| Recently, with the sequent occurrence of food safty issues in china, the food safty has been regarded more and more. The causes of food issues are generally. divided into two parts; one is the abuse of food additives and the illegal added of chemical substances, the other is drug residues in food raw. Generally, reasonable amounts of food additives added in food are harmless even beneficial for human, but excessive added would cause health problems after human consumption, such as the melamine scare in China events. Generally, some veterinary drugs and feed additives are used in animal feeding such as feather. The overproof section of additives would stay unavoidablely and produce severely impair on animal and human body over the food chain. In order to effectively monitor the food safety, it has a great social significance to set up rapidly and convenient assay methods.Gatifloxacin is a kind of broad-spectrum antibiotic which is often added to feed in order to increase the disease resistance of livestock for its excellent antibacterial ability. But the gatiflixacin is very easily absorbated by human body through the food chain and affecting people's health heavily. Melamine is a sort of chemical raw materials. Unfortunately, many illegal companies adopted melamine as food addictives due to its high nitrogen content and low price to enhance the nitrogen content during the Food Testing. Since the cases of pets died of melamine in the United States and babies suffering from stone event in China have broken out, more and more people have recognized the harm of the illegal applied. Therefore, many countries have provided the maximum detecting limit with gatifloxacin and melamine in food. At present, in order to detect the residues of gatifloxacin and melamine accurately in food, many simple and advantageous methods have established. Same of them even have been recognized as the national standards. Instrumental detection methods include liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), high performance liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC-MS), gas chromatography-mass spectrometry/mass spectrometry Law (GC-MS/MS) and so on. Those methods have many advantages of specific, stable and accurate, but they require expensive equipments, long time-consuming sample clean-up process and trained operator in the implementation process. Immunoassays including CL-ELISA, EICA, ELISA, etc. Those methods are sensitive, convenient and exact, which overcome the shortcomings of these methods mentioned above. Moreover, they have the ability for the real-time detection of a large number of samples without any other large instruments. In a word, these methods have extensive prospects for development based on their merits.For electrochemical detection of gatifloxacin studies, the immunogen and coating antigen were obtained by EDC method. We prepared polyclonal antibodies for gatifloxacin and the carbon electrode to develop an electrochemical immunoassay in this study. The immunoassay based on the antibody for gatifloxacin showed great specificity and sensitivity with IC50 8.9 ng/mL and the detection limit 0.01 ng/mL. Using this electrochemical ELISA method for the determination of gatifloxacin in pig urine has an excellent effect. The recoveries rates were in range from 93.72% to 117.36%, and the coefficients of variation were between 2.16% to 13.38%.For the preparation of melamine ELISA kit, we synthesized the immunogen and coating antigen via the amido scheme on the basis of the structure of melamine. Through the animal immunization and monoclonal antibody technique, we obtained a lot of specificity monoclonal antibodies for melamine. At the same time, we have utilized the monoclonal antibidies to prepare the direct competition ELISA kit for melamine. The test dimension of the melamine kit shows high sensitivity, with IC50 value of 134.8 ng/mL and the detective limit value of 20 ng/mL toword melamine.There is small cross-reaction with most related species and compounds. We also have preceded the accelerated stability test, and the results showed the ELISA kit can keep long hours. The recovery rates of the kit were in the range of 73-94% for milk,53-73% for milk powder and 52-65% for animal feed. The coefficients of variation of the kit were 9-15.4% for milk,9-15.3% for powder milk and 12.4-18.3% for animal feed.In conclusion, we have successfully synthesized immunogen and coating antigen of gatifloxacin, and also obtained polyclonal antibody for gatifloxacin by animal immunization. Using the antibodies of gatifloxacin, we have successfully established sensitive electrochemical immunoassay to detect the residues of gatifloxacin in pig urine. Simultaneity, this study prepared the monoclonal antibody of melamine and the ELISA kit for the determination of melamine. The ELISA kit showed a great effect for detecting melamine in creamery, powered mik and animal feed. These results in this study can provide a feasible basis for the development of drug testing kits and open the way for application of the ELISA technology. |
PDF Full Text Request