Rapid and homogeneous electrochemical detection by fabricating a high affinity bispecific antibody-enzyme complex using two Catcher/Tag systems

Antibody-enzyme complexes (AECs) with binding ability to specific targets and catalytic activities to gain signals are known to be ideal sensing elements; however, AEC-based universal sensors applicable to point-of-care testing (POCT) have not yet been developed. Here, we achieved rapid and homogene...

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Bibliographic Details
Published in:Biosensors & bioelectronics 2021-03, Vol.175, p.112885-112885, Article 112885
Main Authors: Kimura, Hayato, Miura, Daimei, Tsugawa, Wakako, Ikebukuro, Kazunori, Sode, Koji, Asano, Ryutaro
Format: Article
Language:English
Subjects:
Antibodies, Bispecific
Biosensing Techniques
Bispecific antibody-enzyme complex
Catcher/Tag system
Epidermal growth factor receptor
Epitopes
ErbB Receptors
Glucose dehydrogenase
Homogeneous electrochemical detection
Single-Chain Antibodies
Single-chain Fv
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Summary:Antibody-enzyme complexes (AECs) with binding ability to specific targets and catalytic activities to gain signals are known to be ideal sensing elements; however, AEC-based universal sensors applicable to point-of-care testing (POCT) have not yet been developed. Here, we achieved rapid and homogeneous electrochemical detection by fabricating a high-affinity bispecific AEC (bsAEC) using two Catcher/Tag systems. Recently, we reported a convenient and universal method to fabricate AECs using the SpyCatcher/SpyTag system. The resultant anti-epidermal growth factor receptor (anti-EGFR) AEC worked efficiently as a sensing element; however, the sensitivities did not meet the clinically required detection range of the soluble ectodomain of EGFR (sEGFR). To induce high affinity even to monomeric targets like sEGFR, we designed a convenient fabrication method for bsAEC using two Catcher/Tag systems, which did not express cross-reactivity. The anti-EGFR bsAEC was successfully prepared by constructing glucose dehydrogenase with two different catcher domains at the N- and C-terminus and by combining two corresponding Tag-fused anti-EGFR single-chain Fvs (scFvs), which recognize different epitopes on sEGFR. As expected, bsAEC showed a higher affinity than that of bivalent AEC with two identical anti-EGFR scFvs at low concentrations of sEGFR, and met the clinically required detection range of sEGFR. Further, by combining magnet beads, we established a rapid and wash-free homogeneous electrochemical detection method. This study offers new insights into the fabrication of universal POCT devices. •Fabrication of homogeneous bispecific antibody-enzyme complexes (bsAEC) was established using two Catcher/Tag systems.•BsAEC with two different antibody fragments showed high affinity even to a single monomeric target.•Substantially high sensitivity was achieved using bsAEC.•BsAEC was efficient as an electrochemical sensing element also working in human serum.•Rapid and homogeneous detection method applicable to point of care testing was established using bsAEC and magnet.
ISSN:0956-5663
1873-4235
DOI:10.1016/j.bios.2020.112885