title: Single-cell antibody nanowells: a novel technology in detecting anti-SSA/Ro60- and anti-SSB/La autoantibody-producing cells in peripheral blood of rheumatic disease patients.
abstract: Anti-SSA/Ro60 and anti-SSB/La are essential serological biomarkers for rheumatic diseases, specifically Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). Currently, laboratory detection technology and platforms are designed with an emphasis on high-throughput methodology; therefore, the relationship of sensitivity with specificity remains a significant area for improvement. In this study, we used single-cell antibody nanowells (SCAN) technology to directly profile individual B cells producing antibodies against specific autoantigens such as SSA/Ro60 and SSB/La.
  
  
  
  Peripheral blood mononuclear cells were isolated using Ficoll gradient. Fluorescently labeled cells were added to fabricated nanowells and imaged using a high-speed epifluorescence microscope. The microengraving process was conducted using printed slides coated with immunoglobulins. Printed slides were hybridized with fluorescence-conjugated immunoglobulin G (IgG), SSA/Ro60, and SSB/La antigens. Microarray spots were analyzed for nanowells with single live B cells that produced antigen-specific autoantibodies.
  
  
  
  Our results indicate that SCAN can simultaneously detect high frequencies of anti-SSA/Ro60 and anti-SSB/La with a specific IgG isotype in peripheral blood mononuclear cells of patients, as well as measure their individual secretion levels. The data showed that patients with SS and SLE exhibited higher frequency and greater concentration of anti-SSA/Ro60- and anti-SSB/La-producing B cells in the IgG isotype. Furthermore, individual B cells of patients produced higher levels of IgG-specific anti-SSA/Ro60 autoantibody, but not IgG-specific anti-SSB/La autoantibody, compared with healthy control subjects.
  
  
  
  These results support the application of SCAN as a robust multiparametric analytical bioassay that can directly measure secretion of autoantibody and accurately report antigen-specific, autoantibody-producing cells.
authors.affiliation: Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, PO Box 110880, Gainesville, FL 32611-0880, USA.
authors.forename: Lida
authors.initials: L
authors.lastname: Esfandiary
authors.affiliation: Department of Pediatric Nephrology, University of Florida, Gainesville, FL, USA.
authors.forename: Nirupama
authors.initials: N
authors.lastname: Gupta
authors.affiliation: Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, PO Box 110880, Gainesville, FL 32611-0880, USA.
authors.forename: Alexandria
authors.initials: A
authors.lastname: Voigt
authors.affiliation: Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, PO Box 110880, Gainesville, FL 32611-0880, USA.
authors.forename: Arun
authors.initials: A
authors.lastname: Wanchoo
authors.affiliation: Department of Oral Biology, University of Florida, Gainesville, FL, USA.
authors.forename: Edward K L
authors.initials: EK
authors.lastname: Chan
authors.affiliation: Rheumatology Section, Department of Pediatrics, University of Arkansas for Medical Sciences, Arkansas Children's Hospital, Little Rock, AR, USA.
authors.forename: Sukesh
authors.initials: S
authors.lastname: Sukumaran
authors.affiliation: Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, PO Box 110880, Gainesville, FL 32611-0880, USA. nguyenc@ufl.edu.|Center of Orphaned Autoimmune Diseases, University of Florida, Gainesville, FL, USA. nguyenc@ufl.edu.
authors.forename: Cuong Q
authors.initials: CQ
authors.lastname: Nguyen
mesh_terms: D000293:Adolescent; D001323:Autoantibodies / Q000032:analysis* / Q000276:immunology; D001324:Autoantigens / Q000276:immunology; D015415:Biomarkers / Q000032:analysis*; D002648:Child; D005260:Female; D057166:High-Throughput Screening Assays; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008297:Male; D008875:Middle Aged; D036103:Nanotechnology / Q000379:methods*; D010446:Peptide Fragments / Q000276:immunology; D020733:RNA, Small Cytoplasmic / Q000276:immunology; D012216:Rheumatic Diseases / Q000097:blood / Q000276:immunology*; D012261:Ribonucleoproteins / Q000276:immunology; D000099207:SS-A Antigen
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D015415:Biomarkers; D010446:Peptide Fragments; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins; C506261:SSB protein, human; C116006:RO60 protein, human; D000099207:SS-A Antigen
keywords: Anti-SSA/Ro60; Anti-SSB/La; Antinuclear autoantibody; Single-cell analysis; Sjögren’s syndrome; Systemic lupus erythematosus