2.2. Cell-based assay of human IFN-α neutralization in HEK-blueTM cells

The IFN-neutralizing capacity of S95021 and benchmark antibodies was tested with the help of reporter HEK-BlueTM IFN-α/β cells (InvivoGen) that express alkaline phosphatase (AP) under the inducible ISG54 promoter after ISGF binding to the IFN-stimulated response elements (ISRE) in the promoter as previously reported [33]. The cells were grown in DMEM (Naxo), containing 10% heat inactivated FBS and supplemented with 30 ​μg/mL blasticidin (InvivoGen) and 100 ​μg/mL Zeocin (InvivoGen). IFN-α2a was purchased from Miltenyi Biotech (Bergisch Gladbach, Germany) and all other subtypes were from PBL Assay Science and used at the following final concentrations: IFN-α2a 200 U/ml, IFN-α1 50 U/ml, IFN-α4, IFN-α5, IFN-α7 and IFN-α21 25 U/ml, IFN-α10 and IFN-α17 12.5 U/ml, IFN-α8 6.25 U/ml, IFN-α6 3 U/ml, IFN-α16 1.5 U/ml and IFN-α14 0.6 U/ml. Cells were stimulated with optimized concentrations of type I IFNs that were preincubated for 2 ​h with serial dilutions of monoclonal antibodies. QUANTI-Blue TM (InvivoGen) colorimetric enzyme assay was used to determine AP in the cell culture supernatants after 21 ​h of incubation. OD was measured at 620 ​nm with a Multiscan MCC/340 (Labsystems) ELISA reader. IC₅₀ values were calculated from the dose-response curves.