Fc-specific and covalent conjugation of a fluorescent protein to a native antibody through a photoconjugation strategy for fabrication of a novel photostable fluorescent antibody.

Fluorophore-antibody conjugates with high photobleaching resistance, high chemical stability, and Fc-specific attachment is a great advantage for immunofluorescence imaging. Here, an Fc-binding protein (Z-domain) carrying a photo-cross-linker (p-benzoylphenylalanine, Bpa) fused with enhanced green fluorescent protein (EGFP), namely photoactivatable ZBpa-EGFP recombinant, was directly generated using the aminoacyl-tRNA synthetase/suppressor tRNA technique without any further modification. By employing the photoactivatable ZBpa-EGFP, an optimal approach was successfully developed which enabled EGFP to site-selectively and covalently attach to native antibody (IgG) with approximately 90% conjugation efficiency. After characterizing the Fc-specific and covalent manner of the EGFP-photoconjugated antibody, its excellent photobleaching resistance for immunofluorescence imaging was demonstrated in a model study by monitoring the toll-like receptor 4 (TLR4) expression in HepG2 cells. The proposed approach here for the preparation of a novel fluorescent antibody is available and reliable, which would play an important role in fluorescence immunoassay, and is expected to be extended to the generation of other biomolecule-photoconjugated antibodies, such as other fluorescent proteins for multiplex immunofluorescence imaging or reporter enzymes for highly sensitive enzyme immunoassays.Graphical abstract.

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 0.1-100 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.

Development of site-specific antibody-conjugated immunoliposomes for sensitive detection of disease biomarkers.

Liposome-based immunoassay (LIA) is an attractive protocol for amplifying the detection signals because of the excellent ability of liposomes to encapsulate signal marker compounds. The antigen-binding activity of the conjugated antibodies on the liposomal surface is crucial for the specificity and sensitivity of LIA. We present here a general platform to ensure that antibodies can conjugate onto the surface of liposomes in a site-specific and oriented manner. A His-handle-modified antibody with Fc region-specific and covalent conjugation was first fabricated using a photoactivatable ZBpa-His tag that was engineered using the aminoacyl-tRNA synthetase/suppressor tRNA technique. Based on the high affinity between the His tag and divalent metal ions, the novel His-modified antibody was oriented onto the surface of nickel ion-modified liposomes encapsulating horseradish peroxidase. With the prostate-specific antigen as a model, the detection efficiency of the new immunoliposomes was evaluated by chemiluminescence immunoassay. The immunoliposomes exhibited a limit of detection of 0.2 pg mL-1, which was a six time improvement compared with that of the chemical-coupled antibody-liposome conjugates. Thus, the proposed immunoliposomes are expected to hold potential applications for the sensitive detection of various biomarkers in complicated serum samples.