The Development Of The Antigen And Polyclonal Antibody To The Hapten Monensin

Monensin belongs to a class of compounds known as the polyether monocarboxylic acid ionophores. Because of its specific physical and chemical characteristics and the effect mechanism, this antibiotic has already applied for the production. At present, Monensin has been widely used as a feed additive to control coccidiosis in poultry and rabbits. In addition, It is used to increase the food conversion efficiency and accelerate the growth of cattle and pigs. However, using this antibiotic irrationally will lead to the remedy residue, which will damage to animals and human beings. The toxicity of Monensin is different for kinds of animals. In the mass, Monensin is harmful to growth and reproduction performance of animals , and will lead to the muscle and heart disease of the human being. Consequently, how to assay monensin rapidly and efficiently becomes the aspect of research at home and abroad. There are many methods of detecting the antibiotic such as classical colorimetric method, the TLC method, the microbiological assay, the bioautographic method, the liquid chromatography method, the immunoassay method and so on. In these methods, Immunoassay method which has developed in these years is simple and quick. The basis of immunoassay method is the development of the polyclonal or monoclonal antibody. The present research includes the transformation of the hapten Monensin and the development of the polyclonal antibody. At the same time, we appraised the antibody. The research establishes the basis of immunoassay of Monensin. The results are shown as follows: 1. The transformation of the hapten Monensin Sodium SaltIn production Monensin is existed as Monensin sodium salt. However, Monensin sodium salt has not any group which are directly coupled with protein conjugations such as BSA,OVA. So it can not synthesize immugen directly and must be modified to the substance contained of carboxyl to produce antibody. We applied three methods including of hydrochloric acid soaking method, cation exchange method, succinate acid anhydride method for transformation to monensin acid and monensin-succinate acid anhydridederivative. The materials of the transformation were appraised by thin layerchromatography and Infrared spectrum. The result of appraising is approved successfully.The materials of the transformation provide the basis for the development of polyclonalantibody.2. The Development of Polyclonal Antibody to MonensinThe materials (Monensin acid, Mon-HS) were coupled with protein (BSA, OVA) by the mixed acid anhydride reaction and EDC method. The conjugation included the immunogen(Mon-HS-BSA, Mon-OVA) and the coating antigen(Mon-HS-OVA, Mon-BSA). The synthesized artificial antigen were appraised by DATA and SDS-PAGE. The protein-hapten conjugate rate was 1:18(Mon-HS-BSA), 1:7(Mon-HS-OVA), l:ll(Mon-OVA),l:20(Mon-BSA) separately. High tiler polyclonal antibodies against Monensin were produced from rabbits immunized with the synthesized immunogen conjugates Mon-HS-BSA and Mon-OVA. The results show the rising trend of the antibodies to the 3rd , 4th and 5lh immunity. The final liter of antibodies were l:12800(ear No.0799), l:6400(ear No.0791) and l:25600(ear No.0569) respectively. Through the indirect competitive enzyme-linked immunosorbent assay(ELISA), there is the one to monensin among the antibodies.