title: Well-oriented ZZ-PS-tag with high Fc-binding onto polystyrene surface for controlled immobilization of capture antibodies.
abstract: The site specificity and bioactivity retention of antibodies immobilized on a solid substrate are crucial requirements for solid phase immunoassays. A fusion protein between an immunoglobulin G (IgG)-binding protein (ZZ protein) and a polystyrene-binding peptide (PS-tag) was constructed, and then used to develop a simple method for the oriented immobilization of the ZZ protein onto a PS support by the specific attachment of the PS-tag onto a hydrophilic PS. The orientation of intact IgG was achieved via the interaction of the ZZ protein and the constant fragment (Fc), thereby displayed the Fab fragment for binding antigen. The interaction between rabbit IgG anti-horseradish peroxidase (anti-HRP) and its binding partner HRP was analyzed. Results showed that the oriented ZZ-PS-tag yielded an IgG-binding activity that is fivefold higher than that produced by the passive immobilization of the ZZ protein. The advantage of the proposed immunoassay strategy was demonstrated through an enzyme-linked immunosorbent assay, in which monoclonal mouse anti-goat IgG and HRP-conjugated rabbit F(ab')2 anti-goat IgG were used to detect goat IgG. The ZZ-PS-tag presented a tenfold higher sensitivity and a wider linear range than did the passively immobilized ZZ protein. The proposed approach may be an attractive strategy for a broad range of applications involving the oriented immobilization of intact IgGs onto PS supports, in which only one type of phi-PS (ZZ-PS-tag) surface is used.
authors.affiliation: School of Pharmacy & Biology, Weifang Medical University, Weifang 261053, PR China. tangjinbao@yahoo.com.cn
authors.forename: Jin-Bao
authors.initials: JB
authors.lastname: Tang
authors.forename: Xi-Feng
authors.initials: XF
authors.lastname: Sun
authors.forename: Hong-Ming
authors.initials: HM
authors.lastname: Yang
authors.forename: Bao-Gang
authors.initials: BG
authors.lastname: Zhang
authors.forename: Zhi-Jian
authors.initials: ZJ
authors.lastname: Li
authors.forename: Zhi-Juan
authors.initials: ZJ
authors.lastname: Lin
authors.forename: Zhi-Qin
authors.initials: ZQ
authors.lastname: Gao
mesh_terms: D000818:Animals; D055910:Antibodies, Immobilized / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D011137:Polystyrenes / Q000737:chemistry*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D013499:Surface Properties
chemical_list: D055910:Antibodies, Immobilized; D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D011137:Polystyrenes; D011993:Recombinant Fusion Proteins; D006735:Horseradish Peroxidase