Phage displayed anti-idiotypic nanobody mediated immuno-PCR for sensitive and environmentally friendly detection of mycotoxin ochratoxin A
Phage displayed anti-idiotypic nanobody mediated immuno-PCR for sensitive and environmentally friendly detection of mycotoxin ochratoxin A
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噬菌体展示的抗独特型纳米抗体介导的免疫 PCR 用于灵敏且环保地检测霉菌毒素赭曲霉毒素 A
DOI：
10.1039/c6ay01264g
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发表时间：
2016-01-01
影响因子：
3.1
通讯作者：
Liu, Yuanyuan
中科院分区：
文献类型：
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作者：
Ji, Yanwei;He, Qinghua;Liu, Yuanyuan
抗独特型纳米抗体（Anti-idiotypic nanobodies，AId-Nbs）是一种新型的抗原，可以取代传统的小分子毒素的半抗原-蛋白偶联物，在竞争性免疫分析中发挥同样的作用。此外，纳米抗体显示来自初始（未免疫）噬菌体展示文库的增加的灵敏度，因为获得的那些具有比靶分析物的结合亲和力更低的亲和力。在这里，我们展示了一种新的方法，用于发展敏感和环境友好的免疫PCR（IPCR）的OTA的基础上噬菌体展示AId-Nbs抗OTA单克隆抗体。在这项研究中，从天然纳米抗体文库中选择噬菌体展示的AId-Nb。在四个淘选循环后，使用竞争结合生物淘选策略分离噬菌体展示的AId-Nb。半抑制浓度（IC_（50））为300 pg/mL，是常规ELISA的8.83倍。此外，还建立了一种基于噬菌体展示AId-Nbs的农产品中赭曲霉毒素A的无毒素定量IPCR检测方法。该方法的检测限（LOD）为4.17 pg/mL（-1），比噬菌体-ELISA法提高了9倍。所开发的方法被成功地验证使用OTA污染的农产品。此外，新的和环境友好的IPCR可能有潜在的应用，在一个通用的方法，用于各种有毒小分子的免疫分析。
Anti-idiotypic nanobodies (AId-Nbs) are novel antigens that can replace the conventional hapten-protein conjugates of small molecule toxins, serving the same function in the competitive immunoassay. Furthermore, nanobodies show increased sensitivity from naive (non immunized) phage display libraries because those obtained have a lower affinity than the binding affinity of the target analytes. Here, we demonstrated a new approach for the development of sensitive and environmentally friendly immuno-PCR (IPCR) for OTA based on phage displayed AId-Nbs against anti-OTA monoclonal antibodies. In this study, a phage displayed AId-Nb was selected from a naive nanobody library. After four cycles of panning, a phage displayed AId-Nb was isolated using a competition-binding biopanning strategy. The half-inhibition concentration (IC50) of the phage-ELISA was 300 pg mL(-1), which was 8.83-fold better than that of a conventional ELISA. Furthermore, a non-toxin quantitative IPCR assay was also developed for the detection of ochratoxin A in agri-products based on phage displayed AId-Nbs. The limit of detection (LOD) of the assay is 4.17 pg mL(-1), which exhibits a 9-fold improvement over the phage-ELISA. The developed method was successfully validated using OTA contaminated agri-products. Furthermore, novel and environmentally friendly IPCR might have potential applications in a general method for the immunoassay of various toxic small molecules.