A versatile protein microarray platform enabling antibody profiling against denatured proteins

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

Research output: Contribution to journalArticle

30 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)378-383
Number of pages6
JournalProteomics - Clinical Applications
Volume7
Issue number5-6
DOIs
StatePublished - Jun 1 2013

All Science Journal Classification (ASJC) codes

  • Clinical Biochemistry

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    Wang, J., Barker, K., Steel, J., Park, J., Saul, J., Festa, F., Wallstrom, G., Yu, X., Bian, X., Anderson, K. S., Figueroa, J. D., Labaer, J., & Qiu, J. (2013). A versatile protein microarray platform enabling antibody profiling against denatured proteins. Proteomics - Clinical Applications, 7(5-6), 378-383. https://doi.org/10.1002/prca.201200062