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

Details

• Alternative Title:
  Anal Chem
• Personal Author:
  Liu, Xing ; Xu, Yang ; Wan, De-bin ; Xiong, Yong-hua ; He, Zhen-yun ; Wang, Xian-xian ; Gee, Shirley J. ; Ryu, Dojin ; Hammock, Bruce D.
• 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.
• Subjects:
  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

  More +
• 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
• Volume:
  87
• Issue:
  2
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:001be8094882fd3c2531f2d89621b5a9cedacbc9638863b32a7f38ac2b05ff86
• Download URL:
  https://stacks.cdc.gov/view/cdc/31736/cdc_31736_DS1.pdf
• File Type:
  [PDF - 2.41 MB ]