title: Site-specific covalent attachment of an engineered Z-domain onto a solid matrix: an efficient platform for 3D IgG immobilization.
abstract: Immobilized antibodies with oriented and homogeneous patterns are crucial to solid-phase molecular recognition assay. Antibody binding protein-based immobilization can effectively present the desired antibodies. However, steadily installing the stromatoid protein with site-specific attachment manner onto a matrix surface remains to be elucidated. In this study, we present an optimal protocol to tightly attach an immunoglobulin G (IgG)-binding protein (Z-domain) through covalent incorporation of Cys-tag and maleimide group onto polystyrene surface to guarantee site-specific, oriented, and irreversible attachment, resulting in a highly efficient platform for three-dimensional IgG immobilization. The actual IgG-binding characteristic of immobilized Z-Cys was investigated by employing affinity chromatography and size exclusion chromatography. And the efficacy and potential of this platform was demonstrated by applying it to the analysis of interaction between rabbit anti-HRP IgG and its binding partner HRP. The proposed approach may be an attractive strategy to construct high performance antibody arrays and biosensors given that the antibody is compatible with the Z-domain.
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: Yuan-Zheng
authors.initials: YZ
authors.lastname: Cheng
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: D055910:Antibodies, Immobilized / Q000737:chemistry*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003545:Cysteine / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry*; D008301:Maleimides / Q000737:chemistry; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics; D011137:Polystyrenes / Q000737:chemistry; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D013499:Surface Properties
chemical_list: D055910:Antibodies, Immobilized; D007074:Immunoglobulin G; D008301:Maleimides; D010455:Peptides; D011137:Polystyrenes; C043592:maleimide; D003545:Cysteine
keywords: 3D Immobilization; Immunoglobulin G; Site-specific covalent attachment; Z-Domain