Preparation And Application Of Monoclonal Antibody Against Enrofloxacin

Enrofloxacin (ENR),a fluoroquinolone, is specific for treatment and prevention of animal disease. Recently, however, due to wide-ranging use of ENR resulting in residue is animal tissue, it has been given great interest. And the use of ENR in veterinary medicine has been prohibited in many countries. In addition, it has approved by European Community that the Maximum Residue limits (MRL) of ENR in edible animal tissue are not beyond 30ug/kg. The purpose of our study is to prepare of Monoclonal Antibody, to establish the method of indirect competitive ELISA(Ci-ELISA) detecting ENR residue and to make foundation for exploration and application of the ENR residue test kit.In this study, ENR was coupled to BSA and OVA carrier protein respectively by N-hydroxysuccinimide activated ester method by which ENR-BSA and ENR-OVA were prepared. Both antigens were identified by colour reaction of FecL and UV scan. The results were as follows: exterior solution in dialysis became light yellow while interior solution appeared red result from reaction of FecL. The maximum absorbance peaks of ENR-BSA and ENR-OVA were at 319nm and 320nm wavelength, respectively. And there were a common peak at 332nm wavelength for the two antigens. The detection indicated whether absorbance spectrum of ENR-BSA or ENR-OVA was integration of spectrum of ENR and protein, which included the characteristic absorbance peak of ENR.ENR-BSA was used as immunogen to immunize 8-week-old BALB/C mice for obtaining of antisera. ENR-OVA was used as coating antigen. The positive mice of high titre and antisera against ENR were selected by the Indirect Enzyme linked Immunosorbent Assay (ELSA). Splenocytes from the mice were fused with sp2/0 myeloma cells. Through determining, cloning and selecting, B₆ and G₁2 secreting monoclonal antibodies against ENR were selected. The identification of subclasses indicated that the monoclonal antibodies secreted by B₆ and G₁2 were antibodies of IgG2b group. Mice were injected with by hybridoma to produce ascites, which was purified. SDS-PAGE figure appeared two clear strips, among which one was heavy chain and another was light chain.