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

• Personal Author:
  Gee SJ ; Hammock BD ; He Z-Y ; Liu X ; Ryu D ; Wan D-B ; Wang X-X ; Xiong Y-H ; Xu Y
• 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. [Description provided by NIOSH]
• Subjects:
  Energy Metabolism Fluorometry Genetics Mycoses Occupational Health Phosphoric Acids Safety
• Keywords:
  Metabolism Mycotoxins Phosphates Proteins
• ISSN:
  0003-2700
• Document Type:
  Text
• Funding:
  Cooperative Agreement
• Genre:
  Journal Article
• Place as Subject:
  California ; Idaho ; OSHA Region 10 ; OSHA Region 9
• CIO:
  National Institute for Occupational Safety and Health (NIOSH)
• Topic:
  Workplace Safety & Health
• Location:
  North America
• Volume:
  87
• Issue:
  2
• NIOSHTIC Number:
  nn:20046976
• Citation:
  Anal Chem 2015 Jan; 87(2):1387-1394
• Email:
  xuyang@ncu.edu.cn
• Federal Fiscal Year:
  2015
• NORA Priority Area:
  Agriculture, Forestry and Fishing
• Performing Organization:
  University of California - Davis
• Peer Reviewed:
  True
• Start Date:
  20010930
• Source Full Name:
  Analytical Chemistry
• End Date:
  20270929
• Collection(s):
  National Institute for Occupational Safety and Health
• Main Document Checksum:
  urn:sha-512:a4db555ce2edad3013463e77b8a969f3497c0e7495e6385b446dbd61fcd0d86551d8da558600d04c911517934eb0731c28f4feca2affa09f709955cdf6187c07
• Download URL:
  https://stacks.cdc.gov/view/cdc/202222/cdc_202222_DS1.pdf
• File Type:
  [PDF - 3.62 MB ]