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Title 

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

 

새로운 면역분석용 Alkaline phosphatase

Authors 

H D SeoJ J LeeYu Jung KimO HantschelSeung Goo LeeHak-Sung Kim

Publisher 

Elsevier

Issue Date 

2016

Citation 

Analytica Chimica Acta, vol. 950, no. 0, pp. 184-191

Keywords 

Genetic fusionImmuno-reagentImmunoassayMonomeric alkaline phosphataseRepebody

Abstract 

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-α (hTNF-α) 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-α. 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-α in a sandwich immunoassay. Furthermore, this repebody-mAP fusion protein enabled the detection of hTNF-α 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.

ISSN 

0003-2670

Link 

http://dx.doi.org/10.1016/j.aca.2016.11.013

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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