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Author:
Date:
2014-11-05
[Abstract] Surface Plasmon Resonance (SPR) is widely used to generate kinetic and affinity information on specific interactions between biomolecules. This technique is label-free and monitors the binding event in real-time. It is generally used for characterization of monoclonal antibody - antigen interactions. This protocol describes specifically the use of SPR with a Biacore T100 instrument to measure the affinity of crude hybridoma samples to a protein. For that purpose an anti-IgG antibody was firstly covalently immobilized onto a CM5 chip by amide coupling (Canziani et al., 2004; Schraml and Biehl, 2012). Then the antibodies from hybridoma supernatants were captured non-covalently onto the surface via their Fc region providing an optimal analyte-binding orientation. Finally, the ...
[摘要] 表面等离子体共振（SPR）被广泛用于产生关于生物分子之间的特异性相互作用的动力学和亲和力信息。这种技术是无标签的，并实时监控绑定事件。它通常用于表征单克隆抗体 - 抗原相互作用。该方案特别描述了使用SPR与Biacore T100仪器测量粗杂交瘤样品对蛋白质的亲和力。为此，首先通过酰胺偶联将抗IgG抗体共价固定在CM5芯片上（Canziani等人，2004; Schraml和Biehl，2012）。然后来自杂交瘤上清液的抗体通过其Fc区非共价地捕获到表面上，提供最佳的分析物结合方向。最后，通过与EDC/NHS交联来稳定所得的复合物，以避免在测量和再生期间的基线漂移（Pope等人，2009）。然后在若干浓度下监测与蛋白质的相互作用，并且用从经典动力学分析获得的相应K D确定其对固定化抗体的亲和力。这种设置避免了二价抗体的亲合力效应，允许使用具有未知浓度的非纯化分析物和以类似的稳定的共价定向方式特异性捕获抗体。
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