间接竞争酶联免疫吸附法快速测定黄曲霉毒素B1、T-2毒素、玉米赤霉烯酮

Rapid determination of aflatoxin B1, T-2 toxin and zearalenone by indirect competitive enzyme-linked immunosorbent assay

中文关键词:  黄曲霉毒素B1  T-2毒素  玉米赤霉烯酮  间接竞争ELISA

英文关键词:aflatoxin B1  T-2 toxin  zearalenone  indirect competitive enzyme-linked immunosorbent assay

中文摘要:

      目的 建立间接竞争酶联免疫吸附测定(indirect competitive enzyme-linked immunosorbent assay, ic-ELISA)法快速检测农产品中常见且毒性较强的黄曲霉毒素B1 (aflatoxin B1, AFB1)、T-2毒素和玉米赤霉烯酮(zearalenone, ZEA)的方法。方法 本研究以高亲和力单克隆抗体为识别元件, 基于ic-ELISA原理, 系统优化了包被抗原类型与浓度、抗体稀释倍数、抗原稀释液组成及竞争孵育时间等关键参数。分别建立AFB1、T-2毒素与ZEA的检测体系, 并通过加标回收实验验证方法学性能。结果 3种毒素完全抗原偶联成功。单克隆抗体效价分别为AFB1: 2.56×104; T-2毒素: 8.19×105; ZEA: 2.56×104, 且与其他真菌毒素无交叉反应。最优检测条件为: AFB1[包被抗原AFB1-牛血清白蛋白(bovine serum albumin, BSA), 质量浓度8 μg/mL, 一抗稀释1:6400, 抗原稀释液为含10%甲醇+0.010%吐温-20的0.010 mol/L 磷酸盐缓冲液(phosphate-buffered saline, PBS), pH 9.0, 竞争孵育45 min]、T-2毒素(包被抗原T-2-BSA, 质量浓度8 μg/mL, 一抗稀释1:25600, 抗原稀释液为含10%甲醇+0.005 mol/L PBS, pH 7.4, 竞争孵育30 min)、ZEA[包被抗原ZEA-鸡卵清白蛋白(ovalbumin, OVA), 质量浓度0.50 μg/mL, 一抗稀释1:1600, 抗原稀释液为含10%甲醇+0.010 mol/L PBS, pH 6.0, 竞争孵育60 min]。方法性能: AFB1检出限0.0273 ng/mL, 线性范围0.047~0.588 ng/mL, 加标回收率104.0%~110.0%; T-2毒素检出限0.183 ng/mL, 线性范围0.288~7.493 ng/mL, 加标回收率107.3%~108.3%; ZEA检出限1.090 ng/mL, 线性范围2.319~69.533 ng/mL, 加标回收率97.9%~112.3%, 所有指标变异系数均小于15%。结论 本研究建立的ic-ELISA方法灵敏度高、重复性好、特异性强, 可实现对谷物样品中AFB1、T-2毒素和ZEA的快速筛查, 为农产品真菌毒素污染监测与风险评估提供技术支撑。

英文摘要:

      Objective To establish a method for the rapid detection of 3 kinds of common and highly toxic mycotoxins in agricultural products—aflatoxin B1 (AFB1), T-2 toxin and zearalenone (ZEA) by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). Methods High-affinity monoclonal antibodies were used as recognition elements, based on the ic-ELISA principle, and key ic-ELISA parameters—including coating antigen type and concentration, antibody dilution ratio, antigen diluent composition and competition incubation time—were systematically optimized. Independent detection systems for AFB1, T-2 toxin and ZEA were established and their performance was validated through spiked recovery experiments. Results The complete antigens for the 3 kinds of mycotoxins were successfully conjugated. The titers of the corresponding monoclonal antibodies were 2.56×104 for AFB1, 8.19×105 for T-2 toxin and 2.56×104 for ZEA, with no cross-reactivity observed with other mycotoxins. The optimal detection conditions were as follows: For AFB1—coating antigen AFB1-bovine serum albumin (BSA) at 8 μg/mL, primary antibody dilution of 1:6400, antigen diluent comprising 0.010 mol/L phosphate-buffered saline (PBS) (pH 9.0) containing 10% methanol and 0.010% Tween-20 and a competition incubation time of 45 min; for T-2 toxin—coating antigen T-2-BSA at 8 μg/mL, primary antibody dilution of 1:25600, antigen diluent comprising 0.005 mol/L PBS (pH 7.4) containing 10% methanol and a competition incubation time of 30 min; for ZEA—coating antigen ZEA-ovalbumin (OVA) at 0.5 μg/mL, primary antibody dilution of 1:1600, antigen diluent comprising 0.010 mol/L PBS (pH 6.0) containing 10% methanol and a competition incubation time of 60 min. The method performance parameters were as follows: Limits of detection of 0.0273 ng/mL (AFB1), 0.183 ng/mL (T-2 toxin) and 1.09 ng/mL (ZEA), with corresponding linear ranges of 0.047–0.588, 0.288–7.493 and 2.319–69.533 ng/mL. Spiked recoveries were 104.0%–110.0%, 107.3%–108.3% and 97.9%–112.3%, respectively and all coefficient of variations were below 15%. Conclusion The established ic-ELISA method demonstrates high sensitivity, good reproducibility and strong specificity, enabling the rapid screening of AFB1, T-2 toxin and ZEA in grain samples. It provides reliable technical support for the monitoring and risk assessment of mycotoxin contamination in agricultural products.