Journal
TALANTA
Volume 164, Issue -, Pages 154-158Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.talanta.2016.11.039
Keywords
Nanobody; Ochratoxin A; Immunoassay; Cereal; Matrix interference
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Funding
- Scientific Research Foundation of Hainan University [kyqd1631]
- Academic Innovation Project for Youth Science Talents, Hainan Provincial Association for Science and Technology [201504]
- NIEHS Superfund Research Program [P42 ES04699]
- Guangxi talent highland of preservation and deep processing research in fruit and vegetables
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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. coil 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 degrees C for 5 min or to 90 degrees C for 75 min. 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 IC50 of 0.64 ng/mL and a linear range (IC20-IC80) of 0.27-1.47 ng/mL. Cereal samples were analyzed following a 2.5 fold dilution of sample extracts, showing the good resistance to matrix interference of the Nb-ELSLk. 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.
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