赤霉毒素脱氧雪腐镰刀菌烯醇(DON)酶联免疫检测方法研究

Enzyme-Linked Immunosorbent-Assay for Deoxynivalenol(DON)

【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∶6400,而小鼠混合血清的效价则为1∶12800。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>1000μg·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1/1500,小鼠血清工作浓度为1/1600。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。

[Objective] To detect Fusarium toxin Deoxynivalenol （DON）, a trichothecene mycotoxin occurring in FHB contaminated grain, an indirect competitive enzyme-linked immunosorbent assay （ELISA） was developed. [Method]Conjugation of DON to carrier proteins was facilitated by conversion of DON to 3-O-hemisuccinyl-DON（3-HS-DON）. 3-HS-DON-bovine serum albumin （3-HS-DON-BSA） and 3-HS-DON-ovalbumin （3-HS-DON-OVA） were allowed to produce DON-specific mice antisera. 3HS-DON-OVA was coated onto the microplate, followed by incubation with DON and anti-DON antibody. [Result] The indirect competitive ELISA revealed that the optimai concentration of mice serum and the coated antigen were 1/1 600 and 1/1 500, respectively. The antiserum cross-reacted with 3-AC-DON and T-2 at about 55.2% and 6.3% as much as they did with DON. Studies on the effect of methanol on reaction between the antiserum and DON showed that the assay could be performed effectively by using an extraction buffer containing less than 15% methanol. Recovery of DON was 82%-93% in grains with coefficients of variation of 4.65% to 21.3% at the range of tested dosage. The linear detection range was between 0.01 and 100 μg·ml^-1. [Conclusion] This ELISA can be applied for the detection of DON in grains.