Development of a nanobody-alkaline phosphatase fusion protein and its application in a highly sensitive direct competitive fluorescence enzyme immunoassay for detection of ochratoxin a in cereal

Xing Liu, Yang Xu, De Bin Wan, Yong Hua Xiong, Zhen Yun He, Xian Xian Wang, Shirley J. Gee, Dojin Ryu, Bruce D. Hammock

Research output: Contribution to journalArticle

57 Scopus citations

Abstract

A rapid and sensitive direct competitive fluorescence enzyme immunoassay (dc-FEIA) for ochratoxin A (OTA) based on a nanobody (Nb)-alkaline 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-AP construct was transformed into Escherichia coli BL21(DE3)plysS, and soluble expression in bacteria was confirmed by sodium dodecyl sulfate-polyacrylamide 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 0.06-0.43 ng/mL. This assay was compared with LC-MS/MS, and the results indicated the reliability of Nb-AP fusion protein-based dc-FEIA for monitoring OTA contamination in cereal.

Original languageEnglish (US)
Pages (from-to)1387-1394
Number of pages8
JournalAnalytical Chemistry
Volume87
Issue number2
DOIs
StatePublished - Jan 20 2015

ASJC Scopus subject areas

  • Analytical Chemistry

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