| With the development of international trade in food, the need for the animal-origin food is increasing greatly, so animal husbandry industry has developed rapidly. However, due to the lack of scientific knowledge and wanting to make more money, the phenomenon that some farmers use veterinary drug illegally is widespread in the breeding industry, and the situation is particularly serious in China. In recent years, Chinese food exports were blocked repeatedly because of the problem of drug residues. It caused huge economic losses, and damaged the image of Chinese food seriously. Drug residues lead to not only human acute and chronic toxic directly and bacterial resistance, but also potential harm to human health indirectly through the food chain, such as allergy, deformity, mutation, cancer and so on. Therefore, the safety issue of drug residues in animal-origin food has been highly concerned in the world.Food safety work is not only the need to the development of animal husbandry and Chinese foreign trade is also the need to the political and economic stability. Therefore, it becomes important to control drug residues level in animal-origin food.Many developed countries attach great importance to monitoring of drug residue; they issued a series of regulations, drawn up detailed standards for drug residue limits, and strictly implement the monitoring, supervision and inspection of drug residue. In recent years, our government also has a high regard to this issue, strict residue standards and off-drug period have been formulated to limit and effectively control drug residues in animal-origin food. They take law, economy, administration and technology to monitoring, draw lessons from foreign advanced experience and technology, and screen a set of feasible detection method about drugs residues on the basis of our nationality.At present the analytical techniques to detect drug residues mainly include physical and chemical detection methods (such as HPLC,LC-MS/MS), microbial detection method, immunosorbent assay (including ELISA, RIA, EIA, FIA, MAIA). Physical and chemical detection methods can be considered as the standard method to detect drug residues, because it is accurate, stable and reliable, but the equipment used in these methods is expensive, plus complex, time-consuming and laborious sample treatment procedure. So it is difficult to popularize. The results measured by the microbial detection methods are usually higher than the Maximum Residue Limits (MRLs). A rapid, sensitive and effective detection method must be established. Enzyme-linked immunoassay (including ELISA, CL-ELISA, and MAIA) overcomes the shortcomings of the methods mentioned above and is a rapid, sensitive, convenient, easy universal detection method. This paper aims at establishing chemiluminescence enzyme-linked immunoassay (CL-ELISA) to detect ractopamine, magnetic affinity immunoassay (MAIA) to detect chloramphenicol and enzyme-linked immunoassay (ELISA) to detect melamine.For ractopamine, first, we synthesized the immunogen (Rac-cBSA) via the Para amino benzoic acid scheme on the basis of the structure of ractopamine. The immunogen was injected into New Zealand white rabbits and we obtained polyclonal antibodies to develop an immunoassay in this study. At the same time, we prepared coating antigen by the BDE scheme. Then using the indirect CL-ELISA, we obtained the results:The anti-ractopamine shows high sensitivity, with an IC50 value of 0.45 ppb toward ractopamine; the antibody has a detective limit of 0.001 ppb and a linear range of 5.0-405 ppb; with high specificity, there is no detectable cross-reactivity with the most other similar drugs. Recovery rates from the ractopamine in milk samples were in the range of 70-125%, while the intra-assay and inter-assay coefficients of variation were<12.0% and<13.0% respectively.For Chloramphenicol, through the direct MAIA, we obtained the results:The anti-Chloramphenicol shows high sensitivity, with an IC50 value of 0.05 ppb toward Chloramphenicol; the antibody has a linear range of 0.005-5.0 ppb; with high specificity, there is only lower cross-reactivity with Chloramphenicol Succinate (6.25%)and no detectable cross-reactivity with the most other similar drugs. Recovery rates from the Chloramphenicol in milk samples were in the range of 85.2-113.7%, while the coefficients of variation were in the range of 9.9-22%.For melamine, first, we synthesized the immunogen (MEL-TG) via the EDC scheme on the basis of the structure of melamine. The immunogen was injected into mice and we obtained monoclonal antibody to develop an immunoassay in this study. At the same time, we prepared coating antigen by the same scheme. Then using the indirect ELISA, we obtained the results:The anti-melamine shows high sensitivity, with an IC50 value of 22.6 ppb toward melamine; the antibody has a detective limit of 5.0 ppb and a linear range of 0.01-10.0 ppb; with high specificity, there is no detectable cross-reactivity with the most other similar drugs. Recovery rates from the ractopamine in milk samples were in the range of 78-120%, while the intra-assay and inter-assay coefficients of variation were<18.1% and<24.7% respectively.Through this study, we have successfully prepared specific polyclonal antibody for ractopamine, and monoclonal antibody for melamine. Using these antibodies, we established rapid sensitive methods to detect the residues of ractopamine Chloramphenicol and melamine, at the same time, we identified CL-ELISA and MAIA shows a significant improvement in sensitivity and detestability compared with traditional ELISA method. There researches can be used to develop various local immunoassay kits and open the way for detection kits of other drugs... |
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