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%.

Figure 1

Schematic of the immunofluorometric IFN-a autoantibody assay.

Figure 2

Index of IFN-a Ab’s in different patients groups. Anti-IFN a autoantibodies showed significant specificity in APS1 patients. Serum was diluted in 1:10 and measured by europium-based fluorescence. Each point presents the mean index of the duplicated well for that individual sample. The bar indicates the mean value for each cohort. APS1, n=7; non-APS1, n=6; normal controls, n=100; Addison’s Disease, n =71; Type 1 diabetes, n=141.

Figure 3

IFN- a Ab assay results with and without competition for the five different patient groups. Shown are CPS results for individual samples with and without competition for IFN-α(8 μg/ml). Un: without competition assay results; C: competition assay results; NC: normal controls.

Figure 4

A) Competition assay of inhibition by different concentrations of fluid phase IFN-a protein (ranging from 0.0016 to 1.6 μg/ml) in the initial APS1 patient sample. Results are average CPS of duplicated wells. B) Scatchard plot of IFN-a protein binding to antibodies from the initial APS1 patient. The Scatchard analysis was performed with competition concentrations of IFN-a protein ranging from 0.0016 to 1.6 μg/ml. Results are the average of duplicate wells.