Convenient and Universal Fabrication Method for Antibody-Enzyme Complexes as Sensing Elements Using the...

Area of Science:

• Biochemistry
• Immunotechnology
• Biosensing

Background:

• Antibody-enzyme complexes (AECs) are crucial for electrochemical biosensors, particularly those utilizing oxidoreductases.
• Conventional AEC fabrication methods often lead to insoluble aggregates and lack homogeneity.
• Developing homogeneous AECs is essential for reliable and sensitive electrochemical measurements.

Purpose of the Study:

• To develop a convenient, universal, and efficient method for fabricating homogeneous antibody-enzyme complexes (AECs).
• To utilize the SpyCatcher/SpyTag system for AEC construction, overcoming limitations of traditional methods.
• To demonstrate the applicability of the fabricated AECs in detecting soluble epidermal growth factor receptor (EGFR).

Main Methods:

• Engineered fusion proteins: SpyTag-fused anti-EGFR variable domain of heavy chain antibody (VHH) and SpyCatcher-fused Aspergillus flavus glucose dehydrogenase (AfGDH).
• Escherichia coli expression system for preparing fusion proteins.
• Simple mixing of fusion proteins to form homogeneous anti-EGFR AECs; preparation of bivalent AECs.
• Detection of soluble EGFR using immobilized anti-EGFR IgG and the bivalent AEC.

Main Results:

• Successfully produced homogeneous anti-EGFR AECs via SpyCatcher/SpyTag ligation.
• Prepared bivalent AECs demonstrating enhanced binding affinity.
• Achieved dose-dependent detection of soluble EGFR using the fabricated AECs.
• Confirmed the universality of the method by applying it to another VHH.

Conclusions:

• The SpyCatcher/SpyTag system provides a convenient and universal platform for fabricating homogeneous AECs.
• This method overcomes aggregation issues and simplifies AEC production for electrochemical sensing.
• The developed AECs show promise for sensitive and specific detection in biosensing applications.