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

• Published in: Analytica chimica acta Vol. 950; pp. 184 - 191
• Main Authors: Seo, Hyo-Deok, Lee, Joong-jae, Kim, Yu Jung, Hantschel, Oliver, Lee, Seung-Goo, Kim, Hak-Sung
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.01.2017; Elsevier BV
• Subjects: Affinity; Alkaline phosphatase; Alkaline Phosphatase - chemistry; Antibodies; Antibodies - chemistry; Binding; Bioengineering; Conjugation; Crosslinking; Enzymatic activity; Enzyme activity; Format; Fusion protein; Genetic fusion; Humans; Immuno-reagent; Immunoassay; Immunoassays; Immunoglobulins; Indicators and Reagents; Libraries; Modular engineering; Monomeric alkaline phosphatase; Phage display; Phages; Repebody; Reproducibility; Reproducibility of Results; Tumor Necrosis Factor-alpha - analysis; Tumor necrosis factor-α; Repebody; Immunoassay; Immuno-reagent; Monomeric alkaline phosphatase; Genetic fusion
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2016.11.013

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. [Display omitted] •A human TNF-α (hTNF-α)-specific repebody was selected using a phage display.•A monomeric alkaline phosphatase (mAP) was genetically fused to the repebody.•mAP-fused repebody enabled detection of hTNF-α with high sensitivity and accuracy.•mAP-fused repebody can be widely used as a new immuno-reagent in immunoassays.