title: 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.
abstract: 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.
authors.affiliation: State Key Laboratory of Food Science and Technology and ‡Sino-Germany Joint Research Institute, Nanchang University , No. 235 Nanjing East Road, Nanchang 330047, People's Republic of China.
authors.forename: Xing
authors.initials: X
authors.lastname: Liu
authors.forename: Yang
authors.initials: Y
authors.lastname: Xu
authors.forename: De-bin
authors.initials: DB
authors.lastname: Wan
authors.forename: Yong-hua
authors.initials: YH
authors.lastname: Xiong
authors.forename: Zhen-yun
authors.initials: ZY
authors.lastname: He
authors.forename: Xian-xian
authors.initials: XX
authors.lastname: Wang
authors.forename: Shirley J
authors.initials: SJ
authors.lastname: Gee
authors.forename: Dojin
authors.initials: D
authors.lastname: Ryu
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Hammock
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D002523:Edible Grain / Q000737:chemistry / Q000382:microbiology*; D004926:Escherichia coli / Q000235:genetics; D005470:Fluorometry / Q000379:methods; D007124:Immunoenzyme Techniques / Q000379:methods*; D057230:Limit of Detection; D009183:Mycotoxins / Q000032:analysis*; D009793:Ochratoxins / Q000032:analysis* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D061905:Single-Domain Antibodies / Q000235:genetics / Q000276:immunology*
chemical_list: D009183:Mycotoxins; D009793:Ochratoxins; D011993:Recombinant Fusion Proteins; D061905:Single-Domain Antibodies; C025589:ochratoxin A; D000469:Alkaline Phosphatase