Development of a Nanobody鈥揂lkaline Phosphatase Fusion Protein and Its Application in a Highly Sensitive Direct Competitive Fluorescence Enzyme Immunoassay for Detection of Ochratoxin A in Cereal

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• 作者：Xing Liu ; Yang Xu ; De-bin Wan ; Yong-hua Xiong ; Zhen-yun He ; Xian-xian Wang ; Shirley J. Gee ; Dojin Ryu ; Bruce D. Hammock
• 刊名：Analytical Chemistry
• 出版年：2015
• 出版时间：January 20, 2015
• 年：2015
• 卷：87
• 期：2
• 页码：1387-1394
• 全文大小：446K
• ISSN：1520-6882

文摘

A rapid and sensitive direct competitive fluorescence enzyme immunoassay (dc-FEIA) for ochratoxin A (OTA) based on a nanobody (Nb)鈥揳lkaline phosphatase (AP) fusion protein was developed. The VHH (variable domain of heavy chain antibody) gene of Nb28 was subcloned into the expression vector pecan45 containing the AP double-mutant gene. The Nb28鈥揂P construct was transformed into Escherichia coli BL21(DE3)plysS, and soluble expression in bacteria was confirmed by sodium dodecyl sulfate鈥損olyacrylamide gel electrophoresis and Western blot. Both the Nb properties and AP enzymatic activity were validated by colorimetric and fluorometric analysis. The 50% inhibitory concentration and the detection limit of the dc-FEIA were 0.13 and 0.04 ng/mL, respectively, with a linear range of 0.06鈥?.43 ng/mL. This assay was compared with LC鈥揗S/MS, and the results indicated the reliability of Nb鈥揂P fusion protein-based dc-FEIA for monitoring OTA contamination in cereal.