Preparation And Characterization Of Kanamycin-resistant Monoclonal Antibodies

Kanamycin was a kind of aminoglycoside antibiotics, which was widely added toanimal feed to prevent bacterial infections. However, kanamycin could not becompletely metabolized, thus formed residues in animal tissues, if the long-termingestion of food which contained kanamycin residues would cause severe ototoxicityand renal toxicity to humans. Therefore, the establishment of ELISA method to detectkanamycin residues in food of animal origin was necessary. The complete antigenpreparation and the monoclonal antibodies were the core steps of ELISA detection.Thetwo steps had a great influence on the results of ELISA detection.In the experiment we analysed the structural characteristics and immunologicalproperties of kanamycin, using two methods to prepare the kanamycin artificialantigen, and the preparation of the results were identified by SDS-PAGE; usinghybridoma technology to prepare anti-kanamycin monoclonal antibodies,thenidentified it. The work laid a solid foundation for the detection of kanamycin. Thisstudy included the contents as follows:1. Preparation and identification of the kanamycin artificial antigenFirstly, kanamycin standard was reacted with bovine serum albumin (BSA) orovalbumin (OVA) to prepare kanamycin artificial antigens. Two different couplingmethods:carbodiimide (EDC) and glutaraldehyde (GDA) were respectively preparedKAN-BSA (as an immunogen) and KAN-GDA-OVA (as the coating antigen). Both ofthe SDS-PAGE gel electrophoresis and the animal immunization experiment wereconduct to identified the antigen-coupled case. The results showed that: the preparedkanamycin complete antigen could stimulate the bodies to produce antibodies in mice,and the antiserum titer reached1:2.56×104, The IC50of the antiserum was22.28ng/mL. The results illustrated the immunogenicity of the complete antigens were goodand the preparations of the antigens were successful.2. Preparation and identification of kanamycin-resistant monoclonal antibodiesHybridomas were prepared by cell fusion techniques. Positive clones werescreened by methods of indirect ELISA and indirect competitive ELISA, clonal cellswere amplified subsequently.Positive hybridomas which could stably passage by limiting dilution cloning were selectied.At last,2subclones named1E1and2B12wereobtained.Using mice induced ascites method to product a large number of monoclonalantibodies. The checkerboard ELISA test was conducted to select the best packageconcentrations, the result showed that the optimal coating concentration ofKAN-GDA-OVA was2.5μg/mL. Microtiter plates were coated with optimal coatingconcentration, indirect ELISA and indirect competitive ELISA were used to test thetiter and sensitivity of the prepared anti kanamycin monoclonal antibodies.Accordingto the data on the results of the indirect competitive ELISA performed1E1standardcurve, The regression equation was y=-0.3793x+0.8084. The IC50was6.5024ng/mL.The affinity constant(Ka) was1.06×1011L/mol. Besides TOB (58.06%), the crossreactions of the other antibodies were less than0.65%.In this study, the anti-kanamycin monoclonal antibody was obtained which washigh affinity and high specificity.