Journal
ANALYTICAL CHEMISTRY
Volume 87, Issue 2, Pages 1387-1394Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac504305z
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Funding
- National Basic Research Program of China [2013CB127804]
- National Natural Science Foundation of China [NSFC-31471648, NSFC-31171696]
- National Institute of Occupational Safety and Health [2U50OH007550]
- National Institute of Environmental Health Sciences Superfund Research Program [P42ES04699]
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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.
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