Evaluation of homo- and hetero-functionally activated glass surfaces for optimized antibody arrays.
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.
Centro de Investigación del Cáncer/IBMCC (USAL/CSIC)-IBSAL, Departamento de Medicina and Servicio General de Citometría, University of Salamanca, 37007 Salamanca, Spain.
María
M
González-González
Centro de Investigación del Cáncer/IBMCC (USAL/CSIC)-IBSAL, Departamento de Medicina and Servicio General de Citometría, University of Salamanca, 37007 Salamanca, Spain.
Raquel
R
Bartolome
ImmunoStep, 37007 Salamanca, Spain.
Ricardo
R
Jara-Acevedo
Spanish National Research Council (CSIC)-Spanish National Biotechnology Centre (CNB), 28049 Madrid, Spain.
Juan
J
Casado-Vela
Centro de Investigación del Cáncer/IBMCC (USAL/CSIC)-IBSAL, Departamento de Medicina and Servicio General de Citometría, University of Salamanca, 37007 Salamanca, Spain.
Noelia
N
Dasilva
Centro de Investigación del Cáncer/IBMCC (USAL/CSIC)-IBSAL, Departamento de Medicina and Servicio General de Citometría, University of Salamanca, 37007 Salamanca, Spain.
Sergio
S
Matarraz
Servicio de Cirugía, Hospital Clínico Universitario de Salamanca-IBSAL, 37007 Salamanca, Spain.
Jacinto
J
García
Servicio de Cirugía, Hospital Clínico Universitario de Salamanca-IBSAL, 37007 Salamanca, Spain.
J A
JA
Alcazar
Centro de Investigación del Cáncer/IBMCC (USAL/CSIC)-IBSAL, Departamento de Medicina and Servicio General de Citometría, University of Salamanca, 37007 Salamanca, Spain.
J M
JM
Sayagues
Centro de Investigación del Cáncer/IBMCC (USAL/CSIC)-IBSAL, Departamento de Medicina and Servicio General de Citometría, University of Salamanca, 37007 Salamanca, Spain. Electronic address: orfao@usal.es.
Alberto
A
Orfao
Centro de Investigación del Cáncer/IBMCC (USAL/CSIC)-IBSAL, Departamento de Medicina and Servicio General de Citometría, University of Salamanca, 37007 Salamanca, Spain. Electronic address: mfuentes@usal.es.
Manuel
M
Fuentes
D000818:Animals; D055910:Antibodies, Immobilized / Q000737:chemistry* / Q000276:immunology; D005898:Glass / Q000737:chemistry*; D006680:HLA Antigens / Q000276:immunology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000276:immunology; D040081:Protein Array Analysis / Q000379:methods*; D015203:Reproducibility of Results; D013499:Surface Properties
D055910:Antibodies, Immobilized; D006680:HLA Antigens
Antibody arrays; Antibody immobilization; Functionalized surfaces; Protein microarrays; Surface functionalization