期刊
ANALYTICA CHIMICA ACTA
卷 887, 期 -, 页码 201-208出版社
ELSEVIER
DOI: 10.1016/j.aca.2015.06.033
关键词
Nanobody; Immunoassay; Deoxynivalenol; Mimetics
资金
- National Natural Science Funds [NSFC-31360386, NSFC-31201360, NSFC-31171696, NSFC-31301479]
- Jiangxi Province Key Technology R D Program [20141BBG70090]
- major program of Natural Science Foundation of Jiangxi, China [20143ACB21008, 20152ACB2005]
- Natural Science Foundation of Jiangxi, China [20143ACB21008, 20132BAB214005]
- Education Department of Jiangxi Province [GJJ13095, GJJ12142]
In this study, using mycotoxin deoxynivalenol (DON) as a model hapten, we developed a nanobody-based environmental friendly immunoassay for sensitive detection of DON. Two nanobodies (N-28 and N-31) which bind to anti-DON monoclonal antibody (MAb) were isolated from a naive phage display library. These nanobodies are clonable, thermally stable and mycotoxin-free products and can be served as coating antigen mimetics in heterologous immunoassay. The half inhibition concentration (IC50) of the immunoassay developed with N-28 and N-31 was 8.77 +/- 0.41 ng mL(-1) and 19.97 +/- 0.84 ng mL(-1), respectively, which were 18- and 8-fold more sensitive than the conventional coating antigen (DON-BSA) based immunoassay. In order to better understand the molecular mechanism of antigen mimicry by nanobody, the 3D structure of nanobody (N-28) - anti-DON MAb complex was presented and verified by molecular modeling and alanine-scanning mutagenesis. The results showed that hydrogen bond and hydrophobic interaction formed between Thr 102 - Ser 106 of N-28 and CDR H3 residues of anti-DON antibody may contribute to their binding. This novel concept of enhancing sensitivity of immunoassay for DON based on nanobody may provide potential applications in a general method for immunoassay of various food chemical contaminants. (C) 2015 Elsevier B.V. All rights reserved.
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