Development of a horseradish peroxidase-nanobody fusion protein for visual detection of ochratoxin A by dot immunoassay.
Development of a horseradish peroxidase-nanobody fusion protein for visual detection of ochratoxin A by dot immunoassay.
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开发辣根过氧化物酶-纳米抗体融合蛋白，用于通过斑点免疫分析视觉检测赭曲霉毒素A
作者：
赭曲霉毒素A（OTA）是一种常见的谷物霉菌毒素，严重威胁食品安全和公众健康。在此，经过包涵体变性、复性以及酶重组后，获得了一种保留抗体和酶活性的辣根过氧化物酶 - 纳米抗体融合蛋白（HRP - Nb），它既作为一抗又作为报告酶，被用于开发一种基于膜的斑点免疫分析法（HN - DIA），以实现对OTA的可视化检测。基于最佳实验条件，HN - DIA可在10分钟内完成，肉眼观察大米和燕麦样品的检测限为50μg/kg。HN - DIA对OTA显示出高选择性，在回收率实验中具有良好的准确性和重现性。HN - DIA和高效液相色谱法（HPLC）的加标样品分析结果相互之间具有良好的相关性。因此，所提出的HN - DIA具有通过肉眼对食品和环境中的OTA及其他小分子污染物进行快速筛选的潜力。
辣根过氧化物酶 - 纳米抗体融合蛋白被开发为一种有前景的用于食品中有毒低分子量化合物的免疫诊断试剂。
Ochratoxin A (OTA) is a common cereal mycotoxin that seriously threatens food safety and public health. Herein a horseradish peroxidase-nanobody fusion protein (HRP-Nb) retaining antibody and enzyme activity was obtained after inclusion body denaturation and renaturation and enzyme reconstitution, which served both as the primary antibody and reporter enzyme and was applied to develop a membrane-based dot immunoassay (HN-DIA) for OTA visual detection. Based on the optimal experimental conditions, the HN-DIA could be finished in 10 min with a cut-off limit of 50 μg kg−1 in rice and oat samples by eye. The HN-DIA showed high selectivity for OTA and had good accuracy and reproducibility in the recovery experiments. Spiked sample analysis results of the HN-DIA and high performance liquid chromatography (HPLC) correlated well with each other. Therefore, the proposed HN-DIA has the potential for rapid screening of OTA and other small molecule pollutants in food and the environment by naked eye. The horseradish peroxidase-nanobody fusion protein was developed as a promising immunological diagnostic reagent for toxic low-molecular-weight compounds in food.
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