Alkaline phosphatase-fused repebody as a new format of immuno-reagent for an immunoassay

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Enzyme-linked immunoassays based on an antibody-antigen interaction are widely used in biological and medical sciences. However, the conjugation of an enzyme to antibodies needs an additional chemical process, usually resulting in randomly cross-linked molecules and a loss of the binding affinity and enzyme activity. Herein, we present the development of an alkaline phosphatase-fused repebody as a new format of immuno-reagent for immunoassays. A repebody specifically binding to human TNF-alpha (hTNF-alpha) was selected through a phage display, and its binding affinity was increased up to 49 nM using a modular engineering approach. A monomeric alkaline phosphatase (mAP), which was previously isolated from a metagenome library, was genetically fused to the repebody as a signal generator, and the resulting repebody-mAP fusion protein was used for direct and sandwich immunoassays of hTNF-alpha. We demonstrate the utility and potential of the repebody-mAP fusion protein as an immuno-reagent by showing the sensitivity of 216 pg mL(-1) for hTNF-alpha in a sandwich immunoassay. Furthermore, this repebody-mAP fusion protein enabled the detection of hTNF-alpha spiked in a serum-supplemented medium with high accuracy and reproducibility. It is thus expected that a mAP-fused repebody can be broadly used as an immuno-reagent in immunoassays. (CB.V) 2016 Elsevier B.V. All rights reserved.
Publisher
ELSEVIER SCIENCE BV
Issue Date
2017-01
Language
English
Article Type
Article
Keywords

NECROSIS-FACTOR-ALPHA; SINGLE-DOMAIN ANTIBODY; FUSION PROTEINS; FACTOR RECEPTOR; AFFINITY; BINDER; ASSAYS; SCFV

Citation

ANALYTICA CHIMICA ACTA, v.950, pp.184 - 191

ISSN
0003-2670
DOI
10.1016/j.aca.2016.11.013
URI
http://hdl.handle.net/10203/220384
Appears in Collection
BS-Journal Papers(저널논문)
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