Philips Research, High Tech Campus, 5656 AE Eindhoven, The Netherlands.
Analyst. 2017 Nov 6;142(22):4247-4256. doi: 10.1039/c7an01424d.
The accessibility of particle-coupled antibodies is important for many analytical applications, but comprehensive data on parameters controlling the accessibility are scarce. Here we report on the site-specific accessibility of monoclonal antibodies, immobilized on magnetic nanoparticles (500 nm) by the widely used covalent EDC coupling method, with the variation of four key coupling parameters (surface activation and immobilization pH, crosslinker and antibody concentration ratios). By developing quantitative radio-labelled assays, the number of immobilized antibodies, the Fab domain accessibility (in a sandwich immunoassay), and the Fc domain accessibility (in a Protein G assay) were determined. For sub-monolayer surface coverage, the observed numbers of accessible Fab and Fc domains are equal and scale linearly with the antibody density. For above monolayer coverage, the fractions of accessible Fab and Fc domains decrease, in an unequal manner. The results show that the antibody accessibility is primarily determined by the antibody surface density, rather than by chemical reactivity or the charge state, and that crowded conditions affect Fab and Fc accessibility in an unequal manner.
颗粒偶联抗体的可及性对于许多分析应用很重要，但控制可及性的参数的综合数据却很少。在这里，我们报告了通过广泛使用的共价 EDC 偶联方法固定在磁性纳米颗粒（500nm）上的单克隆抗体的位点特异性可及性，其中变化了四个关键的偶联参数（表面活化和固定 pH 值、交联剂和抗体浓度比）。通过开发定量放射性标记分析，确定了固定的抗体数量、Fab 结构域的可及性（在三明治免疫测定中）以及 Fc 结构域的可及性（在 Protein G 测定中）。对于亚单层表面覆盖率，观察到的可及 Fab 和 Fc 结构域的数量相等，并与抗体密度呈线性关系。对于超过单层覆盖，可及的 Fab 和 Fc 结构域的分数以不同的方式减少。结果表明，抗体的可及性主要取决于抗体的表面密度，而不是化学反应性或电荷状态，并且拥挤条件以不同的方式影响 Fab 和 Fc 的可及性。
