Preparation And Identification Of Polyclonal Antibody And Monoclonal Antibodies Against The Clavibacter Michiganensis Subsp. Michiganensis

Objective:Bacterial canker of tomato is a serious hazard of bacterial diseases which spreaded widely in the major tomato producing areas.The bacteria is named Clavibacter michiganensis subsp.michiganensis(Cmm).Cmm can infect and destroy the tomato vascular bundle and caused the die of tomoto plant.Cmm can caused great loss to the quantity, as well as the quality of tomoto production for its strong pathogenicity、long latent period. How to detect Cmm is the most important tache for the control of Bacterial canker of tomato. Traditional methods contains biological assay、serological method and molecular biology testing. Serological method such as ELISA (Enzyme linked immunosorbent assay) is used more often than other methods because it’s good specificity、high sensitivity and easily to using. But almost all of the antiboies used in China depends on import from foreign countries,hereby the very high cost makes this method hard to popularization in grass roots of China. In order to solve this problem we tried to prepare the polyclonal antibody and monoclonal antibody with good quality by ourselves, for the purpose to produce the ELISA test kit and break the monopolization of foreign companies in this field and reduce the cost of Cmm detection.Finally we hope we can get the monoclonal antibody and ELISA test kit with our own independent intellectual property rights.Method:Used Cmm as the antigen to immune New Zealand rabbits subcutaneouly.Tested the antibody titer by the method of ELISA. Collected the blood of the rabbits from carotid artery and got blood serum from blood.Identified the specificity of the antibody and finally purified the antibody. We used Cmm as the antigen to immune BALB/c mouse and the method of hybridoma technique to prepare monoclonal antibody. After the process of immunity、cell fusion、indirect ELISA screening and cloning,we got the positive hybridoma cell which can secrete antibody againest Cmm.Finally we got the antibody againest Cmm successfully and Indentified the titer、specificity and affinity of the antibody. Result:We prepared the polyclonal antibody against Cmm and the antibody titer is 1: 256000.The result of western blotting showed that the polyclonal antibody can recognize Cmm specially.We used Cmm as antigen to immune BALB/C mouse and got three hybridoma cell strains which can secrete monoclonal antibody againest Cmm. We named them IA4、IC3 and IB7.Through the method of subclass analysis we confirmed that IA4 antibody is IgM while IC3 antibody and IB7 antibody are IgGl. We purified the ascites to got the monoclonal antibodies and uesd the method of ELISA to teste the antibody titer.The antibody titer of 1A4 antibody 1C3 antibody and 1B7 antibody are 1:25600、1:12800 and 1:12800 respectively.The monoclonal antibody we prepared have no cross-reaction with other Clavibacter michiganensis;subspecies.Conclusion:Both the polyclonal antibody and monoclonal antibody have strong specificity and sensitivity,so they can used as the detector of Cmm.The antibody secreteed by IA4 is better than anther two strains for its high specificity.Because of the high-quailty of the antibodies we get,it is reasonable for us to believe that the kit which is used to detect Cmm will be produced soon.