title: Fc-specific biotinylation of antibody using an engineered photoactivatable Z-Biotin and its biosensing application.
abstract: 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.
authors.affiliation: School of Pharmacy, Weifang Medical University, Weifang, 261053, Shandong Province, China.
authors.forename: Hong-Ming
authors.initials: HM
authors.lastname: Yang
authors.affiliation: School of Pharmacy, Weifang Medical University, Weifang, 261053, Shandong Province, China.
authors.forename: Ru-Meng
authors.initials: RM
authors.lastname: Bao
authors.affiliation: School of Pharmacy, Weifang Medical University, Weifang, 261053, Shandong Province, China.
authors.forename: Chang-Mei
authors.initials: CM
authors.lastname: Yu
authors.affiliation: School of Pharmacy, Weifang Medical University, Weifang, 261053, Shandong Province, China.
authors.forename: Yan-Na
authors.initials: YN
authors.lastname: Lv
authors.affiliation: School of Pharmacy, Weifang Medical University, Weifang, 261053, Shandong Province, China.
authors.forename: Wei-Fen
authors.initials: WF
authors.lastname: Zhang
authors.affiliation: School of Pharmacy, Weifang Medical University, Weifang, 261053, Shandong Province, China. Electronic address: tangjb@wfmc.edu.cn.
authors.forename: Jin-Bao
authors.initials: JB
authors.lastname: Tang
mesh_terms: D001360:Avidin; D015374:Biosensing Techniques; D001710:Biotin; D019921:Biotinylation; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*
chemical_list: D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D001360:Avidin; D001710:Biotin
keywords: Biosensing; Biotin; Fc-specific covalent conjugation; Immunoglobulin G; Photoactivatable Z–Biotin