Preparation And Characterization Of Monoclonal Antibody Against Recombinant Human Endostatin And Establish ELISA Method To Detect Human Endostatin

Objective To prepare mouse anti-recombinant human endostatin monoclonal antibodies and identify it,establish the double antibody sandwich ELISA using human endostatin check.Methods Recombinant human ES was used as antigen to immune BALB/c mouse, and apply hybridoma technique to fuse immunized spell cells of the mouse with the source mouse myeloma cells (SP2/0),Indirect ELISA method was used to screen supernatant of positive wells. The extensive development can stabilize the secretive cells of anti-recombinant ES. The ascites developed by injecting the hybridoma cells into abdomen cavity of the BALB/c mouse and then identification of antibodies was performed. Adopting Sigma antibody sub-model check test box determine monoclonal antibodies cell lines, Western-Blotting check and it,s immunology characteristic,by Protein G Sepharose 4FF pureed antibody and marking. We initially set up the double antibody sandwich ELISA using anti-ES monoclonal and polyclonal antibodies by Punnett Square,using optimization concentration for study human,s ES expression.Results We have successfully obtained four hybridoma cell lines 3B4,1D10,1G5 and 2D10. With repetitious generation,the hybridoma cell lines are all still could secrete high titer antibodies,and then identified 3B4 and 1D10.The subtypes of the monoclonal antibodies were IgG2a and IgG3,others two hybridoma cell lines were IgG2b , The titer of ascites was form10-6 to 10-4. Western blotting and immunohistochemistry showed these antibodies have specialty and sensitivity. By Protein G Sepharose 4FF pureed antibody and checked they concentration. By enzyme marking antibody brief easy sodium periodate method distinctly excelled glutaraldehyde two steps methods brief easy Sodium periodate method the titers of high 1:6400.We initially set up the double antibody sandwich ELISA using anti-ES monoclonal and polyclonal antibodies for tumors person,s ES experiment method.Conclusion Succeeded in preparing four hybridoma cell strains of anti-recombinant human endostatin monoclonal antibodies.By Protein G Sepharose 4FF pureed antibody and marking , we initially set up the double antibody sandwich ELISA using anti-ES monoclonal and polyclonal antibodies given a good tool for behind experiment,which lay the foundation for study human,s ES expression level and related realm is effective enough complementation.