期刊
TALANTA
卷 143, 期 -, 页码 388-393出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.talanta.2015.05.010
关键词
Anti-idiotypic antibodies; Nanobodies; Phage display library
资金
- National Basic Research Program of China [2013CB127804]
- National Natural Science Foundation of China [NSFC-31471648, NSFC-31360386, NSFC-31201360, NSFC-31171696]
- Jiangxi Province Key Technology R D Program [2014BBG70090]
- Natural Science Foundation of Jiangxi, China [20132-BAB214005, 20143ACB21008]
- Education Department of Jiangxi Province [GJJ13095]
Nanobodies that are small and thermally stable, as well as have high expression level, are leading alternative to produce anti-idiotypic antibodies. These antibodies have the advantage of replacing mycotoxins and their conjugates for immunoassays. In this work, anti-fumonisin B-1 (FB1) monoclonal antibody (mAb) was used as the target for biopanning from a naive alpaca nanobody (Nb) phage display library. After three cycles of panning, one anti-idiotypic nanobody (Ab2 beta Nb) was isolated and subjected to a Nb-ELISA for the detection of FB1. Surface plasmon resonance was used to analyze the reaction kinetics between the Ab2 beta Nb and anti-FB1 mAb. The developed assay showed a half inhibitory concentration (IC50) of 0.95 +/- 0.12 ng/mL, a limit of detection of 0.15 ng/mL, a linear range of 0.27-5.92 ng/mL, and a low cross-reactivity toward FB2 of 4.93%. The sensitivity was enhanced approximately 20-fold compared with that of the chemosynthetic FB1-BSA conjugates. The equilibrium dissociation constant (K-D) measured for Ab2 beta Nb: anti-FB1 mAb was 164.6 nM. The assay was compared with conventional ELISA (the commercial ELISA kit), and the results indicated the reliability of Ab2 beta Nb replacing the antigen-carrier protein conjugates. The use of biotechnology in developing the surrogate is an ideal strategy for replacing conventional synthesized antigens. (C) 2015 Elsevier B.V. All rights reserved.
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