Fc-specific biotinylation of antibody using an engineered photoactivatable Z–Biotin and its biosensing application

• Published in: Analytica chimica acta 2017-01, Vol.949, p.76-82
• Main Authors: Yang, Hong-Ming, Bao, Ru-Meng, Yu, Chang-Mei, Lv, Yan-Na, Zhang, Wei-Fen, Tang, Jin-Bao
• Format: Article
• Language: English
• Subjects: Amino acids; Aminoacyl-tRNA ligase; Antibodies; Avidin; Binding; Biomarkers; Biosensing; Biosensing Techniques; Biosensors; Biotin; Biotinylation; Carcinoembryonic antigen; Conjugates; Covalence; Crosslinking; Digestion; Fc-specific covalent conjugation; Gel electrophoresis; Immunoassay; Immunoassays; Immunoglobulin Fc Fragments - chemistry; Immunoglobulin G; Immunoglobulin G - chemistry; Immunoglobulins; Irradiation; Pepsin; Photoactivatable Z–Biotin; Sodium; Sodium dodecyl sulfate; Sodium lauryl sulfate; Surface plasmon resonance; tRNA; Ultraviolet radiation
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2016.10.039

The development of a site-specific and covalent attachment methodology is crucial for antibody–biotin conjugates to preserve the antigen-binding ability of antibodies and yield homogeneous products. In this study, an engineered photoactivatable Z-domain variant [an UV-active amino acid benzoylphenylalanine (Bpa) was genetically incorporated into the Z-domain] carrying one biotin molecule (ZBpa–Biotin) was prepared by employing aminoacyl-tRNA synthetase/suppressor tRNA and Avitag/BirA techniques. The site-specific and covalent attachment of IgG–biotin conjugates, viz. photo-biotinylated IgG, was successfully achieved after UV exposure by combining the inherent Fc-binding capability of the Z-domain with the formation of covalent bond by the photo-crosslinker. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay showed that more than 90% of IgGs conjugated with ZBpa–Biotin molecules suffered 3 h UV irradiation. Further pepsin digestion analysis confirmed that the ZBpa–Biotin was conjugated to the Fc fragment of IgG without interference. We took the tumor biomarker carcinoembryoic antigen (CEA) as model to evaluate the detection efficiency of the site-specific photo-biotinylated IgG in biosensing application using surface plasmon resonance (SPR) technology. The photo-biotinylated IgG coated surface gave a limit of detection (LOD) of 2 ng mL-1, is 5-fold lower than that of the randomly NHS-biotinylated IgG (10 ng mL-1). Given that the (strept)avidin–biotin complex is extensively used in immunoassays, the proposed method for biotinylated IgG provides a powerful approach to further expand related applications. [Display omitted] •A photoactivable ZBpa–Biotin was fabricated by aaRS/tRNA and Avitag/BirA techniques.•A approach for Fc-specific photo-biotinylated IgG via ZBpa–Biotin was proposed.•The photo-biotinylated IgG was used to fabricate an immunosensor for detecting CEA.•It gave a LOD of 2 ng mL-1 CEA, was 5-fold lower than that of NHS-biotinylated IgG.