Centro de Investigación del Cáncer/IBMCC (USAL/CSIC)-IBSAL, Departamento de Medicina and Servicio General de Citometría, University of Salamanca, 37007 Salamanca, Spain.
ImmunoStep, 37007 Salamanca, Spain.
Anal Biochem. 2014 Apr 1;450:37-45. doi: 10.1016/j.ab.2014.01.002. Epub 2014 Jan 15.
Antibody arrays hold great promise for biomedical applications, but they are typically manufactured using chemically functionalized surfaces that still require optimization. Here, we describe novel hetero-functionally activated glass surfaces favoring oriented antibody binding for improved performance in protein microarray applications. Antibody arrays manufactured in our facility using the functionalization chemistries described here proved to be reproducible and stable and also showed good signal intensities. As a proof-of-principle of the glass surface functionalization protocols described in this article, we built antibody-based arrays functionalized with different chemistries that enabled the simultaneous detection of 71 human leukocyte membrane differentiation antigens commonly found in peripheral blood mononuclear cells. Such detection is specific and semi-quantitative and can be performed in a single assay under native conditions. In summary, the protocol described here, based on the use of antibody array technology, enabled the concurrent detection of a set of membrane proteins under native conditions in a specific, selective, and semi-quantitative manner and in a single assay.
抗体微阵列在生物医学应用中具有广阔的前景，但它们通常是使用化学功能化表面制造的，这些表面仍需要优化。在这里，我们描述了新型的杂功能化玻璃表面，有利于定向抗体结合，从而提高蛋白质微阵列应用的性能。我们在实验室中使用这里描述的功能化化学方法制造的抗体微阵列具有重现性和稳定性，并且显示出良好的信号强度。作为本文中描述的玻璃表面功能化方案的原理验证，我们构建了基于不同化学方法的抗体微阵列，这些微阵列能够同时检测外周血单个核细胞中常见的 71 个人白细胞膜分化抗原。这种检测是特异性和半定量的，可以在单个测定中在天然条件下进行。总之，这里描述的基于抗体微阵列技术的方案，能够以特异性、选择性和半定量的方式，在单个测定中，在天然条件下同时检测一组膜蛋白。
