title: Immobilization of unraveled immunoglobulin G using well-oriented ZZ-His protein on functionalized microtiter plate for sensitive immunoassay.
abstract: Highly efficient protein immobilization is extremely crucial for solid-phase immunoassays. We present a strategy for oriented immobilization of functionally intact immunoglobulin G (IgG) on a polystyrene microtiter plate via iminodiacetic acid (IDA)-Ni(2+) and ZZ-His protein interaction. We immobilized a ZZ-EAP (Escherichia coli alkaline phosphatase)-His fusion protein, which exhibits Fc binding, His tag, and intrinsic AP activities, and analyzed it against the interaction between rabbit IgG anti-horseradish peroxidase (anti-HRP) and its binding partner HRP to investigate the specificity and efficacy of this method. We compared the IDA-Ni(2+)-(ZZ-His) method with ZZ-EAP random immobilization using sandwich enzyme-linked immunosorbent assay, and the results showed that the former method had an enhanced signal, 10-fold higher sensitivity, and a wider linear range. Thus, the proposed method allows a broad range of oriented immobilized functionally intact IgG antibodies on polystyrene plates using only one type of IDA-Ni(2+) chelate surface because the ZZ protein can bind to the Fc region of various IgGs.
authors.affiliation: School of Basic Medicine, Weifang Medical University, Weifang 261053, China.
authors.forename: Hong-Ming
authors.initials: HM
authors.lastname: Yang
authors.forename: Shu-Juan
authors.initials: SJ
authors.lastname: Liang
authors.forename: Jin-Bao
authors.initials: JB
authors.lastname: Tang
authors.forename: Yong
authors.initials: Y
authors.lastname: Chen
authors.forename: Yuan-Zheng
authors.initials: YZ
authors.lastname: Cheng
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000096:biosynthesis / Q000235:genetics; D006735:Horseradish Peroxidase / Q000378:metabolism; D007098:Imino Acids / Q000737:chemistry; D055767:Immobilized Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism; D007074:Immunoglobulin G / Q000032:analysis / Q000276:immunology*; D009532:Nickel / Q000737:chemistry; D009842:Oligopeptides / Q000096:biosynthesis / Q000235:genetics; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011137:Polystyrenes / Q000737:chemistry; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*
chemical_list: C471213:His-His-His-His-His-His; D007098:Imino Acids; D055767:Immobilized Proteins; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D009842:Oligopeptides; D011137:Polystyrenes; D011993:Recombinant Fusion Proteins; D006639:Histidine; D009532:Nickel; D006735:Horseradish Peroxidase; D000469:Alkaline Phosphatase; C008109:iminodiacetic acid