclonal antibodies 9A5 and 5A5 supernatant secreted antibody titers did not change,3C10,5H2 and 10A1 at the 10 th generation the ability to secrete antibodies decreased,but between the second generation to 10 generations the antibody secretion more stable,indicating that these five hybridomas can be more stable secretion of antibodies;specific experimental results show that 9 monoclonal antibodies only react with CSBV but no CBPV,DWV and BQCV.After the purified 9 ascites monoclonal antibodies were respectively interacted with CSBV,inoculated 2~3 days old Chinese honey bee larvae,in which 5A5,10A1 and 9A5 monoclonal antibodies had the ability to neutralize CSBV.On this basis,an anti-CSBV monoclonal antibody was used as a diagnostic antibody to establish an agar diffusion method for detection of CSBV.By optimizing the agar diffusion test conditions and observation time,the optimal dilution of antibody was 1: 4000,the optimal dilution of CSBV was 1:2,and the best observation time of agar diffusion was 48 h,the stability,specificity and sensitivity of this assay were validated,which showed that there was a good cross immunity reaction between CSBV and anti-CSBV monoclonal antibody.Agar diffusion detection method has the advantages of easy operation,fast,suitable for grass-roots applications,laid the foundation for the prevention and treatment of CSBV research.In conclusion1.Three anti-CSBV monoclonal antibodies 5A5,10A1 and 9A5 with virus-neutralizing ability were successfully prepared,which provided a material reserve for the prevention and detection of CSBV.2.The anti-CSBV monoclonal antibody was used as a diagnostic antibody,a agar diffusion method for the detection of CSBV has established.The detection method is specific,stable,easy to operate and fast.