| Tetrodotoxin mainly exists in the puffer fish, and it is a deadly marine toxins. Because of its low molecular weight and the unique chemical structure, it can be combined with sodium ion channel specificly, blocking nerve conduction nerve, causing muscle paralysis, can make the Organization eventually causes to death. In recent years, tetrodotoxin food poisoning accidents occur frequently and the study of pharmacological effect of tetrodotoxin develops, because of its unique role, TTX has became a hot topic on pharmacology studies. China as a rich country of marine resources, there is a sharp increasing need of tetrodotoxin. So there is a urgent need to establish a new detection method which meet the requirements of simpleness, quickness,accuracy.Fluorescence polarization immunoassay as a homogeneous immunoassay method, compared with the traditional immunoassay like ELISA, FPIA method operation procedure does not need the separation and washing steps, so to shorten the time needed for the whole process of analysis. And FPIA is simple, sensitive and fast, which could realize the automatic analysis, and it is suitable for high-throughput screening applications, suitable for environmental monitoring, drug analysis, food safety control.1. In this research we used the prepared anti-tetrodotoxin hybridoma cell which is prepared by hybridoma cell fusion technology, we injected 2.5×105 hybridoma cells to each of Balb/c mice by intraperitoneal injection which had stimulated with liquid paraffin for 7 d, the mice abdomen increased significantly after 10 d, produced a lot of abdominal water.2. Ascites was purified by method of SAS to purify the monoclonal antibody initially, then we used immuno-affinity chromatography to purify the monoclonal antibody further by the immuno-chromatography column of Hitrap rProtein A, the antibody titer was measure by the method of indirect ELISA method for ascites, antibody purified by SAS, and antibody purified by immuno-affinity, after purifying by the immuno-affinity chromatography,the antibody titer decreased significantly, from which 2.6×105 decreased to 7×104.3. In this study we used fluorescence polarization immunoassay, developed fluorescence polarization immunoassay method for detecting tetrodotoxin. Through the condition of EDC and DMAP, the nanomaterial quantum dot and tetrodotoxin was conjugated, and with the purified tetrodotoxin FPIA tracer, we obtained the optimal tracer dilution, and optimal anti-tetrodotoxin monoclonal antibody dilution, and then we developed fluorescence polarization immunoassay method for the detection of tetrodotoxin successfully. The detection range of the method for detection of TTX was from 1.36 ng/mL to 101.27 ng/mL, with the IC50 value is 11.76 ng/mL. The limit of detection was 0.67 ng/mL. The recovery test result was 99.91±2.60%, with the CV value of 2.60%.4. we compared FPIA method with competitive ELISA method, determined the value of competitive ELISA method as the independent variable parameter x, detection value of FPIA as the parameter y, after linear regression analysis, the regression equation formula obtained was: y=0.991x-0.299, R2=0.999. Through which we indicated that the FPIA method and the competitive ELISA method showed a good correlation. And we determined that the accuracy and sensitivity of tetrodotoxin FPIA method was great. The FPIA method which was developed could provide a new direction of tetrodotoxin detection in clinical diagnosis, food safety detection. |
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