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Functional peptide microarrays for specific and sensitive antibody diagnostics

  • Peptide microarrays displaying biologically active small synthetic peptides in a high-density format provide an attractive technology to probe complex samples for the presence and/or function of protein analytes. We present a new approach for manufacturing functional peptide microarrays for molecular immune diagnostics. Our method relies on the efficiency of site-specific solution-phase coupling of biotinylated synthetic peptides to NeutrAvidin (NA) and localized microdispensing of peptide-NA-complexes onto activated glass surfaces. Antibodies are captured in a sandwich manner between surface immobilized peptide probes and fluorescence-labeled secondary antibodies. Our work includes a total of 54 peptides derived from immunodominant linear epitopes of the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c-myc protein, and three domains of the Human coronavirus polymerase polyprotein and their cognate mAbs. By using spacer molecules of different type and length for NA-mediated peptide presentation, we show that thePeptide microarrays displaying biologically active small synthetic peptides in a high-density format provide an attractive technology to probe complex samples for the presence and/or function of protein analytes. We present a new approach for manufacturing functional peptide microarrays for molecular immune diagnostics. Our method relies on the efficiency of site-specific solution-phase coupling of biotinylated synthetic peptides to NeutrAvidin (NA) and localized microdispensing of peptide-NA-complexes onto activated glass surfaces. Antibodies are captured in a sandwich manner between surface immobilized peptide probes and fluorescence-labeled secondary antibodies. Our work includes a total of 54 peptides derived from immunodominant linear epitopes of the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c-myc protein, and three domains of the Human coronavirus polymerase polyprotein and their cognate mAbs. By using spacer molecules of different type and length for NA-mediated peptide presentation, we show that the incorporation of a minimum spacer length is imperative for antibody binding, whereas the peptide immobilization direction has only secondary importance for antibody affinity and binding. We further demonstrate that the peptide array is capable of detecting low-picomolar concentrations of mAbs in buffered solutions and diluted human serum with high specificityshow moreshow less

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Metadaten
Author:Heiko Andresen, Carsten Grotzinger, Kim Zarse, Oliver Johannes Kreuzer, Eva Ehrentreich-Förster, Frank Fabian BierORCiDGND
URL:http://www3.interscience.wiley.com/cgi-bin/jhome/76510741
DOI:https://doi.org/10.1002/pmic.200500343
ISSN:1615-9853
Document Type:Article
Language:English
Year of first Publication:2006
Year of Completion:2006
Release Date:2017/03/25
Source:Proteomics. - ISSN 1615-9853. - 6 (2006), 5, S. 1376 - 1384
Organizational units:Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie
Peer Review:Referiert