Medical College, Dalian University, Xuefu Avenue No.10, Dalian Economical and Technological Development Zone, Liaoning 116622, China.
Biomacromolecules. 2013 Jan 14;14(1):153-9. doi: 10.1021/bm301518p. Epub 2012 Dec 21.
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.
抗体衍生片段在固相检测，如生物标志物检测和蛋白质纯化等方面具有巨大的潜在应用。固定化抗体分子的定向控制是提高此类检测灵敏度和效果的关键要求。我们提出了一种在固相载体上进行 scFv 抗体片段共价、正确定向附着的方法。糖基化 scFv 在大肠杆菌中表达，其 C 末端、结合口袋远端的聚糖标签被氧化，用于与胺功能化珠共价结合。在阻止未糖基化分子非特异性吸附的盐浓度下，可将糖基化 scFv 固定化，并且共价连接的抗体片段比离子吸附的 scFv 表现出高 4 倍的功能活性。在去除超过 90%吸附 scFv 的 NaCl 浓度下，糖基化 scFv 稳定，并且在加速降解试验中，它们表现出比吸附 scFv 和可溶性非固定 scFv 更好的抗原结合稳定性。所报道的简单表达和固定化方法可能会在体外抗体检测中得到广泛应用。
