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.

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.

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.