Preparation Of Monoclonal Antibody To Novel-duck Reovirus σC Protein And Establishment Of Blocking ELISA Antibody Detection Method

Novel duck reovirus disease is an immunosuppressive disease of ducks caused by Novel duck reovirus（NDRV）.Chicks infected with NDRV develop depression,poor appetite,and high mortality.The infection rate of adult waterfowl has no obvious symptoms,but the virus will continue to invade the host’s immune system,causing immunosuppression,and then prone to secondary infection.NDRV has a wide range of infecting hosts,in addition to infecting cherry valley ducks,semi-muscadine ducks,hemp ducks,and geese are also susceptible.The disease is currently widespread in many provinces and cities in my country,causing huge economic losses to the aquaculture industry.Monoclonal antibodies are of great significance to the diagnosis and prevention of diseases,as well as the study of viral pathogenic mechanisms.In this experiment,the NDRV epidemic strain isolated from clinical cases was inoculated with chicken hepatoma cells（LMH）for proliferation,and the concentrated and purified virus was obtained by ultracentrifugation as an antigen to immunize BALB/c mice.The titer was detected by indirect enzyme-linked immunosorbent assay（ELISA）,and then the spleen cells of the mouse with the highest serum titer were selected to fuse with myeloma cells（SP2/0）.After the fusion cells were screened and subcloned by ELISA and Indirect immunofluorescence Assay（IFA）,two hybridoma cells,3H11E5B8 and 3H11E5G8,were finally obtained.The two strains of hybridoma cells were expanded and cultured,and BALB/c mice were injected intraperitoneally to prepare ascites.After IFA identification,the antibodies secreted by the two hybridoma cells obtained in this experiment can effectively recognize the σC protein of NDRV,and the subtypes of the two monoclonal antibodies are Ig M κ type.We further selected 3H11E5G8 monoclonal antibody to initially establish an NDRV blocking ELISA method.The experimental conditions were repeatedly optimized and finally determined as follows: the antigen coating concentration is 5 μg/m L,the blocking solution is10% skim milk,and the dilution ratio of negative and positive serum is 1: 5.The dilution ratio of HRP enzyme-labeled secondary antibody is 1:2,000,and the color development time of TMB substrate is 10 min.Judgment criteria: When PI ≥ 24.06%,serum samples were judged to be positive for NDRV antibodies,and PI ≤ 15.49% were judged to be negative,and when PI was between 15.49% and 24.06%,it was judged to be suspicious.The specificity experiment of the established blocking ELISA showed that the detection method has good specificity only for NDRV,duck plague virus,duck Tembusu virus,duck hepatitis virus type 1and type 3,H7 avian influenza.The positive sera of virus,H9 avian influenza virus and Newcastle disease virus had no blocking effect.Sensitivity experiments showed that when the positive serum was diluted to 400 times,the blocking rate PI was still greater than 24.06%,indicating that the method had good sensitivity.The blocking ELISA method was used to detect some serum samples collected clinically,and at the same time,the IFA method was used to verify the results.In summary,this study successfully prepared a monoclonal antibody targeting the NDRVσC protein,and based on this,a blocking ELISA antibody detection method for the virus was preliminarily established.The preparation of the box lays the foundation for the experiment.