Multiplex Autoantibody Detection in Patients with Autoimmune Polyglandular Syndromes

Abstract

The diagnosis of autoimmune polyglandular syndrome (APS) types 1/2 is difficult due to their rarity and nonspecific clinical manifestations. APS-1 development can be identified with assays for autoantibodies against cytokines, and APS-2 development with organ-specific antibodies. In this study, a microarray-based multiplex assay was proposed for simultaneous detection of both organ-specific (anti-21-OH, anti-GAD-65, anti-IA2, anti-ICA, anti-TG, and anti-TPO) and APS-1-specific (anti-IFN-ω, anti-IFN-α-2a, and anti-IL-22) autoantibodies. Herein, 206 serum samples from adult patients with APS-1, APS-2, isolated autoimmune endocrine pathologies or non-autoimmune endocrine pathologies and from healthy donors were analyzed. The prevalence of autoantibodies differed among the groups of healthy donors and patients with non-, mono- and multi-endocrine diseases. APS-1 patients were characterized by the presence of at least two specific autoantibodies (specificity 99.5%, sensitivity 100%). Furthermore, in 16 of the 18 patients, the APS-1 assay revealed triple positivity for autoantibodies against IFN-ω, IFN-α-2a and IL-22 (specificity 100%, sensitivity 88.9%). No anti-cytokine autoantibodies were found in the group of patients with non-APS-1 polyendocrine autoimmunity. The accuracy of the microarray-based assay compared to ELISA for organ-specific autoantibodies was 88.8-97.6%. This multiplex assay can be part of the strategy for diagnosing and predicting the development of APS.

Keywords: autoantibodies; autoimmune polyglandular syndrome; microarray; multiplex assay.

Figure 1

(a) Microarray configuration; (b) fluorescence image of the microarray; and (c) normalized signals from the microarray elements after assay of the serum sample from patient #49 with APS-1. Designations: IFN-ω—ntierferon omega; IFN-α-2a—interferon-alpha-2a; IL-22—interleukin 22; 21-OH—steroid 21 hydroxylase; GAD-65—glutamic acid decarboxylase 65 kDa; IA-2—tyrosine phosphatase-like autoantigen; ICA—islet cell autoantigen 1; TPO—thyroid peroxidase; TG—thyroglobulin; M—marker; human IgG—human immunoglobulin G; anti-human IgG—mouse anti-human immunoglobulin G; Empty—reference gel elements without immobilized protein.

Figure 2

Normalized signals for autoantibodies against IFN-ω, IFN-α-2a and IL-22 in serum samples from APS-1 patients, as determined by the microarray-based assay.

Figure 3

Normalized signals for autoantibodies against (a) IFN-ω; (b) IFN-α-2a; and (c) IL-22 (c) in serum samples from non-APS-1 patients, as determined by the microarray-based assay.

Figure 4

Frequencies of positive autoantibodies among (a) all patients; (b) healthy donors and patients without autoimmune polyendocrine syndromes; and (c) patients with autoimmune polyendocrine syndromes.

Figure 5

Frequency of positivity for organ-specific autoantibodies against (a) GAD-65, IA2, and ICA and against (b) TG and TPO among healthy controls and patients with APS-associated or isolated diseases. Designations: ‘+’—confirmed disease; ‘-’—absence of disease.