Development of a luciferase-based system for the detection of ZnT8 autoantibodies.
Appearance of autoantibodies represents the first detectable sign of autoimmune destruction of beta cells in type 1 diabetes (T1D). In addition, autoantibody levels represent an important predictive marker regarding the development of an autoimmune process. Recently, the zinc transporter (ZnT8) protein was identified as an autoimmune target in T1D; therefore, there is a need for reliable and simple methods for detection of ZnT8 autoantibodies. This report describes an assay designed to detect anti-ZnT8 autoantibodies in the serum of patients with T1D. This was carried out by generating a ZnT8 C-terminal dimer fused to the N-terminus of the Gaussia princeps luciferase that was overexpressed in insect cells using the baculovirus expression system. Recombinant protein was semi-purified and used as the target antigen in the liquid-phase luciferase immunoprecipitation system assay (LIPS), and results were compared to data obtained using a commercial ELISA designed to detect ZnT8 autoantibodies in T1D patient sera, particularly among adults. LIPS was less effective in detecting antibodies in children probably due to the relatively high prevalence of IgM anti-ZnT8 antibodies in children with T1D.
Department of Immunology, Institute of Biomedicine and Translational Medicine, Faculty of Medicine, University of Tartu, Estonia; Centre of Excellence for Translational Medicine, University of Tartu, Estonia.
Julia
J
Ustinova
Institute of Technology, Faculty of Science and Technology, University of Tartu, Estonia.
Eva
E
Zusinaite
Department of Immunology, Institute of Biomedicine and Translational Medicine, Faculty of Medicine, University of Tartu, Estonia; Centre of Excellence for Translational Medicine, University of Tartu, Estonia.
Meeme
M
Utt
Department of Immunology, Institute of Biomedicine and Translational Medicine, Faculty of Medicine, University of Tartu, Estonia.
Kaja
K
Metsküla
Department of Immunology, Institute of Biomedicine and Translational Medicine, Faculty of Medicine, University of Tartu, Estonia; Centre of Excellence for Translational Medicine, University of Tartu, Estonia.
Koit
K
Reimand
Department of Immunology, Institute of Biomedicine and Translational Medicine, Faculty of Medicine, University of Tartu, Estonia.
Vimala
V
Huchaiah
Institute of Technology, Faculty of Science and Technology, University of Tartu, Estonia.
Andres
A
Merits
Department of Immunology, Institute of Biomedicine and Translational Medicine, Faculty of Medicine, University of Tartu, Estonia; Centre of Excellence for Translational Medicine, University of Tartu, Estonia; Immunotron OÜ, Estonia. Electronic address: raivo.uibo@ut.ee.
Raivo
R
Uibo
D000328:Adult; D000818:Animals; D001323:Autoantibodies / Q000097:blood / Q000276:immunology*; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D002648:Child; D003922:Diabetes Mellitus, Type 1 / Q000097:blood / Q000175:diagnosis / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D008156:Luciferases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D008297:Male; D055503:Protein Multimerization; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D061987:Sf9 Cells; D018411:Spodoptera; D000075703:Zinc Transporter 8
D001323:Autoantibodies; D027682:Cation Transport Proteins; C492522:SLC30A8 protein, human; D000075703:Zinc Transporter 8; D008156:Luciferases
Autoantibodies; Luciferase immunoprecipitation system (LIPS); Type 1 diabetes; Zinc transporter 8 (ZnT8)