Development Of New Techniques Of Fast Detection For Okadaic Acid In Marine Products And Drug Residue In Fishery Products And Their Application

As the gradual expantion of China’s mariculture and fresh water aquaculture, the safty problems of aquatic products are getting more and more attention by people. People who eat the contaminative marine products would be resulted in food-poisoning caused by the shellfish toxicity generated by seaweed gathered in marine products through food chain. And diarrhetic shellfish toxicity (DSP) Poisoning with Okadaic Acid(OA) as its main component caused most damaging Otherwise, with the large-scale seawater and freshwater aquaculture, the phenomenon of drug residues is also a growing concern. Drug residues has become the social focus as it not only affect the quality of the fishery products for export trade, but also threaten the human’healthy seriously. Therefore, in this study chemiluminescence immunoassay assay of okadaic acid in marine products was done and compared with commercially available ELISA kit from other countries and domestic standard methods. In order to accomplish the multi-residue analysis of many banned drugs, aquatic protein chip detection technology was also studied in this paper.1. Study on Enzyme Enhancement Chemiluminescence Immunoassay of okadaic acid in marine products1) Preparation of monoclonal antibody OA The immune antigen of OA-BSA and detection antigen of OA-OVA were prepared by activated-ester method and evaluated by ultraviolet spectrometry, SDS-PAGE and MALDI TOF-MS/MS methods, respectively. The results showed that the immune antigen was synthesized successfully. The conjugate ratio of OA and BSA was4.1:1, which indicated that OA was combined with BSA successfully. Then BALB/c mice were immunized by OA-BSA and the spleen cells of the immune mice were fused with SP2/0cells. After that supernatants from the wells were screened for the production of antibody using an indirect ELISA method and three strain hybridoma cells (4H4、4H7、5C10) secreting antibody were selected.4H4was the best with the IC50value of monoclonal antibody to OA2.15ng/mL. Then, the monoclonal antibody in ascitic fluid was purified using a DEAE cellulose chromatographic column and the titer of the monoclonal antibody was above1:64000.2) Chemiluminescent ELISA proceduresMonoclonal antibody generated by hybridoma cells of4H4was selected and an indirect competitive enzyme-linked immunosorbent assay with chemiluminescent (CL-ELISA) detection for okadaic acid (OA) in mussel muscle was developed. The optimal working concentrations of monoclonal antibody and coating antigen were1:80000and0.5μg/mL, respectively. At the concentration range of0.08125-10ng/mL of OA, a regression equation (y=-0.2436x+0.3721, R2=0.9977) was established to obtain the best fit of the points over the entire range of the assay. Detection limit of the sample was1.232ng/kg, intra-assay and inter-assay coefficient of variation were2.64%and3.14%, respectively. The detection limit was0.175ng/g. The range of average fortified recovery was86-97%and85%-93%when OA was spiked in mussel and clam muscle at levels of50μg/kg,160μg/kg and800μg/kg, respectively. Specificity experiments indicated that the cross-reactivity with dinophysistoxinl and saxitoxin was51.82%and0%, respectively.3) Comparison with foreign ELISA kit and domestic standard methods and preliminary applicationThe chemiluminescent ELISA kit prepared in this study was compared with the commercial ABRAXIS kit in these characters of sensitivity, accuracy and precision. The results indicated that sensitivity of our chemiluminescent ELISA kit was higher than ABRAXIS kit. There were no significant differences in accuracy and precision between them, but the sample pre-treatment process of our kit was simpler and faster than ABRAXIS kit. The results of nine real samples determined by CL-ELISA kit and the ABRAXIS kit had good correlation. Compared with LC-MS SC/T2269-2009method, the detection limit of chemiluminescent ELISA was2.5ug/kg while LC-MS was100μg/kg. The recoveries of the two methods were similar when OA was spiked at the concentrations of100-800μg/kg.125samples were detected by our chemiluminescent ELISA, and43of samples were considered to be positive. The test results of these samples in which the concentrations of OA exceeding100μg/kg, had great correlation between LC-MS method and our CL-ELISA kit. The results showed that our laboratory-made CL-ELISA could meet the detection limit needs of China’s import and export of seafood of OA.2. Study on protein chip analytical technique in detecting drug residues in aquatic productsIn order to study protein chips of four drugs, NH2-LMG, DES-MCPE, MPA-3-(O-carboxymethyl)-oxime and CPAOZ were synthesized. Then these compounds were coupled with BSA (RSA) and OVA as immunizing antigen and coating antigen. The complete antigen was identified by ultraviolet scanning and MALDI-TOF-MS. The results showed that the compounds were coupled with proteins successfully and the conjugate ratios of drug and protein were between1:15and1:30. Four monoclonal antibodies were prepared using the hybridoma monoclonal antibody preparative technique. We found that the monoclonal antibodies of LMG, DES, MPA and AOZ had high titers and low50%inhibition of control values (ICso) of the drugs through the titration and inhibition test. The IC50values of LMG, MPA, DES and AOZ were19.7ng/mL,3.8ng/mL,9.6ng/mL and0.374ng/mL, respectively. And the detection limits of LMG, MPA, DES and AOZ were5.95ng/mL、1.30ng/mL、1.97ng/mL and0.286ng/mL. The sensitivity of MPA, DES and AOZ achieved the needs of detecting banned Drugs. The recoveries of LMG, MPA and DES were between48.7%and94.5%when they were spiked in shrimp and fish samples. The results of the Preliminary practical application of protein chips for detection of drug residues in fish show that the technology can achieve fast, high-throughput, precise quantitative requirements.