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首页> 外文期刊>Analytical methods >Phage displayed anti-idiotypic nanobody mediated immuno-PCR for sensitive and environmentally friendly detection of mycotoxin ochratoxin A
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Phage displayed anti-idiotypic nanobody mediated immuno-PCR for sensitive and environmentally friendly detection of mycotoxin ochratoxin A

机译：噬菌体展示抗独特型纳米抗体介导的免疫PCR技术，用于灵敏且环保的霉菌毒素to曲霉毒素A检测

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摘要

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.
机译：抗独特型纳米抗体（AId-Nbs）是一种新型抗原，可以取代小分子毒素的常规半抗原-蛋白质结合物，在竞争性免疫测定中发挥相同的功能。此外，由于获得的纳米抗体的亲和力比目标分析物的结合亲和力要低，因此纳米抗体从天然（未免疫）噬菌体展示库中显示出更高的灵敏度。在这里，我们展示了一种新的方法，用于开发基于针对抗OTA单克隆抗体的噬菌体展示AId-Nbs的OTA敏感且环保的免疫PCR（IPCR）。在这项研究中，噬菌体展示AId-Nb是从幼稚的纳米抗体库中选择的。淘选四个周期后，使用竞争结合生物淘选策略分离展示噬菌体的AId-Nb。噬菌体ELISA的半抑制浓度（IC50）为300 pg mL（-1），是常规ELISA的8.83倍。此外，基于噬菌体展示的AId-Nbs，还开发了一种非毒素定量IPCR分析法来检测农产品中的och曲霉毒素A。该方法的检测极限（LOD）为4.17 pg mL（-1），与噬菌体ELISA相比，显示出9倍的提高。所开发的方法已使用OTA污染的农产品成功验证。此外，新型且环境友好的IPCR在各种有毒小分子免疫测定的通用方法中可能具有潜在的应用。

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