Preparation and application of a fluorescein-labeled peptide for determining the affinity constant of a monoclonal antibody-hapten complex by fluorescence polarization.
Preparation and application of a fluorescein-labeled peptide for determining the affinity constant of a monoclonal antibody-hapten complex by fluorescence polarization.
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DOI：
10.1016/0003-2697(91)90488-f
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发表时间：
1991-08
影响因子：
2.9
通讯作者：
W. Jiskoot;P. Hoogerhout;E. Beuvery;J. Herron;D. Crommelin
中科院分区：
文献类型：
--
作者：
W. Jiskoot;P. Hoogerhout;E. Beuvery;J. Herron;D. Crommelin
提出了一种在平衡条件下测定单克隆抗体-肽复合物的亲和常数的简单快速的方法。合成了与脑膜炎球菌菌株 MC50 1 类外膜蛋白序列 178-185 相对应的肽，该肽可被单克隆抗体 MN12（小鼠 IgG2a）识别。荧光素与肽偶联后，肽-荧光素缀合物用于与 MN12 的结合研究，利用荧光素标记的荧光偏振来探测肽的结合部分。 Scatchard 分析表明亲和常数与 pH 值相关。 MN12 在碱性条件下储存会导致抗原结合位点丢失，但不会改变亲和常数。 Sips 图显示了统一的同质性指数。
A simple and rapid method for determining the affinity constant of a monoclonal antibody-peptide complex under equilibrium conditions is presented. A peptide corresponding to sequence 178–185 of meningococcal strain MC50 class 1 outer membrane protein, which is recognized by monoclonal antibody MN12 (mouse IgG2a), was synthesized. After fluorescein was coupled to the peptide, the peptide-fluorescein conjugate was used for binding studies with MN12, employing fluorescence polarization of the fluorescein label to probe the bound fraction of the peptide. Scatchard analysis showed that the affinity constant was pH dependent. Storage of MN12 under alkaline conditions resulted in a loss of antigen-binding sites, but did not alter the affinity constant. Sips plots showed a homogeneity index of unity.