Details:

Alternative Title:
Anal Chem
Publisher's site:
http://dx.doi.org/10.1021/ac504305z
Personal Author:
Liu, Xing ; Xu, Yang ; Wan, De-bin ; Xiong, Yong-hua ; He, Zhen-yun ; ... More +
Liu, Xing ; Xu, Yang ; Wan, De-bin ; Xiong, Yong-hua ; He, Zhen-yun ; Wang, Xian-xian ; Gee, Shirley J. ; Ryu, Dojin ; Hammock, Bruce D. ; Less -
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.
Subject:
[+]
Alkaline Phosphatase
Article
Edible Grain
Escherichia Coli
Fluorescence Enzyme Immunoassay
Fluorometry
Fusion Protein
Immunoenzyme Techniques
Limit Of Detection
Mycotoxins
Nanobody
Ochratoxin A
Ochratoxins
Recombinant Fusion Proteins
Single-Domain Antibodies

Source:
Anal Chem. 87(2):1387-1394.
Pubmed ID:
25531426
Pubmed Central ID:
PMC4476795
Document Type:
Journal Article ;
Funding:
2U50OH007550/OH/NIOSH CDC HHS/United States ; P42 ES004699/ES/NIEHS NIH HHS/United States ; P42ES04699/ES/NIEHS NIH HHS/United States ;
2U50OH007550/OH/NIOSH CDC HHS/United States ; P42 ES004699/ES/NIEHS NIH HHS/United States ; P42ES04699/ES/NIEHS NIH HHS/United States ; Less -
Collection(s):
CDC Public Access
Main Document Checksum:
[+]
urn:sha256:001be8094882fd3c2531f2d89621b5a9cedacbc9638863b32a7f38ac2b05ff86
File Type:

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