ELISAs were performed as previously described (Puel et al., 2022 (link)). In brief, 96-well ELISA plates (MaxiSorp; Thermo Fisher Scientific) were coated by incubation overnight at 4°C with 2 μg/ml rhIFN-α and rhIFN-ω (R&D Systems). Plates were then washed (PBS/0.005% Tween), blocked by incubation with the same buffer supplemented with 5% nonfat milk powder, washed, and incubated with 1:50 dilutions of plasma samples from the patients or controls for 2 h at room temperature (or with specific mAbs as positive controls). Each sample was tested once. Plates were thoroughly washed. HRP-conjugated Fc-specific IgG fractions from polyclonal goat antiserum against human IgG or IgA (Nordic Immunological Laboratories) were added to a final concentration of 2 μg/ml. Plates were incubated for 1 h at room temperature and washed. Substrate was added and OD was measured.
Rhifn α
Manufactured by R&D Systems
Sourced in United States
RhIFN-α is a recombinant human interferon alpha product manufactured by R&D Systems. It is a protein that is involved in the regulation of the immune system and cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Rhifn ω, by R&D Systems (2 mentions)
Maxisorp, by Thermo Fisher Scientific (2 mentions)
Anti phosphostat1 bv421 tyr701 clone 4a, by BD (1 mentions)
Anti phosphostat3 pe tyr705 clone 4 5 stat3, by BD (1 mentions)
Anti hla dr alexa700 clone l243, by BioLegend (1 mentions)
Rhil 27, by Thermo Fisher Scientific (1 mentions)
Rhil 6, by R&D Systems (1 mentions)
Facsaria 2, by BD (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Anti cd14 pedy594 clone mem 15, by Exbio (1 mentions)
Anti cd11c apc clone bu15, by Exbio (1 mentions)
Rhifn λ1, by R&D Systems (1 mentions)
Recombinant human m csf, by Thermo Fisher Scientific (1 mentions)
4 protocols using rhifn α
1
ELISA Detection of IFN-α/ω Autoantibodies
2
Intracellular Signaling Pathway Analysis
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Whole blood was stimulated with 100 ng/ml rhIL-27 (Peprotech, New York, USA) for 15 minutes, 10 ng/ml rhIL-6, 500 ng/ml rhIFNα (both from R&D Systems) when indicated or left untreated at 37 °C. The optimal stimulation time was estimated after analyzing the effects of 5, 15 and 30 minutes of rhIL-27 stimulation on 8 healthy controls (Suppl. Fig. 2A ). Intracellular signaling was prevented by using 4% paraformaldehyde without methanol (Sigma Aldrich) for 10 minutes at room temperature. Erythrocytes were lysed using 0, 1% Triton-X for 20 minutes (Sigma Aldrich) at 37 °C, leukocytes were permeabilized with ice-cold 80% methanol for 30 minutes and stained with a FITC-conjugated lineage antibody cocktail (CD3 clone MEM-57, CD19 clone LT19, CD20 clone LT20, CD16 clone LNK16, and CD56 clone MEM-188), anti-CD14-PEDy594 (clone MEM-15) (Exbio), anti-HLA-DR-Alexa700 (clone L243), anti-CD123-PC7 (clone 6H6) (BioLegend), anti-CD11c-APC (clone BU15) (Exbio), anti-phosphoSTAT1-BV421 (Tyr701) (clone 4a) and anti-phosphoSTAT3-PE (Tyr705) (clone 4/5-STAT3) (both from BD Bioscience). The samples were acquired on FACSAria II (BD Biosciences), and data analysis was performed using FlowJo (TreeStar).
3
Monocyte-Derived Macrophage Polarization
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Peripheral blood mononuclear cells were isolated from buffy coats obtained from healthy donors using a Ficoll density gradient as previously described.34 (link) Monocytes were separated from the peripheral blood mononuclear cells using anti-CD14 magnetic beads (Miltenyi Biotechnology, UK) following the manufacturer’s instructions. Resting Mφs were obtained by culturing 106 /mL CD14+ monocytes for six days in RPMI 1640 containing 10% FBS with 25 ng/mL recombinant human M-CSF (PeproTech, USA). Mφs were polarized by stimulating them with 10 ng/mL recombinant human IFN-α (rhIFN-α, R&D Systems, USA) or 100 ng/mL recombinant human IFN-λ1 (rhIFN-λ1, R&D Systems, USA; online supplemental table 6 ) for either 6 or 12 hours.
4
Measuring IFN-α and IFN-ω Autoantibodies
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ELISA was performed as previously described (Bastard et al., 2020b (link)). In brief, 96-well ELISA plates (MaxiSorp; Thermo Fisher Scientific) were coated by incubation overnight at 4°C with 2 µg/ml rhIFN-α and rhIFN-ω (R&D Systems). Plates were then washed (PBS/0.005% Tween), blocked by incubation with 5% nonfat milk powder in the same buffer, washed again, and incubated with 1:50 dilutions of plasma from the patients or controls for 2 h at room temperature (or with specific mAbs as positive controls). Each sample was tested once. Plates were thoroughly washed. HRP-conjugated Fc-specific (Fc, fragment crystallizable region) IgG fractions from polyclonal goat antiserum against human IgG or IgA (Nordic Immunological Laboratories) were added to a final concentration of 2 µg/ml. Plates were incubated for 1 h at room temperature and washed. Substrate was added, and the OD was measured. A similar protocol was used to test for Abs against 12 subtypes of IFN-α, except that the plates were coated with cytokines from PBL Assay Science (11002-1).
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