Evaluation of homo- and hetero-functionally activated glass surfaces for optimized antibody arrays

et al.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.This work was supported by grants FIS PI081884 and PI1102114 from the Instituto de Salud Carlos III (ISCIII; Ministerio de Ciencia e Innovación, Madrid, Spain) and grant JCYL-SA198A12-2 from the Consejería de Educación de la Junta de Castilla y León (Spain). María González-González is supported by a PhD scholarship from the ISCIII (FI08/00721), Raquel Bartolome is supported by a PhD scholarship from the Ministerio de Educación (FPU, AP2009-2633), and Noelia Dasilva is supported by a PhD scholarship from JCyL (Orden EDU 346/2013). Juan Casado-Vela is a JAE–DOC (CSIC) holder supported by the Ministerio de Economía y Competitividad (Spain), cofunded by the European Social Fund.Peer Reviewe