基于纳米抗体的可再生免疫亲和柱结合高效液相色谱-串联质谱法测定玉米中的黄曲霉毒素B1

Determination of aflatoxin B1 in maize by high performance liquid chromatography-tandem mass spectrometry coupled with a renewable immunoaffinity column based on nanobodies

中文关键词:  黄曲霉毒素B1  纳米抗体  可再生免疫亲和柱  高效液相色谱串联质谱法  玉米

英文关键词:aflatoxin B1  nanobody  renewable immunoaffinity columns  high performance liquid chromatography-tandem mass spectrometry  corn

中文摘要:

      目的 研究黄曲霉毒素B1 (aflatoxin B1, AFB1)纳米抗体的表达产量的影响因素, 开发具有可再生性的AFB1免疫亲和柱, 构建免疫亲和处理-高效液相色谱-串联质谱法(high performance liquid chromatography-tandem mass spectrometry, HPLC-MS/MS)测定玉米中AFB1污染的分析方法。方法 研究诱导温度、诱导时间和诱导剂浓度等对AFB1纳米抗体表达量的影响, 比对验证AFB1纳米抗体可再生免疫亲和柱的耐受性和可再生使用次数。使用70%甲醇水溶液提取玉米样品中AFB1, 提取液通过免疫亲和柱净化富集后进行HPLC-MS/MS检测, 最后进行方法学验证并应用到实际样品检测。结果 诱导AFB1纳米抗体表达的最优条件分别为诱导温度16℃、诱导剂异丙基-β-D-硫代吡喃半乳糖苷浓度0.5 mmol/L、诱导时间14 h, 在最优条件下产量可达7.8 mg/L。AFB1纳米抗体具有良好灵敏度、亲和性、特异性和甲醇耐受性, 制备的AFB1免疫亲和柱具有极好的可再生性, 重复使用150次以上, 对AFB1回收率仍可达80%以上。同时, 建立的免疫亲和处理-HPLC-MS/MS, 在0.1~100.0 μg/L范围内呈现良好的线性关系, 检出限为0.014 μg/L, 定量限为0.047 μg/L。在3个不同加标浓度下, 回收率在92.0%~104.1%, 变异系数小于3.9%。结论 本研究开发的AFB1纳米抗体免疫亲和柱表现出优秀的再生性和高特异性, 节约了检测成本, 建立的免疫亲和处理-HPLC-MS/MS检测方法操作简便、回收率高、结果准确, 适用于实际玉米样品中AFB1的含量测定。

英文摘要:

      Objective To study the factors influencing the expression yield of aflatoxin B1 (AFB1) nanoantibodies, develop AFB1 immunoaffinity columns with reproducibility, and construct an analytical method for the determination of AFB1 residues in maize by immunoaffinity treatment-high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Methods The expression of AFB1 nanobodies was increased by optimizing the induction temperature, induction time and inducer concentration, and the tolerance and the number of times of use of the regenerable immunoaffinity columns of AFB1 nanoantibodies were also verified comparatively. AFB1 was extracted from the maize samples using 70% aqueous methanol, and the extracts were purified and enriched by immunoaffinity columns and then detected by HPLC-MS/MS, and finally, the methodology was validated and applied to the detection of real samples. Results The optimal conditions for inducing the expression of AFB1 nanoantibodies were the induction temperature of 16℃, the concentration of inducer isopropyl-β-D-thiogalactoside of 0.5 mmol/L, and the induction time of 14 h. The yield of AFB1 nanobodies could reach up to 7.8 mg/L under the optimal conditions. The AFB1 nanobodies had good sensitivity, affinity, specificity and methanol tolerance, and the prepared AFB1 immunoaffinity columns had excellent reproducibility, and the recoveries of AFB1 could still reach more than 80% after reuse for more than 150 times. Meanwhile, the established immunoaffinity treatment-HPLC-MS/MS showed good linearity in the range of 0.1?100.0 μg/L, with the limit of detection of 0.014 μg/L and the limit of quantification of 0.047 μg/L. The recoveries ranged from 92.0% to 104.1% at 3 different spiked concentrations with the coefficient of variation less than 3.9%. Conclusion The immunoaffinity column of AFB1 nanobody developed in this experiment has the advantages of strong regeneration and high specificity, which saves the cost of detection, and the established immunoaffinity treatment-HPLC-MS/MS detection method is easy to operate, with high recovery and accurate results, which is suitable for the continuous monitoring of AFB1 in maize.