title: Nanobody-based enzyme immunoassay for ochratoxin A in cereal with high resistance to matrix interference.
abstract: A sensitive indirect competitive nanobody-based enzyme linked immunosorbent assay (Nb-ELISA) for ochratoxin A (OTA) with high resistance to cereal matrix interference was developed. Nanobodies against OTA (Nb15, Nb28, Nb32, Nb36) were expressed in E. coli cells and their thermal stabilities were compared with that of an OTA-specific monoclonal antibody 6H8. All nanobodies could still retain their antigen-binding activity after exposure to temperature 95°C for 5min or to 90°C for 75min. Nb28 that exhibited the highest sensitivity in ELISA was selected for further research. An indirect competitive ELISA based on Nb28 was developed for OTA, with an IC50of 0.64ng/mL and a linear range (IC20-IC80) of 0.27-1.47ng/mL. Cereal samples were analyzed following a 2.5 fold dilution of sample extracts, showing the good resistance to matrix interference of the Nb-ELSIA. The recovery of spiked cereal samples (rice, oats, barley) ranged from 80% to 105% and the Nb-ELISA results of OTA content in naturally contamined samples were in good agreement with those determined by a commercial ELISA kit. The results indicated the reliablity of nanobody as a promising immunoassay reagent for detection of mycotoxins in food matrix and its potential in biosensor development.
authors.affiliation: College of Food Science and Technology, Hainan University, 58 Renmin Avenue, Haikou 570228, China; Institute of Food Research, Hezhou University, Hezhou 542899, China. Electronic address: xliu@hainu.edu.cn.
authors.forename: Xing
authors.initials: X
authors.lastname: Liu
authors.affiliation: College of Food Science and Technology, Hainan University, 58 Renmin Avenue, Haikou 570228, China.
authors.forename: Zongwen
authors.initials: Z
authors.lastname: Tang
authors.affiliation: Institute of Food Research, Hezhou University, Hezhou 542899, China.
authors.forename: Zhenhua
authors.initials: Z
authors.lastname: Duan
authors.affiliation: Food Science and Technology Program, Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore. Electronic address: zhenyunhe@nus.edu.sg.
authors.forename: Zhenyun
authors.initials: Z
authors.lastname: He
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang 330047, China.
authors.forename: Mei
authors.initials: M
authors.lastname: Shu
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang 330047, China.
authors.forename: Xianxian
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Entomology and Nematology and UCD Comprehensive Cancer Center, University of California, Davis, CA 95616, United States.
authors.forename: Shirley J
authors.initials: SJ
authors.lastname: Gee
authors.affiliation: Department of Entomology and Nematology and UCD Comprehensive Cancer Center, University of California, Davis, CA 95616, United States.
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Hammock
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang 330047, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Xu
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D002523:Edible Grain / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005506:Food Contamination / Q000032:analysis; D009793:Ochratoxins / Q000032:analysis* / Q000276:immunology; D061905:Single-Domain Antibodies / Q000276:immunology*
chemical_list: D009793:Ochratoxins; D061905:Single-Domain Antibodies; C025589:ochratoxin A
keywords: Cereal; Immunoassay; Matrix interference; Nanobody; Ochratoxin A