Summary
Peptide microarrays are useful instruments for miniaturized high-throughput, high-content immunoassays. The substitution of linear epitopes of the protein antigen with short, synthetic peptides is virtually a straightforward approach to capture antigen-specific antibodies from serum samples; however, both the biologically active surface display of peptides and the establishment of a solidly performing peptide microarray immunoassay are often troublesome in detail. The following protocols aim to provide facilitated access to the production of a robust peptide microarray platform and an optimized analytical processing of peptide microarrays in serological diagnosis. The functional surface display is accomplished by site-specific immobilization of peptide probes in a two-step procedure first by coupling of biotinylated peptides to hydrazide-modified streptavidin and then utilizing a subsequent chemoselective reaction between the hydrazide linkers of the streptavidin and an aldehyde-coated substrate for microstructured surface immobilization of the probe complexes. The serological assay is based on the specific capture of primary antibodies in a sandwich complex between the surface-immobilized peptides and a fluorescently labelled secondary antibody. A proposal for the diagnostic evaluation of fluorescence data obtained with the peptide microarray is also made.
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Acknowledgments
The authors greatly acknowledge Dr. C. Grötzinger, Dr. E. Ehrentreich-Förster, and the peptides&elephants GmbH (Potsdam, Germany) for comments and discussions.
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Andresen, H., Bier, F.F. (2009). Peptide Microarrays for Serum Antibody Diagnostics. In: Bilitewski, U. (eds) Microchip Methods in Diagnostics. Methods in Molecular Biology™, vol 509. Humana Press. https://doi.org/10.1007/978-1-59745-372-1_8
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DOI: https://doi.org/10.1007/978-1-59745-372-1_8
Publisher Name: Humana Press
Print ISBN: 978-1-58829-955-0
Online ISBN: 978-1-59745-372-1
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