Covalent and Oriented Immobilization of scFv Antibody Fragments via an Engineered Glycan Moiety
Covalent and Oriented Immobilization of scFv Antibody Fragments via an Engineered Glycan Moiety
复制标题
通过工程聚糖部分共价定向固定 scFv 抗体片段
DOI：
10.1021/bm301518p
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发表时间：
2013-01-01
影响因子：
6.2
通讯作者：
Wall, J. Gerard
中科院分区：
文献类型：
--
作者：
Hu, Xuejun;Hortiguela, Maria J.;Wall, J. Gerard
抗体衍生片段在生物标记物检测和蛋白质纯化等固相分析中具有巨大的应用潜力。固定化抗体分子的受控取向是此类测定的灵敏度和功效的关键要求。我们提出了一种用于将scFv抗体片段共价地、正确定向地附着在固体支持物上的方法。在大肠杆菌中表达糖基化的scFv，并将C-末端结合口袋远端聚糖标签氧化以共价连接至胺官能化的珠。糖基化的scFv可以在排除非糖基化分子的非特异性吸附的盐浓度下固定化，并且共价连接的抗体片段表现出比离子吸附的scFv高4倍的功能活性。糖链连接的scFv在去除大于90%的吸附的scFv的NaCl浓度下是稳定的，并且它们在加速降解测试中表现出比吸附的scFv和可溶性的、非固定化的scFv两者更好的抗原结合稳定性。报道的简单表达和固定化方法可能在体外抗体测试中找到广泛的应用。
Antibody-derived fragments have enormous potential application in solid-phase assays such as biomarker detection and protein purification. Controlled orientation of the immobilized antibody molecules is a critical requirement for the sensitivity and efficacy of such assays. We present an approach for covalent, correctly oriented attachment of scFv antibody fragments on solid supports. Glycosylated scFvs were expressed in Escherichia coli and the C-terminal, binding pocket-distal glycan tag was oxidized for covalent attachment to amine-functionalized beads. The glycosylated scFvs could be immobilized at salt concentrations that precluded nonspecific adsorption of unglycosylated molecules and the covalently attached antibody fragments exhibited 4-fold higher functional activity than ionically adsorbed scFvs. The glyco-tethered scFvs were stable in NaCl concentrations that removed greater than 90% of adsorbed scFvs and they exhibited improved stability of antigen binding over both adsorbed scFvs and soluble, nonimmobilized scFvs in accelerated degradation tests. The simple expression and immobilization approach reported is likely to find broad application in in vitro antibody tests.