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

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-α 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-α incubated with sera with or without competition with fluid phase IFN-α, followed by anti-IgG biotinylated antibody and detection with streptavidin-europium. The index of IFN-α 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-α (Kd 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 ≥ 86% or greater and a specificity of > 99.5%.