title: A robust immunoassay for anti-interferon autoantibodies that is highly specific for patients with autoimmune polyglandular syndrome type 1.
abstract: High titer antibodies to type 1 interferons have been recently reported as being highly specific for patients with autoimmune polyglandular syndrome type 1 (APS1) in Finnish and Norwegian patients with mutations in the AIRE gene. Those studies employed a complex neutralization assay to define the type 1 interferon autoantibodies. Here we have established a competitive europium time resolved fluorescence assay for IFN-alpha autoantibodies and measured sera from subjects with APS1, first degree relatives of APS1 patients, patients with Addison's disease or Type 1 diabetes. The europium-based immunoassay utilizes plate bound human IFN-alpha incubated with sera with or without competition with fluid phase IFN-alpha, followed by anti-IgG biotinylated antibody and detection with streptavidin-europium. The index of IFN-alpha Ab was calculated as (CPS (Counts per second) without competition-CPS with competition)/(CPS positive standard sera without competition-CPS positive standard sera with competition). RESULTS are reported for raw CPS and indices and are compared across the different subjects.
  
  
  RESULTS
  For normal controls (n=100) CPS without competition were 31,237+/-17,328 CPS while after subtracting the competition value, the results were -6563+/-10,303 CPS. The initial APS1 patient (used to create the index as 1.0) gave 394,063 CPS without competition and a delta of 363,662+/-31,587 CPS with competition. Scatchard plot analysis of this patient sample revealed a high avidity for IFN-alpha (K(d) of 0.5 nM). The CPS, delta, and index for 6/7 APS1 patients were strongly positive and 3 standard deviations or more above that of the normal controls. Using a cut-off of 2 standard deviations above normal controls, relatives of APS1 patients were negative for type I interferon autoantibodies as were 71 patients with Addison's disease (non-APS1) and 141 Type 1 diabetes patients. This simple high throughput competitive europium time resolved fluorescence assay had a sensitivity of > or =86% or greater and a specificity of >99.5%.
authors.affiliation: Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center, Box B140 Building M20, 1775 N. Ursula Street, Aurora, CO 80045-6511, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Barker
authors.forename: Sunanda
authors.initials: S
authors.lastname: Babu
authors.forename: Maureen
authors.initials: M
authors.lastname: Su
authors.forename: Matthew
authors.initials: M
authors.lastname: Stenerson
authors.forename: Mickie
authors.initials: M
authors.lastname: Cheng
authors.forename: Anthony
authors.initials: A
authors.lastname: Shum
authors.forename: Ehud
authors.initials: E
authors.lastname: Zamir
authors.forename: Raffaele
authors.initials: R
authors.lastname: Badolato
authors.forename: Adam
authors.initials: A
authors.lastname: Law
authors.forename: George S
authors.initials: GS
authors.lastname: Eisenbarth
authors.forename: Mark S
authors.initials: MS
authors.lastname: Anderson
mesh_terms: D000293:Adolescent; D000328:Adult; D000918:Antibody Specificity; D001323:Autoantibodies / Q000097:blood*; D002648:Child; D002675:Child, Preschool; D005063:Europium / Q000494:pharmacology; D015200:Fluoroimmunoassay / Q000379:methods*; D005470:Fluorometry; D006801:Humans; D016898:Interferon-alpha / Q000276:immunology*; D049408:Luminescent Agents / Q000494:pharmacology; D008875:Middle Aged; D016884:Polyendocrinopathies, Autoimmune / Q000276:immunology*