Preparation Of Monoclonal Antibody Against Tetracycline And Its Preliminary Application Research

Tetracycline (TC) is widely used in treatment and prevention of animal disease since it has the advantages of broad-spetrum and curative effect.Recently, however, due to its acute side-effect to people's healthy resulting from the residue is animal tissue, it has brought people's great attention to the residue in animal food. In order to establish a sensitive method to detect TC residue, monoclonal antibody against TC has been prepared and the Ci-ELISA method has been developed in this study.1. The immunogen(TC-BSA)and coating antigen(TC-OVA) were prepared, and both antigens were identified by UV scan. The results were as follows: The maximum absorbance peaks of TC-BSA and TC-OVA were at 276.6nm and 278nm wavelength respectively. And there were a characteristic absorbance peak for the two antigens. The results showed that TC and carrier proteins condensate successfully.2. TC-BSA was used as immunogen to immunize 8-week-old BALB/C mice for obtaining the antiserum. TC-OVA was used as coating antigen. The positive mice of high antiserum titre against TC were selected by the Indirect Enzyme linked immunosorbent assay (ELISA). Spleen cells from the positive mice were fused with sp2/0 cells by 50%PEG-1500.Through detecting,cloning and selecting, two kinds of hybridization tumor cells that can secrete the monoclonal antibodies against TC was selected. The identification of subclasses indicated that the monoclonal antibodies secreted by E(10) and D₅ were antibodies of IgG2b group. Mice were injected with by hybrid cells to produce ascites which has to be purified, and the purified protein content was 1.326 mg/mL. SDS-PAGE figure appeared two clear strips, among which one was heavy chain and the other was light chain.3. Ci-ELISA method was developed preliminarily with the purified monoclonal antibodies. The working contents of coating antigen and monoclonal antibody were determined, which is 5μg/mL and 1:8000 respectively, and the reaction conditions was optimized. Through analysis of Ci-ELISA, the standard curve was drawed, and its standard equation was y=-20.439x+81.03 (R²=0.9498);LOD was 0.68ng/mL;IC₅₀ was 31.62ng/mL. The crossing reaction results indicated the method of Ci-ELISA has high sensitivity and good specificity.4. A series of concentrations of TC was added into milk, then the standard curve was drawed by milk mediator. Three kinds of TC concentration(high concentration, medium concentration, low concentration)was added to milk to measure TC recovery rate, and the recovery rate was between 61.3% and 76.5 %.The final results showed that the TC residue in milk can be detected by the established Ci-ELISA with the prepared monoclonal antibody against TC.