【摘要】 目的 研究黄曲霉毒素B₁（aflatoxinB₁,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（highperformanceliquidchromatography-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0.1～100.0μg/L范围内呈现良好的线性关系,检出限为0.014μg/L,定量限为0.047μg/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。更多还原

【Abstract】 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 AFB₁nanobodies 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.更多还原