A robust immunoassay for anti-interferon autoantibodies that is highly specific for patients with autoimmune polyglandular syndrome type 1.

Abstract

UNLABELLED:

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: Li Zhang, Jennifer M Barker, Sunanda Babu, Maureen Su, Matthew Stenerson, Mickie Cheng, Anthony Shum, Ehud Zamir, Raffaele Badolato, Adam Law, George S Eisenbarth, Mark S Anderson
• Journal: Clinical immunology (Orlando, Fla.) (Clin Immunol) Vol. 125 Issue 2 Pg. 131-7 (Nov 2007) ISSN: 1521-6616 [Print] United States
• PMID: 17825626 (Publication Type: Comparative Study, Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Autoantibodies
• Interferon-alpha
• Luminescent Agents
• Europium

Topics

• Adolescent
• Adult
• Antibody Specificity
• Autoantibodies (blood)
• Child
• Child, Preschool
• Europium (pharmacology)
• Fluoroimmunoassay (methods)
• Fluorometry
• Humans
• Interferon-alpha (immunology)
• Luminescent Agents (pharmacology)
• Middle Aged
• Polyendocrinopathies, Autoimmune (immunology)