Institut des Biomolécules Max Mousseron-IBMM, Université de Montpellier, CNRS, ENSCM, 34093 Montpellier cedex 5, France.
COLCOM, Cap Alpha, 34830 Clapiers, France.
Talanta. 2018 Feb 1;178:922-927. doi: 10.1016/j.talanta.2017.10.032. Epub 2017 Oct 23.
Antibody-coated surfaces (Ab surfaces) play a key role in bioanalytical tools developed for biosensors and diagnostics. Therefore, a high and well-defined functional activity of the Ab surface is required. The functional activity of the Ab surface depends on its available binding sites i.e. "the active sites" that are able to capture antigen (Ag). The number of active binding sites strongly depends on the immobilization strategy used to fix the Ab on the solid surface. Determination of layer thickness or surface topology are often used to characterize the Ab surfaces but there is no gold standard method for the study of the functionality of the Ab surfaces. For that purpose, we aim at developing an assay allowing to determine the performances of Ab surfaces. In the present study, anti-horseradish peroxidase antibody (anti-HRP Ab) is used as capture Ab covalently bound to the surface and enzyme HRP as Ag. This direct assay permits, in one-step, to generate a signal utilizing the catalytic properties of HRP. The signal is directly proportional to the amount of HRP bound on the Ab surface, and therefore to the active binding sites of immobilized Ab. The HRP/anti-HRP Ab interactions may be a useful indicator to construct accurate and reproducible active Ab surfaces and also to improve their performances in term of stability and sensitivity. Optimization of the assay parameters and quality of the results are presented. A good repeatability and an acceptable inter-day precision (RSD < 10%) are reported.
抗体包被表面（Ab 表面）在生物传感器和诊断开发的生物分析工具中起着关键作用。因此，需要具有高且明确的功能活性的 Ab 表面。Ab 表面的功能活性取决于其可用的结合位点，即能够捕获抗原（Ag）的“活性位点”。活性结合位点的数量强烈依赖于用于将 Ab 固定在固体表面上的固定化策略。通常使用层厚度或表面拓扑结构来表征 Ab 表面，但没有用于研究 Ab 表面功能的金标准方法。为此，我们旨在开发一种能够确定 Ab 表面性能的测定法。在本研究中，辣根过氧化物酶抗体（anti-HRP Ab）用作通过共价键结合到表面的捕获 Ab，酶 HRP 用作 Ag。这种直接测定法可以一步生成利用 HRP 催化特性的信号。该信号与结合在 Ab 表面上的 HRP 的量直接成正比，因此与固定化 Ab 的活性结合位点成正比。HRP/anti-HRP Ab 相互作用可能是构建准确且可重现的活性 Ab 表面的有用指标，并且还可以提高其在稳定性和灵敏度方面的性能。呈现了测定法参数的优化和结果的质量。报道了良好的重复性和可接受的日间精密度（RSD <10%）。
