赤霉毒素脱氧雪腐镰刀菌烯醇(DON)酶联免疫检测方法研究

【目的】建立快速、灵敏、有效的毒素检测方法，以保证麦类作物的安全生产以及谷物食品的安全性。【方法】以主要赤霉病菌毒素脱氧雪腐镰刀菌烯醇(DON)为对象，利用半琥珀酰化脱氧雪腐镰刀菌烯醇的牛血清蛋白偶联物（3-HS-DON-BSA）作免疫原，分别采用腹膜腔注射法和颈、背部多点注射法免疫Balb/c小鼠和豚鼠，获得DON 的多抗血清，建立间接ELISA检测方法。【结果】多抗豚鼠血清的效价达到1∶6 400，而小鼠混合血清的效价则为1∶12 800。引起DON抗体最大结合50%抑制时，所需DON及其类似物3-Ac-DON和T-2毒素的量分别为 63μg•ml-1、114μg•ml-1和＞1 000μg•ml-1；相对交叉反应率分别为 100%，55.2%和6.3%。包被抗原的最适工作浓度为1/1 500，小鼠血清工作浓度为1/1 600。在包被原和小鼠血清的最适工作浓度下，20%以上的甲醇稀释度对DON免疫分析有显著的影响，低于10%浓度的甲醇对DON免疫分析基本无影响。建立的间接竞争ELISA法检测范围为0.01～100μg•ml-1，检出限为0.02μg•ml-1，平均回收率为82%～93%，精密度(CV%)为4.65%～21.3%。【结论】本文提出的毒素检测方法，检测成本低,方便易行,不仅可以应用于小麦赤霉病的病理学研究，也可广泛应用于谷物及其制成品中DON毒素的含量检测，具有较好的应用价值。

Enzyme-Linked Immunosorbent-Assay for Deoxynivalenol (DON)

This paper reported the preparation of antibody of Fusarium toxin Deoxynivalenol (DON) and establishment of indirect ELISA for DON. 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 1/1600 and 1/1500 respectively. Color development in the assay was inhibited 50% by 63μg DON/ml, 114μg 3-Ac-DON/mL and more than 1000μg T-2/mL respectively. The Cross-reactivity to DON, 3-Ac-DON and T-2 of the antiserum were 100%, 55.2% and less than 6.3%. The effect of methanol concentration on the reaction between the antiserum and DON was studied. The result showed that the assay could be performed satisfactorily using an extraction solvent consisting of less than 10% methanol. Meanwhile, indirect competitive ELISA for detection of DON in wheat seeds was developed with the antibody. The range for detection of DON was 0.1-100μg/ml and the minimum detected concentration was 0.1μg/mL. Recoveries averaged 82%-93% with a coefficient of variation of 4.65%-21.3%. Analysis of 4 samples for DON exhibited positive results.