AAN-I-IFN in the patient’s blood neutralized IFN-α and IFN-ω. (A) A549-IFN-reporter (AIR) cells carrying the ISRE reporter were stimulated with IFN-α, -ω, or -β at the concentration indicated, with or without blood from the patient (pt), an AAN-I-IFN–positive control (positive ctrl), an APS-1 patient, or healthy donors (HD). All samples were diluted 1:20. Renilla luciferase activity was measured 24 h after stimulation. The results are expressed as a percentage of the mean value for HDs. Luciferase activity levels <25% that of HDs were considered to indicate neutralizing activity. Two separate blood draws from the patient were sampled. Experiments were done three times. (B) ELISA plates were coated with 1 μg/ml of the IFN subtypes indicated and incubated with blood samples (diluted 1:50). Anti-human IgG-HRP secondary antibodies were then added, and OD was measured at 450 nm. An OD₄₅₀>0.5 was considered to be a positive results. Three separate blood draws from the patient were sampled. Experiment was done once. (C) Multiplex assay beads were incubated with blood samples (diluted 1:1,000), and the MFI was normalized against a beads-only control. Normalized MFI values > 3 were considered positive. Two separate blood draws from the patient were sampled. Experiment was done once. (D) AAN-I-IFN neutralization tested with patient’s blood collected at the indicated time points as described in A. Three separate blood draws from the patient were sampled. Experiment was done once.