Document Detail


A versatile protein microarray platform enabling antibody profiling against denatured proteins.
MedLine Citation:
PMID:  23027520     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
PURPOSE: We aim to develop a protein microarray platform capable of presenting both natural and denatured forms of proteins for antibody biomarker discovery. We will further optimize plasma screening protocols to improve detection.
EXPERIMENTAL DESIGN: We developed a new covalent capture protein microarray chemistry using HaloTag fusion proteins and ligand. To enhance protein yield, we used HeLa cell lysate as an in vitro transcription translation (IVTT) system. Escherichia coli lysates were added to the plasma blocking buffer to reduce nonspecific background. These protein microarrays were probed with plasma samples and autoantibody responses were quantified and compared with or without denaturing buffer treatment.
RESULTS: We demonstrated that protein microarrays using the covalent attachment chemistry endured denaturing conditions. Blocking with E. coli lysates greatly reduced the background signals and expression with IVTT based on HeLa cell lysates significantly improved the antibody signals on protein microarrays probed with plasma samples. Plasma samples probed on denatured protein arrays produced autoantibody profiles distinct from those probed on natively displayed proteins.
CONCLUSIONS AND CLINICAL RELEVANCE: This versatile protein microarray platform allows the display of both natural and denatured proteins, offers a new dimension to search for disease-specific antibodies, broadens the repertoire of potential biomarkers, and will potentially yield clinical diagnostics with greater performance.
Authors:
Jie Wang; Kristi Barker; Jason Steel; Jin Park; Justin Saul; Fernanda Festa; Garrick Wallstrom; Xiaobo Yu; Xiaofang Bian; Karen S Anderson; Jonine D Figueroa; Joshua LaBaer; Ji Qiu
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Intramural; Research Support, Non-U.S. Gov't     Date:  2013-05-17
Journal Detail:
Title:  Proteomics. Clinical applications     Volume:  7     ISSN:  1862-8354     ISO Abbreviation:  Proteomics Clin Appl     Publication Date:  2013 Jun 
Date Detail:
Created Date:  2013-06-19     Completed Date:  2013-11-27     Revised Date:  2014-08-14    
Medline Journal Info:
Nlm Unique ID:  101298608     Medline TA:  Proteomics Clin Appl     Country:  Germany    
Other Details:
Languages:  eng     Pagination:  378-83     Citation Subset:  IM    
Copyright Information:
© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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MeSH Terms
Descriptor/Qualifier:
Antibodies / analysis,  immunology,  metabolism*
Autoantibodies / analysis,  immunology,  metabolism
HeLa Cells
Humans
Protein Array Analysis*
Protein Biosynthesis
Protein Denaturation
Proteins / chemistry,  immunology,  metabolism*
Grant Support
ID/Acronym/Agency:
ZIA CP010126-18/CP/NCI NIH HHS
Chemical
Reg. No./Substance:
0/Antibodies; 0/Autoantibodies; 0/Proteins
Comments/Corrections

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