Studies On Enzyme Immunoassay Of Olaquindox

With the constant improvement of people's material living and the rapid development of animal husbandry, more and more people pay attention to the research and development of the new methods on the testing of drug residues in edible livestock products and the evaluation of food safety. Olaquindox, which is a synthized food additive, was reported that the accumulative toxicity could be caused by the improper use of this drug. It was banned in poultry and fish feed. Thus, it is necessary to detect the content and evaluate safety of OLA in livestock products,feed or feed additive. At present, the residues or concent was measured by HPLC. This method needs the large-scale equipment, high consuming cost, tedious pre-treatment of sample, long time on detecting and so on. Therefore, it is useful and helpful to develop the rapid detection method. In this study, the detection method of ELISA on olaquindox was established, the artificial antigen of OLA was synthized, the polyclonal antibody was prepared. The study provided a rapid measuing way on olaquindox residues or content in edible meat or animal foods.The experimental results showed: By TLC ,MP ,IR ,MS ,1H NMR ,OLA-HS which was synthesized by chemical reaction accords with structural design, and–COOH was successful introduced at–OH site in molecular structure of olaquindox; In UV map, OLA-BSA,OLA-KLH were similar to substrates, but also had differents. With the identification of SDS-PAGE, the M.W of OLA-BSA was greater than BSA, the coupling ratio of OLA-BSA and OLA-KLH was 5.2:1 and 7.8:1; After 6 times immunization of New Zealand white rabbits, the anti-serum was up to 128000; In the establishment of the OLA's ELISA detection methods, the optimal concentration of coating antigen﹑second antibody﹑anti-serum were determined at 10-3mg/ml﹑1:10000﹑1:8000, respectively; The anti-serum was purified by caprylic acid-ammonium sufate precipitation.When the optimal working dilution was 1:2000, the method has higher sensitivity. At the concentration of 0.1 ~ 100μg/ml, between the concentration and inhibition rate have a good linearity. The linear regression equation: I = 12.313LogC +22.662 (R2 = 0.9992), sensitivity of 0.122μg/ml; In the experiment of antibody cross-reaction, the anti-serum and Kuianchun which similar to OLA had cross-reaction, but the inhibition ratio was significantly subordinate to OLA.Chlorine and clenbuterol had no cross-reaction with kuianchun.The preliminary conclusions for this experiment: the target antigen OLA-BSA and OLA-KLH, with the high combined ratio between OLA and carrier protein, was successfully synthized by the Succinic anhydride method and the active esterification method; after the rabbit was repeatedly immuned 6 times by the sythized antigen, the anti-serum with high titer was harvested in the animals. The cross-reaction was observed bweteen this drug and its structural analogues. In next experiments, the specificity of anti-serum should to be improved.