| Deoxynivalenol (DON) is produced by the Fusariwn spp. and is found worldwide in cereal grains. Because DON is a mycotoxin capable of causing disease in animals and human being, it is very important and imperative to detect DON in foodstuffs. This thesis studied the structure modification, antibody preparation and ELISA for DON.1. The conversion of DON to 3-HS-DON was facilitated by protecting two of the three available hydroxyls with a cyclic boronate ester and making DON react with the C3 hydroxyl. The derivatized product was purified with TLC and analyzed by HPLC and ESI-MS, the mass spectrum of derivatized DON had the expected M-H+ peak at m/z 395.1.2. 3-HS-DON-BSA and 3-HS-DON-OVA were prepared by the carbodiimide reaction The conjugates were confirmed by FPLC. Specific antisera were raised in Balb/c mice and guineas by immunizing them with 3-HS-DON-BSA conjugate, and the liters were 12800 and 6400 respectively when tested by indirect ELISA.3. The optimal concentration of mice serum dilutions and the coated antigen required to observe an absorbance of 1.0 in the indirect competitive ELISA were approximately 1/1600 and 1/1500 respectively.4. Color development in the assay was inhibited 50% by 63 g DON/mL, 114 g 3-Ac-DON/mL and more than 1000 M g T-2/mL respectively. The Cross-reactivity to DON, 3-Ac-DON and T-2 of the antisera were 100%, 55.2% and less than 6.3%.5. The effect of methanol concentration on the reaction between the antisera and DON was studied. The result showed that the assay could be performed satisfactorily using anextraction solvent consisting of less than 10% methanol.6. Meanwhile, indirect competitive ELISA for detection of DON in wheat seeds was developed with the antibody. The range for detection of DON was 0.1-100 M g/mL and the minimum detected concentration was 0.1 Mg/mL. Recoveries averaged 82%-93% with a coefficient of variation of 4.65%-21.3%.7. Analysis of 4 samples for DON showed positive results. |
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