Development of a highly sensitive qualitative and quantitative immunochromatographic method for the detection of aflatoxin B1
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摘要:
目的 真菌毒素可污染农产品和动物源性食品，其中黄曲霉毒素B1(AFB1)毒性强、危害大，建立AFB1快速、高灵敏和便捷的检测方法对于监测相关产品中AFB1污染水平，保障人和动物健康均具有重要意义。本研究基于侧向层析技术原理，采用竞争模式，优化建立免疫层析检测方法，以实现AFB1的快速定性检测和定量分析。 方法 通过比较分析不同粒径金颗粒标记抗体效果，优化确定免疫层析各组分材料类型、相关缓冲液配方及最佳使用质量浓度，建立AFB1高灵敏定性定量免疫层析检测方法。 结果 优化建立的AFB1免疫层析检测法在实际样本中的定性和定量检测限分别为2.5和0.5 μg·kg−1，灵敏度高、特异性强，与其他常见真菌毒素无交叉反应，加标回收实验结果显示：该方法准确稳定，且对AFB1天然污染样本的定量检测结果与商品化试剂盒及LC-MS/MS一致性较好。 结论 本研究制备的免疫层析检测法可用于样本中AFB1污染的快速定性检测与定量分析，适合缺乏实验条件的基层检验检疫机构和农产品加工企业对大量样本进行快速筛查，样本检测结果疑似阳性再采用仪器法进行确认，可降低检测成本，提升检测效率，同时为建立其他病原微生物免疫层析检测方法提供参考。图9表2参26 Abstract:Objective Fungal metabolites, commonly known as mycotoxins, can pollute agricultural products and food of animal origin, among which aflatoxin B1 (AFB1) is the most common, toxic and detrimental. Establishing a rapid, highly sensitive and convenient detection method of AFB1 is of great significance for the protection of human and animal health. The objective of this study is to optimize the immunochromatographic detection method based on the principle of lateral-flow chromatography and competitive mode, so as to realize the rapid qualitative detection and quantitative analysis of AFB1. Method A highly sensitive qualitative and quantitative immunochromatographic detection method for AFB1 was established by comparing and analyzing the labeling effects of gold particles of varying sizes, optimizing the material types of each component of immunochromatography, as well as relevant buffer solution and the optimal mass concentration. Result The qualitative and quantitative detection limits of the optimized AFB1 immunochromatographic method in samples were 2.5 and 0.5 μg·kg−1, respectively, with high sensitivity and specificity and no cross reaction with other common mycotoxins. Standard addition recovery experiment showed that the method was accurate and stable, and the quantitative detection results of AFB1 natural contamination samples were in good agreement with commercial kit and LC-MS/MS. Conclusion The immunochromatographic detection method prepared in this study can be used for rapid qualitative detection and quantitative analysis of AFB1 contamination in samples. It is suitable for grass-roots inspection and quarantine institutions and agricultural product processing enterprises that lack experimental conditions to quickly screen a large number of samples. If the sample test result is suspected to be positive, the instrument method can be used for confirmation, which can reduce the test cost, improve the test efficiency and provide reference for the establishment of immunochromatographic detection methods for other pathogenic microorganisms. [Ch, 9 fig. 2 tab. 26 ref.] -
图 1 黄曲霉毒素B1定性定量免疫层析检测法示意图
Figure 1 Schematic diagram of the qualitative and quantitative immunochromatographic assay for the detection of aflatoxin B1
图 3 AFB1完全抗原AFB1-BSA和AFB1-OVA的ELISA鉴定
Figure 3 Absorbance of the AFB1 conjugates by indirect ELISA using their specific monoclonal antibodies
图 4 目测法(A)和透射电镜(B)对金颗粒的鉴定
Figure 4 Colloidal solution of 20 nm and 40 nm diameter gold nanoparticles by visualization by naked eye(A) and images from TEM(B)
图 5 免疫层析法对黄曲霉毒素B1的定性检测限
Figure 5 Detection limits of immunochromatographic assay for aflatoxin B1
图 6 免疫层析法对黄曲霉毒素B1的抑制曲线(A)和定量标准曲线(B)
Figure 6 Tri-parametric curve fitting of log concentration of aflatoxin B1 vs inhibition rate by immunochromatographic assay(A) and the calibration curve for quantification of aflatoxin B1(B)
图 7 定性定量免疫层析法对其他真菌毒素的交叉反应性
Figure 7 Cross-reactivities of the qualitative and quantitative immunochromatographic assay for different mycotoxins
图 8 免疫层析法对黄曲霉毒素B1加标样本的定性检测
Figure 8 Qualitative detection of aflatoxin B1 spiked samples by immunochromatography
图 9 免疫层析法与LC-MS/MS定量结果相关性分析
Figure 9 Correlation of results obtained by immunochromatography assay and LC-MS/MS for AFB1 detection in natural samples
表 1 免疫层析法对黄曲霉毒素B1加标样本的定量检测
Table 1. Quantitative detection of aflatoxin B1 spiked samples by immunochromatography assay
AFB1加标质量浓
度/(μg·kg−1)批内 批间 回收
率/%变异系
数/%回收
率/%变异系
数/%1.0 89.62 ± 5.31 5.93 95.72 ± 8.03 8.39 2.5 96.47 ± 6.35 6.58 103.56 ± 7.54 7.28 5.0 95.16 ± 4.29 4.51 93.25 ± 9.06 9.72 10.0 110.43 ± 6.15 5.57 107.59 ± 8.56 7.96 说明：数据为平均值±标准差，n=3 
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表 2 免疫层析法、商品化试剂盒和LC/MS/MS对天然样本中黄曲霉毒素B1的定量检测结果
Table 2. Quantitative detection of AFB1 in natural samples by the developed immunochromatography assay, commercial ELISA kit and LC-MS/MS
样本编号 AFB1/(μg·kg−1) 样本编号 AFB1/(μg·kg−1) 免疫层析检测法 LC/MS/MS 商品化试剂 免疫层析检测法 LC/MS/MS 商品化试剂 1 5.11 ± 0.32 4.79 ± 0.29 5.65 ± 0.37 8 1.83 ± 0.14 2.15 ± 0.27 2.71 ± 0.19 2 3.17 ± 0.25 2.52 ± 0.16 2.08 ± 0.18 9 2.09 ± 0.17 2.86 ± 0.25 3.57 ± 0.26 3 1.06 ± 0.11 1.34 ± 0.14 2.33 ± 0.32 10 8.65 ± 0.35 7.21 ± 0.62 8.94 ± 0.19 4 6.63 ± 0.47 5.83 ± 0.43 5.36 ± 0.57 11 4.63 ± 0.29 5.93 ± 0.38 4.38 ± 0.23 5 2.69 ± 0.22 3.15 ± 0.29 2.25 ± 0.26 12 5.27 ± 0.41 6.07 ± 0.57 5.66 ± 0.35 6 1.82 ± 0.13 2.57 ± 0.15 3.35 ± 0.18 13 3.28 ± 0.37 2.19 ± 0.18 3.95 ± 0.31 7 7.16 ± 0.51 6.03 ± 0.31 7.82 ± 0.23 说明：数据为平均值±标准差
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