| Trichothecene mycotoxins, mainly produced by fungi, was often found to contaminate grains and foods during their harvest, transportation and storage, causing significant safety hazards. Therefore, it is necessary to establish a convenient, rapid and reliable detection method to detect these toxins in foods and grains. In this study, T-2 toxin and DON (deoxynivalenol) toxin as representative trichothecene mycotoxins were employed as substrates to establish the ELIS A detection method.(1) Firstly, DON was separated and purified from rice solid ferment with Fusarium graminearum by high-speed counter-current chromatography coupled with high performance liquid chromatography.8 mg of DON with the purity of 97.7% were finally obtained from 300 g rice solid ferment. The purified DON was used to conjugate proteins.(2) T-2-HS was synthesized from T-2 using DMAP (dimethylamiopryidine) method. Three completed antigens, namely T-2-HS-BSA, T-2-HS-OVA and T-2-HS-KLH, were prepared by dicyclohexyl carbodiimide method. The conjugate ratio of T-2-HS-BSA was 2.63 measured by MALDI-TOF-MS. Balb/C mice were immunized with T-2-HS-BSA, and high titer of polyclonal antibody was obtained. A competitive indirect ELISA (CI-ELISA) was established. The conditions of this method were:20μg/mL T-2-HS-KLH was coated in plates; blocking buffer is 0.5% OVA; the polyclonal antibody of T-2 toxin was the primary antibody, T-2 toxin standard competed with T-2-HS-KLH. Finally, the detection limit of T-2 toxin was 0.12μg/mL, quantitative detection range was 0.28～30.84μg/mL, IC50 was 1.45μg/mL, the recovery rate of T-2 toxin was 92.0%-104.6% with the coefficients of variation (CVs) 15.1%-34.0%.(3) DON-(HS)n was synthesized from DON by DMAP method. Two completed antigens, namely DON-(HS)n-BSA and DON-(HS)n-KLH, were prepared using dicyclohexyl carbodiimide method. The conjugate of DON-(HS)n-BSA was well identified by MALDI-TOF-MS. Balb/C mice were immunized with DON-(HS)n-BSA. |
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