Well-oriented ZZ-PS-tag with high Fc-binding onto polystyrene surface for controlled immobilization of capture antibodies
Well-oriented ZZ-PS-tag with high Fc-binding onto polystyrene surface for controlled immobilization of capture antibodies
复制标题
定向良好的 ZZ-PS 标签，在聚苯乙烯表面具有高 Fc 结合力，用于捕获抗体的受控固定
DOI：
10.1016/j.aca.2013.03.017
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发表时间：
2013-05-07
影响因子：
6.2
通讯作者：
Gao, Zhi-Qin
中科院分区：
文献类型：
--
作者：
Tang, Jin-Bao;Sun, Xi-Feng;Gao, Zhi-Qin
固定在固相基质上的抗体的位点特异性和生物活性保留是固相免疫测定的关键要求。构建了免疫球蛋白G（IgG）结合蛋白（ZZ蛋白）与聚苯乙烯结合肽（PS-tag）的融合蛋白，并通过将PS-tag特异性连接到亲水性PS上，将ZZ蛋白定向固定到PS载体上。通过ZZ蛋白和恒定片段（Fc）的相互作用实现完整IgG的定向，从而展示用于结合抗原的Fab片段。分析了兔抗辣根过氧化物酶（HRP）IgG与HRP的相互作用。结果表明，定向的ZZ-PS-标签产生的IgG结合活性是ZZ蛋白被动固定化产生的IgG结合活性的5倍。用单克隆鼠抗羊IgG和HRP标记的兔抗羊IgG（F（ab '）（2））进行酶联免疫吸附试验，证明了该方法的优越性。ZZ-PS-tag的灵敏度比被动固定的ZZ蛋白高10倍，线性范围更宽。所提出的方法可能是一个有吸引力的策略，广泛的应用，涉及定向固定的完整的IgG到PS支持，其中只有一种类型的phi-PS（ZZ-PS标签）的表面使用。(C)2013爱思唯尔有限公司版权所有。
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. (C) 2013 Elsevier B.V. All rights reserved.