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

Liu, Xing; Xu, Yang; Wan, De-bin; Xiong, Yong-hua; He, Zhen-yun; Wang, Xian-xian; Gee, Shirley J.; Ryu, Dojin; Hammock, Bruce D.

doi:10.1021/ac504305z

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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

Published Date:

Jan 06 2015

Publisher's site:

http://dx.doi.org/10.1021/ac504305z New Site Icon

Source:

Anal Chem. 87(2):1387-1394.

[PDF-2.41 MB]

Details:

Personal Authors:

Liu, Xing ; Xu, Yang ; Wan, De-bin ; Xiong, Yong-hua ; He, Zhen-yun ; ... More ▼

Keywords:

[+]

Pubmed ID:

25531426

Pubmed Central ID:

PMC4476795

Description:

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.

Document Type:

Journal Article

Collection(s):

CDC Public Access

Funding:

2U50OH007550/OH/NIOSH CDC HHS/United States
P42 ES004699/ES/NIEHS NIH HHS/United States
P42ES04699/ES/NIEHS NIH HHS/United States

Supporting Files:

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