Directional immobilization of antibody onto magnetic nanoparticles by Fc-binding protein-assisted photo-conjugation for high sensitivity detection of antigen

Immobilized antibodies with site-specific, oriented, and covalent pattern are of great significance to improve the sensitivity of solid-phase immunoassay. Here, we developed a novel antibody conjugation strategy that can immobilize antibodies in a directional and covalent manner. In this study, an IgG-Fc binding protein (Z domain) carrying a site-specific photo-crosslinker, p-benzoyl-L-phenylalanine, and a single C-terminal cysteine (Cys) handle was genetically engineered. Upon UV irradiation, the chimeric protein enables the Cys handle to couple with the native antibody in Fc-specific and covalent conjugation pattern, resulting in a novel thiolated antibody. Thus, an approach for the covalent, directional immobilization of antibodies to maleimide-modified magnetic nanoparticles (MNPs) was developed on the basis of the crosslinking between sulfhydryl and maleimide groups. The antibody-conjugated MNPs were applied in MNP-based enzyme-linked immunosorbent assay (ELISA) for the detection of carcinoembryonic antigen. The MNP-based ELISA presented a quantification linear range of 0.1-10 0 ng mL(-1) and detection limit of 0.02 ng mL(-1), which was approximately 100 times more sensitive than the traditional microplate ELISA (2.0 ng mL(-1)). Thus, the proposed antibody immobilization approach can be used in surface functionalization for the sensitive detection of various biomarkers. (C) 2021 Elsevier B.V. All rights reserved.

Automated summary

A novel antibody conjugation strategy was developed to immobilize antibodies in a directional and covalent manner, significantly improving the sensitivity of solid-phase immunoassay. This approach can be used for surface functionalization to detect various biomarkers with increased sensitivity.