| ZEN and DON are the toxic secondary metabolites mainly produced by Fusarium species, such as Fusarium culmorum, Fusarium graminearum and so on. ZEN and DON wich could induce reproduction toxicity, immunotoxicity, cytotoxicity and cancer are common contaminant in maize, barley, wheat and grain. Up to now, the currently used methods to detect ZEN, DON and other mycotoxins are instrument detection methods and immunoasssys. However the instrument detection methods require exorbitant expenses, time consuming, extensive extraction and sample cleanup and immunoasssys have become more and more popular by the analysis of test department, especially by the grassroots inspection department. Due to its rapid, sensitive and low-cost feature, ELISA was widely used in the detection of various mycotoxins, and preparation of good titre, sensitivity and specificity antibody is the key of ELISA.In this study, two different methods, activated ester method and Mannich method, were applied to construct the ZEN complete antigens, respectively. The ZEN complete antigens had been identified and analyzed by ultraviolet spectroscopy. And activated ester method and CDI method were applied to construct the DON complete antigens, and the TNBS was used to identifity and analyze the DON complete antigens. After immunized BALB/c mouse with the ZEN and DON complete antigens, respectively, we analyze the titre, the sensitivity and the specificity of the antiserum by ELISA. The main results were as follows.1. Preparation for artificial antigen of ZENZEN is low molecule substance with 318.4 molecule weight, and hapten, which has reactionogenicity without immunogenicity. ZEN must couple with carrier protein to form complete antigen in order to excite animals to secrect anti-ZEN antibodies. Two different methods, activated ester method and Mannich method, were applied to construct the ZEN complete antigens in this paper. In activated ester method, ZEN was first derived to ZENO (ZEN-oxime). Then ZENO was conjugated with cBSA (cOVA). ZEN was conjugated with BSA (OVA) directly by formaldehyde through Mannich method. From UV spectrum of complete antigens, we could conclude that the coupling of hapten to carrier protein was successful, and the molar ratio of nZENO:ncBSA, nZENO:ncOVA, nZEN:nBSA and nZEN:nOVA were 9.2:1,10.3:1,15.9:1,6.8:1, respectively. 2. Preparation for artificial antigen of DONDON is low molecule substance with 296.3 molecule weight, In this study, two different methods, activated ester method and CDI method were applied to construct the DON complete antigens, In activated ester method, DON was derived to 3-HS-DON, and then 3-HS-DON was conjugated with BSA. In CDI method, DON was conjugated with OVA directly by CDI. TNBS was used to detection free amino of carrier protein and complete antigen, respectively. The results showed that free amino of complete antigen was less than carrier protein, from this we could conclude that the coupling of hapten to carrier protein was successful, and the molar ratio of n3-HS-DON:ncBSA and nDON:oOVA were 9.5:1,7.2:1, respectively.3. Production of anti-ZEN and anti-DON antibodyThe cgonjuate of ZEN-BSA, ZENO-cBSA,3-HS-DON-cBSA were injected into BALB/c mouse with multi-site, respectively. Three mice for each complete antigen, and the mice were sequentially numbered (No.1～No.9). After analyze the production process of antibody, the conclusions could be made that antibody began to obviously after the second immunization. After the 4th immunization, the highest ELISA titer of anti-ZEN antiserum of mouse No.3 reached to 1:12 800, and the ELISA titer of antiserum of mouse No.1 and No.2 reached to 1:6 400. The highest ELISA titer of anti-ZEN antiserum of mouse No.4 and No.5 reached to 1:12 800, and the ELISA titer of antiserum of mouse No.6 reached to 1:6400. The highest ELISA titer of anti-DON antiserum of mouse No.9 reached to 1:6 400, and the ELISA titer of antiserum of mouse No.7 and No.8 reached to 1:3 200. Select the higher ELISA titer of mouse in each group, mouse No.3, mouse No.5 and mouse No.9 to follow experiments.4. Optimization of ELISA conditionsThe optimum working concentration of coating antigen and optimum dilution ratio of antiserum were determined by chequerboard titration test. The antiserum of mouse No.3 was diluted to 1:12 800 by PBST and the optimum concentration of ZEN-OVA was 1μg/mL, The antiserum of mouse No.5 was diluted to 1:12 800 by PBST and the optimum concentration of ZENO-cOVA was 1μg/mL and The antiserum of mouse No.9 was diluted to 1:12 800 by PBST and the optimum concentration of DON-OVA was 4μg/mL. 5. Analysis of the sensitivity and the specificity of the antiserumAccording to optimum conditions, the antiserum of mouse No.3, No.5 and No.9 were analyzed by indirect competitive ELISA. Calibration graphs was formed by the logarithm of the concentration of competing antigen against the inhibitory rate, the linear equation of mouse No.3 was y=-12.92x+100.35 (R2=0.9878), the IC50 of ZEN reached 7.6μg/mL, the limit of detection was 0.075μg/mL, the linear ranges was 0.01～10μg/mL. The linear equation of mouse No.5 was y=-13.61x+100.95 (R2=0.9919), the IC50 of ZEN reached 5.8μg/mL, the limit of detection was 0.093μg/mL, the linear ranges was 0.01～10μg/mL. The linear equation of mouse No.9 was y=-12.35x+101.05 (R2=0.9906), the IC50 of DON reached 14.2μg/mL, the limit of detection was 0.103μg/mL, the linear ranges was 0.01～100μg/mL. According to the cross reaction experiment, the antiserum of mouse No.3, No.5 and No.9 have good specificity. |
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