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( A-B ) Renilla luciferase induction kinetics over 30h following stimulation of 293T cells (A) or AIR cells (B) with IFN-Is (IFNα2 and IFNω: 10 ng/mL, IFNβ: 1.21 ng/mL) or IFN-IIIs (λ1-3; 50 ng/mL). ( C ) Sensitivity analysis of 293T cells and AIR cells to detect neutralization of IFNβ by an anti-IFNβ antibody. The indicated concentration of antibody was incubated with 200 pg/mL IFNβ or 3.6 pg/mL IFNβ prior to stimulation of 293T or AIR cells, respectively. ( D ) Sensitivity analysis of AIR cells to detect neutralization of IFN-IIIs by their respective antibodies. The indicated concentration of antibody was incubated with 10 ng/mL IFNλ1, IFNλ2, or <t>IFNλ3</t> prior to stimulation of AIR cells. ( E ) Use of AIR cells to determine the neutralization of IFNα2 at the indicated concentration by human plasmas. Blue: plasmas with neutralizing IFNα activity. Gray: negative control plasmas. The dashed lines in C-E indicate the 75% neutralization threshold. For A-C, mean values from n=3 replicates are shown and error bars represent standard deviations. For D-E, data are representative of at least n=2 similar experiments.
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( A-B ) Renilla luciferase induction kinetics over 30h following stimulation of 293T cells (A) or AIR cells (B) with IFN-Is (IFNα2 and IFNω: 10 ng/mL, IFNβ: 1.21 ng/mL) or IFN-IIIs (λ1-3; 50 ng/mL). ( C ) Sensitivity analysis of 293T cells and AIR cells to detect neutralization of IFNβ by an anti-IFNβ antibody. The indicated concentration of antibody was incubated with 200 pg/mL IFNβ or 3.6 pg/mL IFNβ prior to stimulation of 293T or AIR cells, respectively. ( D ) Sensitivity analysis of AIR cells to detect neutralization of IFN-IIIs by their respective antibodies. The indicated concentration of antibody was incubated with 10 ng/mL IFNλ1, IFNλ2, or <t>IFNλ3</t> prior to stimulation of AIR cells. ( E ) Use of AIR cells to determine the neutralization of IFNα2 at the indicated concentration by human plasmas. Blue: plasmas with neutralizing IFNα activity. Gray: negative control plasmas. The dashed lines in C-E indicate the 75% neutralization threshold. For A-C, mean values from n=3 replicates are shown and error bars represent standard deviations. For D-E, data are representative of at least n=2 similar experiments.
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( A-B ) Renilla luciferase induction kinetics over 30h following stimulation of 293T cells (A) or AIR cells (B) with IFN-Is (IFNα2 and IFNω: 10 ng/mL, IFNβ: 1.21 ng/mL) or IFN-IIIs (λ1-3; 50 ng/mL). ( C ) Sensitivity analysis of 293T cells and AIR cells to detect neutralization of IFNβ by an anti-IFNβ antibody. The indicated concentration of antibody was incubated with 200 pg/mL IFNβ or 3.6 pg/mL IFNβ prior to stimulation of 293T or AIR cells, respectively. ( D ) Sensitivity analysis of AIR cells to detect neutralization of IFN-IIIs by their respective antibodies. The indicated concentration of antibody was incubated with 10 ng/mL IFNλ1, IFNλ2, or <t>IFNλ3</t> prior to stimulation of AIR cells. ( E ) Use of AIR cells to determine the neutralization of IFNα2 at the indicated concentration by human plasmas. Blue: plasmas with neutralizing IFNα activity. Gray: negative control plasmas. The dashed lines in C-E indicate the 75% neutralization threshold. For A-C, mean values from n=3 replicates are shown and error bars represent standard deviations. For D-E, data are representative of at least n=2 similar experiments.
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( A-B ) Renilla luciferase induction kinetics over 30h following stimulation of 293T cells (A) or AIR cells (B) with IFN-Is (IFNα2 and IFNω: 10 ng/mL, IFNβ: 1.21 ng/mL) or IFN-IIIs (λ1-3; 50 ng/mL). ( C ) Sensitivity analysis of 293T cells and AIR cells to detect neutralization of IFNβ by an anti-IFNβ antibody. The indicated concentration of antibody was incubated with 200 pg/mL IFNβ or 3.6 pg/mL IFNβ prior to stimulation of 293T or AIR cells, respectively. ( D ) Sensitivity analysis of AIR cells to detect neutralization of IFN-IIIs by their respective antibodies. The indicated concentration of antibody was incubated with 10 ng/mL IFNλ1, IFNλ2, or <t>IFNλ3</t> prior to stimulation of AIR cells. ( E ) Use of AIR cells to determine the neutralization of IFNα2 at the indicated concentration by human plasmas. Blue: plasmas with neutralizing IFNα activity. Gray: negative control plasmas. The dashed lines in C-E indicate the 75% neutralization threshold. For A-C, mean values from n=3 replicates are shown and error bars represent standard deviations. For D-E, data are representative of at least n=2 similar experiments.
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( A-B ) Renilla luciferase induction kinetics over 30h following stimulation of 293T cells (A) or AIR cells (B) with IFN-Is (IFNα2 and IFNω: 10 ng/mL, IFNβ: 1.21 ng/mL) or IFN-IIIs (λ1-3; 50 ng/mL). ( C ) Sensitivity analysis of 293T cells and AIR cells to detect neutralization of IFNβ by an anti-IFNβ antibody. The indicated concentration of antibody was incubated with 200 pg/mL IFNβ or 3.6 pg/mL IFNβ prior to stimulation of 293T or AIR cells, respectively. ( D ) Sensitivity analysis of AIR cells to detect neutralization of IFN-IIIs by their respective antibodies. The indicated concentration of antibody was incubated with 10 ng/mL IFNλ1, IFNλ2, or <t>IFNλ3</t> prior to stimulation of AIR cells. ( E ) Use of AIR cells to determine the neutralization of IFNα2 at the indicated concentration by human plasmas. Blue: plasmas with neutralizing IFNα activity. Gray: negative control plasmas. The dashed lines in C-E indicate the 75% neutralization threshold. For A-C, mean values from n=3 replicates are shown and error bars represent standard deviations. For D-E, data are representative of at least n=2 similar experiments.
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( A-B ) Renilla luciferase induction kinetics over 30h following stimulation of 293T cells (A) or AIR cells (B) with IFN-Is (IFNα2 and IFNω: 10 ng/mL, IFNβ: 1.21 ng/mL) or IFN-IIIs (λ1-3; 50 ng/mL). ( C ) Sensitivity analysis of 293T cells and AIR cells to detect neutralization of IFNβ by an anti-IFNβ antibody. The indicated concentration of antibody was incubated with 200 pg/mL IFNβ or 3.6 pg/mL IFNβ prior to stimulation of 293T or AIR cells, respectively. ( D ) Sensitivity analysis of AIR cells to detect neutralization of IFN-IIIs by their respective antibodies. The indicated concentration of antibody was incubated with 10 ng/mL IFNλ1, IFNλ2, or <t>IFNλ3</t> prior to stimulation of AIR cells. ( E ) Use of AIR cells to determine the neutralization of IFNα2 at the indicated concentration by human plasmas. Blue: plasmas with neutralizing IFNα activity. Gray: negative control plasmas. The dashed lines in C-E indicate the 75% neutralization threshold. For A-C, mean values from n=3 replicates are shown and error bars represent standard deviations. For D-E, data are representative of at least n=2 similar experiments.
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( A-B ) Renilla luciferase induction kinetics over 30h following stimulation of 293T cells (A) or AIR cells (B) with IFN-Is (IFNα2 and IFNω: 10 ng/mL, IFNβ: 1.21 ng/mL) or IFN-IIIs (λ1-3; 50 ng/mL). ( C ) Sensitivity analysis of 293T cells and AIR cells to detect neutralization of IFNβ by an anti-IFNβ antibody. The indicated concentration of antibody was incubated with 200 pg/mL IFNβ or 3.6 pg/mL IFNβ prior to stimulation of 293T or AIR cells, respectively. ( D ) Sensitivity analysis of AIR cells to detect neutralization of IFN-IIIs by their respective antibodies. The indicated concentration of antibody was incubated with 10 ng/mL IFNλ1, IFNλ2, or <t>IFNλ3</t> prior to stimulation of AIR cells. ( E ) Use of AIR cells to determine the neutralization of IFNα2 at the indicated concentration by human plasmas. Blue: plasmas with neutralizing IFNα activity. Gray: negative control plasmas. The dashed lines in C-E indicate the 75% neutralization threshold. For A-C, mean values from n=3 replicates are shown and error bars represent standard deviations. For D-E, data are representative of at least n=2 similar experiments.
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( A-B ) Renilla luciferase induction kinetics over 30h following stimulation of 293T cells (A) or AIR cells (B) with IFN-Is (IFNα2 and IFNω: 10 ng/mL, IFNβ: 1.21 ng/mL) or IFN-IIIs (λ1-3; 50 ng/mL). ( C ) Sensitivity analysis of 293T cells and AIR cells to detect neutralization of IFNβ by an anti-IFNβ antibody. The indicated concentration of antibody was incubated with 200 pg/mL IFNβ or 3.6 pg/mL IFNβ prior to stimulation of 293T or AIR cells, respectively. ( D ) Sensitivity analysis of AIR cells to detect neutralization of IFN-IIIs by their respective antibodies. The indicated concentration of antibody was incubated with 10 ng/mL IFNλ1, IFNλ2, or <t>IFNλ3</t> prior to stimulation of AIR cells. ( E ) Use of AIR cells to determine the neutralization of IFNα2 at the indicated concentration by human plasmas. Blue: plasmas with neutralizing IFNα activity. Gray: negative control plasmas. The dashed lines in C-E indicate the 75% neutralization threshold. For A-C, mean values from n=3 replicates are shown and error bars represent standard deviations. For D-E, data are representative of at least n=2 similar experiments.
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( A-B ) Renilla luciferase induction kinetics over 30h following stimulation of 293T cells (A) or AIR cells (B) with IFN-Is (IFNα2 and IFNω: 10 ng/mL, IFNβ: 1.21 ng/mL) or IFN-IIIs (λ1-3; 50 ng/mL). ( C ) Sensitivity analysis of 293T cells and AIR cells to detect neutralization of IFNβ by an anti-IFNβ antibody. The indicated concentration of antibody was incubated with 200 pg/mL IFNβ or 3.6 pg/mL IFNβ prior to stimulation of 293T or AIR cells, respectively. ( D ) Sensitivity analysis of AIR cells to detect neutralization of IFN-IIIs by their respective antibodies. The indicated concentration of antibody was incubated with 10 ng/mL IFNλ1, IFNλ2, or IFNλ3 prior to stimulation of AIR cells. ( E ) Use of AIR cells to determine the neutralization of IFNα2 at the indicated concentration by human plasmas. Blue: plasmas with neutralizing IFNα activity. Gray: negative control plasmas. The dashed lines in C-E indicate the 75% neutralization threshold. For A-C, mean values from n=3 replicates are shown and error bars represent standard deviations. For D-E, data are representative of at least n=2 similar experiments.

Journal: bioRxiv

Article Title: Highly-sensitive reporter cell line for detection of interferon types I-III and their neutralization by antibodies

doi: 10.1101/2024.06.18.599537

Figure Lengend Snippet: ( A-B ) Renilla luciferase induction kinetics over 30h following stimulation of 293T cells (A) or AIR cells (B) with IFN-Is (IFNα2 and IFNω: 10 ng/mL, IFNβ: 1.21 ng/mL) or IFN-IIIs (λ1-3; 50 ng/mL). ( C ) Sensitivity analysis of 293T cells and AIR cells to detect neutralization of IFNβ by an anti-IFNβ antibody. The indicated concentration of antibody was incubated with 200 pg/mL IFNβ or 3.6 pg/mL IFNβ prior to stimulation of 293T or AIR cells, respectively. ( D ) Sensitivity analysis of AIR cells to detect neutralization of IFN-IIIs by their respective antibodies. The indicated concentration of antibody was incubated with 10 ng/mL IFNλ1, IFNλ2, or IFNλ3 prior to stimulation of AIR cells. ( E ) Use of AIR cells to determine the neutralization of IFNα2 at the indicated concentration by human plasmas. Blue: plasmas with neutralizing IFNα activity. Gray: negative control plasmas. The dashed lines in C-E indicate the 75% neutralization threshold. For A-C, mean values from n=3 replicates are shown and error bars represent standard deviations. For D-E, data are representative of at least n=2 similar experiments.

Article Snippet: For detection of IFN activity by A549-IFN-reporter (AIR) cells, 30,000 cells per well were seeded in a 96-well white-bottomed tissue culture plate and stimulated 24h later with IFNα2 (Novusbio; NPB2-34971), IFNβ (pbl assay science; 11420-1), IFNω (Novusbio; NBP2-35893), IFNγ (Novusbio; NBP2-34992), IFNλ1 (Novusbio; NBP2-34996), IFNλ2 (R&D systems; 8417-IL), or IFNλ3 (R&D systems; 5259-IL) at the indicated concentrations.

Techniques: Luciferase, Neutralization, Concentration Assay, Incubation, Activity Assay, Negative Control