Preparation And Preliminary Application Of Monoclonal Antibody Against Streptococcus Agalactiae Isolated From Tilapia

Fish-derived Streptococcus agalactiae,a kind of high virulent gram-positive bacterium,can infect many freshwater and seawater fishes and bring about billion of dollars losses to global aquiculture each year.In recent year,the disease let by this bacteria spread across Tilapia fish raising zones in south China and severely hinders the development of China'stilapia aquaculture.On account of the advantages that monoclonal antibody has high specificity and sensitivity and plays an outstanding role in pathogen detection.In this experiment we have prepared monoclonal antibody against Streptococcus agalactiae of Tilapia and utilized the monoclonal antibody to establish Sandwish ELISA for detecting this bacteria.1.The 6 to 8 weeks old BALB/c mouse were immuned by the inactivated bacteria antigen of Tilapia-derived Streptococcus agalactiae GD09.When the titer reached above 1:12800 after immuned 3 to 4 times,cell fusion can be done.The fusion cells was filtrated by ELISA and 9 strains hybridoma,named 4C12,4B5,3A9,3A10,3A12,3C5,1F1,4D12,3D2 respectively,against bacteria antigen of Tilapia-derived Streptcoccus agalactiae were gained.By judgement of the chromosome it show that the chromosome of 9 strains hybridoma were numbered 90 to 110 and the chromosome number of 9 strain hybridoma were more than their parent cell.The identification of monoclonal antibody subclass show 4C12 and 3A10 belonge to IgG2 subclass,4B5 and 4D12 belonged to IgG1 subclass and 3A9,3C5,1F1,3A12 and 3D2 belonged to IgM subclass.By detecting the cell culture fluid of 9 strain hybridoma the titer of 4C12,4B5,3A9,3A10,3A12,3C5,1F1,4D12,3D2 are 1:214,1:210,1:212,1:28,1:212,1:212,1:211,1:210,1:210 respectively.The ascites titer of 4C12,3A10 and 3A9 are 1:224,1:224 and 1:216 respectively.After frozen hybridoma cell for 1 month the recovery experiments showed that 9 strains hybridoma can steadily secrete monoclonal antibody.2.Ascites monoclonal antibodies of 3A9 and 4C12 were purified and 3A9 monoclonal antibody was used as capture antigen to coat ELISA board and HRP,which was used as enzyme labeled antibody,was marked to purified 4C12 monoclonal antibody through Sodium periodate oxidation method to set up double antibody sandwich ELISA method for detecting Streptococcus agalactiae.The result showed the best reaction conditions of double antibody sandwich ELISA method were coated concentration of 3A9 monoclonal antibody was 10.24?g/mL,working concentration of 4C12 monoclonal antibody marked by HRP was 1:1000.The critical value mensurating by enzyme labeled antibody was OD450nm ? 0.210.The established ELISA method was used to detecting vibrio anguillarum,aeromonas hydrophila,pseudomonas fluorescens,Aeromonas caviae,pseudomonas aeruginosa,Molisch pseudomonas bacteria suspension and the result showed no cross reaction appearance.The experiment showed detection limit of Streptococcus agalactiae by this ELISA was 3.2×104 CFU/mL.The above results account for the established ELISA method has significant peculiarity and sensitivity.In this study 9 strains hybridoma that can steadily secrete monoclonal antibodies against bacteria antigen of Tilapia-derived Streptococcus agalactiae were successfully prepared,and a new double antibody sandwich ELISA method for detecting Streptococcus agalactiae was established by using the monoclonal antibodies of the strains hybridoma secretion,and the established ELISA method showed favorable detecting effect.Immune diagnosis and prevention of Tilapia-derived Streptococcus agalactiae disease are based on these research findings.The harvest of this study has set up an base for further researching new diagnose methods and can strongly provide technic support for prevention and treatment of Streptococcus agalactiae disease.