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Chemiluminescence And Labeled-avidin-biotin Enzyme-Linked Immunosorbent Assay For ZEN Determination In Corn

Posted on:2012-12-01Degree:MasterType:Thesis
Country:ChinaCandidate:J S ChuFull Text:PDF
GTID:2214330338469117Subject:Fermentation engineering
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Zearalenone (ZEN) (previously known as F-2 toxin) is a non-steroidal oestrogenic mycotoxin biosynthesized through a polyketide pathway by a variety of Fusarium fungi which are common soil fungi,in temperate and warm countries, and are regular contaminants of cereal crops worldwide.Because of its structure similar to oestrogenic, ZEN could cause a series of domino offect by interaction with the estrogen receptors. In studies,the toxicity include reproductive and developmental toxicity, genotoxicity, immunotoxicity, chronic toxicity and carcinogenicity. At present, there are several methods to detect ZEN, such as microorganism detection, instrument detetion and immunoassays. ELISA which belonging to immunoassay is a sensitive, rapid method for screening large number of samples and it is the most commonly used methods in immunoassays because of its simple operation. Chemiluminescence and biotin-avdin system are two effective ways to improve the sensitivity of immunological detection methods. In this study, chemiluminescence analysis and biotin-avidin system were used to establish CLEIA and BA-ELISA for detecting ZEN in corn. The results show as follows:1 The establishment of CLEIA to detect ZEN in corn.The horseradish peroxidase (HRP)-luminol-H2O2 chemiluminescent system with high sensitivity was chosen as the detetion system. To obtain maximized sensitivity enhancement in chemiluminescent detection, assay conditions were optimized. The optimized CLEIA gave a limit detetion of 0.09 ng/mL and the linear working range was 0.104-1.69 ng/mL, the total reaction time was 60 min. The mean intraassay and interassay of coefficients of variation were 9.1% and 14.7%, respectively. The recoveries of ZEN added to corn were range from 79.6% to 97.6%.40 samples were also analyzed by this CLEIA and the results show a good correlation to ELISA kit.2 The establishment of DC-CLEIA to detect ZEN in corn.HRP was successfully coupled to antibody(IgG) at different molar ratio by glutaraldehyde method and periodate method, respectively. And the HRP-IgG conjugates were confirmed by UV-scanning. The ELISA results shown that the titer conjugates at molar ratio of 1:20 is best in glutaraldehyde method compared with 1:1.5 in periodate method. And the titer of conjugates in periodate was higher than the conjugates in glutaraldehyde method.Luminol solution was used as the substrate of horseradish peroxidase. The detection limit was 0.084 ng/mL. The DC-CLEIA was 3 times more sensitive compared to the colorimetric-ELISA. When ZEN was spiked in corn at levels of 50-500g/kg, recoveries ranged from 77.0 to 95.3%. In an actual residue study, the results obtained by DC-CLEIA correlated well with those obtained by ELISA kit with a correlative coefficient of 0.925.3 The establishment of BA-ELISA to detect ZEN in corn.Activated to reactive ester (BNHS), the biotin was successfully labeled to antibody (IgG). After optimization, the reaction conditions of BNHS and IgG is that: the BNHS was added dropwise to a stirred solution of IgG using molar ratio of 100:1 and adjust the pH to 7.5, then the reaction mixture was kept 1 hour at 37℃.The HRP was successfully coupled to avidin and the diluent of this HRP-avidin conjugate was selected. The results shown that the titer of HRP-avidin conjugate at molar ratio 2:1 is higher than that of 3:1.And the non-specific adsorption is stronger. 5×PBS was selected as the diluent of HRP-avidin conjugate in BA-ELISA.By checherboard titration, the working concentration of antigen was selected 1.6 g/mL and antibody was diluted 1:4000. After optimization, the detection limit of the method was found to 0.42 ng/mL ng/mL, which is 6-fold more sensitive than the traditional competitive ELISA using the same antibody and coating antigen and the linear range is 0.54-7.99 ng/mL. Samples could be tested with a simle and rapid extraction procedure, and recoveries (86.6-93.7%) were got. This method was successfully applied to determine ZEN in 27 corn samples and good correlation with the ELISA Kit (0.9701) were obtained. The result indicated that the biotin-avidin system may be a valuable tool to improve the specific detection of ZEN contamination.
Keywords/Search Tags:chemiluminescence enzyme immunoassay, biotin-avidin system, zearelenone
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