Autoantibody measurements were adopted from the protocol described above with a few modifications as below. In brief, 384-well plates were coated with 2 μg/mL of recombinant IFN-α2 (Miltenyi Biotech), U1-small nuclear ribonucleoprotein (U1-snRNP) (Diarect), Ribosomal Phosphoprotein P1 (P1) (Diarect), Ro/SS-A (Diarect), La/SS-B (Diarect), or histidyl-transfer ribonucleic acid synthetase (Jo-1) (Diarect), followed by incubating with 1:50 dilutions of plasma samples in duplicates. End-point OD at 450nm was measured and recorded. Since it is common for healthy people to have detectable anti-nuclear antibody titers (Pisetsky, 2011 (link); Slight-Webb et al., 2016 (link); Tan et al., 1997 (link)), two methods were adopted to analyze the autoantibody data. Observation in Figure 2 B was made only using datapoints that had a value greater than mean standard deviations of healthy controls. Other observations associated with autoantibodies were made using all datapoints.
Recombinant ifn α2
Manufactured by Miltenyi Biotec
Recombinant IFN-α2 is a cytokine produced through recombinant technology. It is a type I interferon that exhibits antiviral, antiproliferative, and immunomodulatory activities.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using recombinant ifn α2
1
Autoantibody Measurement Protocol for IFN-α2, U1-snRNP, P1, Ro/SS-A, La/SS-B, and Jo-1
2
Immunoblotting and IFN-I ELISA Protocols
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For immunoblotting, cells were washed with PBS and lysed in RIPA buffer with added protease and phosphatase inhibitors (Roche). Samples were sonicated, diluted in Laemmli sample buffer and separated by SDS‐PAGE before transferred to the PVDF membrane. Membranes were probed with antibodies listed in Table S1 and a secondary antibody conjugated to horseradish peroxidase, before developing using enhanced chemiluminescence substrate. For IFN‐I ELISA, 96 well plates were coated with a mixture of antibodies against IFN‐α (Mabtech, MT1/3/5) overnight at 4°C, and blocked with PBS supplemented with 10% FCS. Samples along with recombinant IFN‐α2 (Miltenyi), for construction of the standard curve, were added in triplicate and incubated overnight at 4°C. The plate was washed and IFN‐I assayed using a mixture of biotinylated detection antibodies against IFN‐α (Mabtech, MT2/4/6). Avidin‐conjugated alkaline phosphatase (Sigma, ExtrAvidin), and p‐Nitrophenyl phosphate substrate (Sigma, SigmaFast tablets) were used to develop the ELISA. Absorbance was read at 405 nm on a Multiskan EX plate reader (Thermo Fisher).
3
Immunoblotting and ELISA Techniques
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Immunoblotting and ELISA assays: For immunoblotting, cells were washed with PBS and lysed in RIPA buffer with added protease and phosphatase inhibitors (Roche). Samples were sonicated, diluted in Laemmli sample buffer and separated by SDS-PAGE, before transfer to PVDF membrane. Membranes were probed with antibodies listed in Supplementary Table 1 and a secondary antibody conjugated to horseradish peroxidase, before developing using enhanced chemiluminesence substrate. For IFN-I ELISA, 96 well plates were coated with a mixture of antibodies against IFN-α (Mabtech, MT1/3/5) overnight at 4°C, and blocked with PBS supplemented with 10% FCS. Samples along with recombinant IFN-α2 (Miltenyi), for construction of the standard curve, were added in triplicate and incubated overnight at 4°C. The plate was washed and IFN-I assayed using a mixture of biotinylated detection antibodies against IFN-α (Mabtech, MT2/4/6). Avidin-conjugated alkaline phosphatase (Sigma, ExtrAvidin), and p-Nitrophenyl phosphate substrate (Sigma, SigmaFast tablets) were used to develop the ELISA. Absorbance was read at 405nm on a Multiskan EX plate reader (Thermo Fisher).
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