title: A versatile protein microarray platform enabling antibody profiling against denatured proteins.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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
  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.affiliation: Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, AZ 85287-6401, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Wang
authors.forename: Kristi
authors.initials: K
authors.lastname: Barker
authors.forename: Jason
authors.initials: J
authors.lastname: Steel
authors.forename: Jin
authors.initials: J
authors.lastname: Park
authors.forename: Justin
authors.initials: J
authors.lastname: Saul
authors.forename: Fernanda
authors.initials: F
authors.lastname: Festa
authors.forename: Garrick
authors.initials: G
authors.lastname: Wallstrom
authors.forename: Xiaobo
authors.initials: X
authors.lastname: Yu
authors.forename: Xiaofang
authors.initials: X
authors.lastname: Bian
authors.forename: Karen S
authors.initials: KS
authors.lastname: Anderson
authors.forename: Jonine D
authors.initials: JD
authors.lastname: Figueroa
authors.forename: Joshua
authors.initials: J
authors.lastname: LaBaer
authors.forename: Ji
authors.initials: J
authors.lastname: Qiu
mesh_terms: D000906:Antibodies / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D001323:Autoantibodies / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D040081:Protein Array Analysis; D014176:Protein Biosynthesis; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*
chemical_list: D000906:Antibodies; D001323:Autoantibodies; D011506:Proteins