Use of cloneable peptide-MBP fusion protein as a mimetic coating antigen in the standardized immunoassay for mycotoxin ochratoxin A.
Use of cloneable peptide-MBP fusion protein as a mimetic coating antigen in the standardized immunoassay for mycotoxin ochratoxin A.
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DOI：
10.1021/jf5028922
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发表时间：
2014-08
影响因子：
6.1
通讯作者：
Yang Xu;Zhenyun He;Qing-hua He;Yulou Qiu;Bo Chen;Jing Chen;Xing Liu
中科院分区：
文献类型：
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作者：
Yang Xu;Zhenyun He;Qing-hua He;Yulou Qiu;Bo Chen;Jing Chen;Xing Liu
霉菌毒素结合物的质量对发展真菌毒素免疫分析的可靠性至关重要。然而，传统的霉菌毒素结合物通常是通过化学方法合成的，这不仅对环境有害，而且会产生不必要的交叉反应。在本研究中，以赭曲霉毒素A(OTA)为模型体系，选择与抗OTA抗体特异结合的OTA模拟表位(噬菌体展示肽)与麦芽糖结合蛋白(MBP)进行可溶性单价融合表达。这些制备的融合蛋白可以作为模拟包被抗原，用于OTA的定量化学发光酶联免疫分析(CLEIA)和定性斑点免疫分析。其中一种基于模拟包被抗原(L12-206-MBP)的CLEIA的半数抑制浓度(IC50)为0.82 ng/m L，工作范围为0.30-2.17 ng/m L，与传统的OTA-OVA结合物免疫分析方法相似。用OTA-OVA结合物和模拟物建立的斑点免疫分析显示，视觉截止值相同，为5 ng/mL。本文提出的模拟包被抗原是一种不含OTA的产品，可以作为均一产品重复性制备，并有助于真菌毒素OTA免疫检测的标准化。
The quality of mycotoxin conjugates is essential to the development of reliability of immunoassays for mycotoxins. However, conventional mycotoxin conjugates are usually synthesized by chemical methods, which are harmful to the environment and yield unwanted cross-reactions. In this study, using ochratoxin A (OTA) as a model system, a selected OTA mimotope (phage-displayed peptide) that specifically binds to anti-OTA antibody was expressed as soluble and monovalent fusions to maltose binding protein (MBP). These prepared fusion proteins can serve as a mimetic coating antigen in both a quantitative chemiluminescent enzyme-linked immunoassay (CLEIA) and a qualitative dot immunoassay for OTA. One of the prepared mimetic coating antigen (L12-206-MBP)-based CLEIAs exhibited a half-inhibition concentration (IC50) of 0.82 ng/mL and a working range of 0.30-2.17 ng/mL, which resemble those of the conventional OTA-OVA conjugate-based immunoassay. The dot immunoassay developed with both the OTA-OVA conjugate and the mimetics showed identical visual cutoff values of 5 ng/mL. The mimetic coating antigen proposed here is an OTA-free product and can be prepared reproducibly as a homogeneous product and facilitates standardization of immunoassays for the mycotoxin OTA.