Preparation Of Monoclonal Antibody And Development Of High-throughput Qualitative And Quantitative Analytical Methods For Zearalenone

Zearalenone(ZEN), a secondary metabolites from Fusarium species, is a known nonsteroidal estrogenic mycotoxin. ZEN may induce the reproductive toxicity, teratogenicity, DNA damage and immune suppression in livestock and human. The chromatographic methods(such as high-performance liquid chromatography, liquid chromatography-mass chromatography) have been used as the standard methods. Although these methods have the sensitive and reliable results, but still require expensive equipments and professional operators. The purposes of this study are to obtain the an anti-ZEN monoclonal antibody, and develop the antibody based immunoassay, which is based on the immunochromatographic theory, electrochemical immune sensing, chemiluminescence signal amplification approaches, suspension array technology. These methods could improve the detection of ZEN and other mycotoxin residues in feed or food.According to ZEN structure, the hapten(ZEN-6- carboxymethoxyphenyl oxime) was synthesized firstly. Based on the carbodiimide method, bovine serum albumin(BSA) or ovalbumin(OVA) was conjugated with hapten to prepare the completely conjugation antigens(BSA-ZEN or OVA-ZEN). After the immunization by the immunogen(BSA-ZEN), the immune cells in spleen were fused with SP2/0 cell. After triplicate screening and subcloning, four hybridoma cell lines which secreting anti-ZEN monoclonal antibodies were obtained. After the evaluation of titer, sensitivity, and stability, the optimum cell line(2C9) was chosen to prepare the indirect competitive ELISA. In the optimum situations of ELISA, the detection limit was 0.051 ng/mL. The cross-reactivity for ZEN structural analogs was 16%, and no cross reactivity for other mycotoxins.Qualitative and quantitative colloidal gold immunochromatographic strips were employed for the rapid detection(less than 30 min) and screening for large scale samples. The anti-ZEN monoclonal antibody and antifumonisin B1 monoclonal antibody, which preparing in our laboratory, were labeled with 25 nm colloidal gold nanoparticles respectively. BSA-ZEN and OVA-FB1 were sprayed on the nitrocellulose membrane as two test lines, and horseradish peroxidase labeled goat anti-mouse antibody was sprayed as control line. The materials of test strip, the types and pH of buffers, types and concentrations of additives, sample dilution ratio, concentration of coating antigen and gold labeled antibody were optimized. The detection limits of dual qualitative immunochromatography strip were 6 and 50 ng/mL for ZEN and FB1 respectively. For the dual quantitative strip, the detection limits were 0.35 and 5.23 ng/mL for ZEN and FB1 respectively. The sensitively of these immunochromatographic strips were significantly better than the previous studies.Highly sensitive detection method is urgently needed for the effective regulation of ZEN in specific foods. Based on the principle of alkaline phosphatase(ALP) converting the substrate p-nitrophenylphosphate(pNPP, colorless, non-electrochemical activity) to the p-nitrophenol(pNP, yellow product, electrochemical activity), electrochemical sensing and ELISA were prepared to detect the ZEN. The type of reaction buffer, p H, electrochemical parameters, concentrations of coating antigen and antibody were optimized. Combination with the ELISA, the sensitivity can be achieved as 0.002 ng/mL, the detection range was 0.004-9.5 ng/mL. Comparison with previous electrochemical method, this study can reach the purpose to broaden the detection range, also highly sensitive, accurate and rapid.Based on the chemiluminescence signal amplification systems which could improve the sensitivity of the detection, three types direct competitive chemiluminescence immunoassay methods were prepared using two signal amplification strategies(biotin-streptavidin system, gold nanoparticle system). The concentrations of reagents, methanol and incubation time were optimized. Comparing the sensitivity of the three methods, the detection limit was gradually increased with the adding of signal amplification systems. And the detection limit was eventually increased to 0.008 ng/mL. This method was the first report for the use of two signal amplification strategies at the same time, and also the first report for the use of double codified gold nanoparticles technology in the field of agriculture and food. This study provides a rapid and accurate platform for the trace detection in the samples.In order to detect a variety of mycotoxins(ZEN, FB1, deoxynivalenol, aflatoxin B1) in cereals which often co-exist, a new multiple suspension arrays was prepared. Four anti-mycotoxin monoclonal antibodies were coated on the different encoding polyphenylene ethylene microspheres surface(No. 49, 39, 37, 19), and four mycotoxins complete antigen were conjugated with biotin respectively. The streptavidin-phycoerythrin was employed as a signal reporter protein finally. The concentrations of the reaction reagents were optimized. The results show that the detection limit of ZEN, FB1, DON, of aflatoxin B1 were 0.51, 6.0, 4.3 and 0.56 ng/mL in this method. This method has a good prospect for application in the future.In this study, different analytical methods, including ELISA, dual qualitative colloidal gold strip, dual quantitative colloidal gold strip, electrochemical immunoassay, chemiluminescence immunoassay and multiple suspension array detection method were prepared. These methods meet the demands of cost-less, high sensitivity, multiplex detection, rapid and high-throughput detection. This study provides technical reserve for the mycotoxins and other low weight analytes detection, and builds a broad detection platform.