Quantification of Anti-Type I IFN Auto-Antibodies Using Gyros Platform

Gyros was used for the detection of anti-type I IFN auto-Abs, as described by Bastard et al. [8 (link)]. Cytokines, recombinant human (rh)IFN-α2 (Miltenyi Biotec, reference number 130–108-984) and rhIFN-ω (Merck, reference number SRP3061) were first biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, catalog number A39257), according to the manufacturer’s instructions, with a biotin-to-protein molar ratio of 1:12. The detection reagent contained a secondary antibody [Alexa Fluor 647 goat anti-human IgG (Thermo Fisher Scientific, reference number A21445)] diluted in Rexxip F (Gyros Protein Technologies, reference number P0004825; 1:500 dilution of the 2 mg/mL stock to yield a final concentration of 4 μg/mL). Phosphate-buffered saline, 0.01% Tween 20 (PBS-T) and Gyros Wash buffer (Gyros Protein Technologies, reference number P0020087) were prepared according to the manufacturer’s instructions. BAL or plasma samples were then diluted 1:100 in 0.01% PBS-T and tested with the Bioaffy 1000 CD (Gyros Protein Technologies, reference number P0004253) and the Gyrolab xPand (Gyros Protein Technologies, reference number P0020520). Cleaning cycles were performed in 20% ethanol.