Interferon alpha (IFN-α) protein levels were quantified with a digital-ELISA assay (Simoa, Quanterix) developed by Rodero et al.36 (link), in accordance with the instructions provided by the manufacturer of the Homebrew Simoa assay kit, with two autoantibodies specific for IFN-α isolated and cloned from two patients with autoimmune polyendocrinopathy syndrome type 1 (APSl)/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED)50 (link). The 8H1 antibody clone was used to coat paramagnetic beads (0.3 mg/ml), as a capture antibody, and the 12H5 antibody was biotinylated (biotin-to-antibody ratio 30: 1) and used as the detector. SBG was used at concentrations of 0.3 μg/ml and 150 pM as a detector and revelation enzyme, respectively. Recombinant IFN-α17/-αI (PBL Assay Science) was used to generate a standard curve, after we had tested for cross-reactivity. Each sample was analyzed in duplicate and at a 1:3 dilution. The limit of detection (LOD) was calculated as the mean value +3SD (positivity with 99% confidence) of reactivity for all blank runs. The LOD, taking the dilution factor into account, was 0.19 fg/ml.