258 tnf α duoset elisa kits  (R&D Systems)

Journal: bioRxiv

Article Title: Human antibodies against West Nile and related orthoflaviviruses

doi: 10.64898/2026.04.02.715800

Figure Lengend Snippet: (A) IgG binding to WNV EDIII by serial serum dilution. ELISA curves for samples from the 72 WND/WNF-suspected individuals and 3 orthoflavivirus naïve controls (black) are shown. The average of two independent experiments is shown. (B) Serum neutralization screening with WNV RVP . Shown is the rank-ordered NanoLuc activity relative to no-serum control for the 72 WND/WNF-suspected individuals and 1 orthoflavivirus naïve control (black); lower values correspond to higher neutralization. Samples with relative NanoLuc signal below 0.3 (dotted line) were selected for neutralization curves in (C). Each bar represents and individual participant sample analyzed at 1:100 dilution. Average signal of triplicate wells from a single experiment. (C) Neutralization of WNV RVP by serial serum dilution. Shown is the NanoLuc activity relative to no-serum control for 36 WND/WNF cases and 1 orthoflavivirus naïve control (black). Mean ± SD of triplicates. Representative of two independent experiments. (D-F) Identification of serum autoantibodies to IFN-α2 and IFN-ω. Plots compare the ability of serum IgG to bind, and of serum to neutralize, IFN-α2 (D) and IFN-ω (E) (n=39; only samples for which sufficient serum was available were assayed). The comparison of IFN-α2 and IFN-ω neutralization is shown in (F). Binding is shown as relative Mean Fluorescence Intensity (MFI) and neutralization as relative ISG15-promoter driven luciferase signal compared to no serum control. The dotted lines indicate the threshold for positivity of the assay. , Representative of 2 independent experiments. (G) Age and gender distribution of the study participants with or without IFN-α2 and/or IFN-ω neutralizing autoantibodies. Welch’s test. (H and I) Serum IgG binding to WNV EDIII and serum neutralization of WNV RVP in study participants with or without IFN-α2 and/or IFN-ω neutralizing autoantibodies. The two groups are compared with respect to (H) IgG binding to WNV EDIII (Area Under the Curve (AUC) of ELISA and (I) neutralization of WNV RVP (NT 50 values). Horizontal lines indicate the mean. Mann-Whitney test. In (A to C), green, blue and red indicate samples from WNV-infected individuals from which antibodies were derived (Figure S2B and C). In (D to I), female is circle, male is square, dark blue neutralizes both IFN-α2 and IFN-ω, light blue neutralizes IFN-α2 only, grey does not neutralize either.

Article Snippet: Serum samples were diluted 1:20 in OptiMEM (Optimized Minimal Essential Medium; Gibco, 31985070) containing 0.01 ng/mL IFN-α2 (Novus Biologicals, NBP2-34971) or 0.02 ng/mL IFN-ω (Novus Biologicals, NBP2-35893), together with a live-cell Renilla luciferase substrate (EnduRen, Promega, E6481; 1:10,000), and incubated for 1 hour with constant shaking at 600 rpm.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Neutralization, Activity Assay, Control, Comparison, Fluorescence, Luciferase, MANN-WHITNEY, Infection, Derivative Assay

Journal: Journal of Virology

Article Title: Porcine hemagglutinating encephalomyelitis virus nucleocapsid protein targets RIG-I and IRF3 to evade IFN immunity

doi: 10.1128/jvi.02112-25

Figure Lengend Snippet: PHEV infection activates the innate immune response. ( A ) Viral load in subcultures harvested at different time points post-infection was quantified by qRT-PCR targeting the viral N gene. All the experiments were performed in triplicate. ( B ) IFN-β levels in PHEV-infected samples were determined by ELISA. Cells infected with VSV (MOI = 1) or treated with Poly(I:C) (20 μM, 24 h) served as positive controls. ( C ) QRT-PCR analysis of IFNA and IFNB1 mRNA expression in N2a cells at various time points (0–48 h) post-PHEV infection. ( D ) QRT-PCR analysis of ISGs (Mx, OAS, GBP, STAT) expression in N2a cells at 24 and 48 h post-PHEV infection. ( E ) QRT-PCR analysis of RIG-I, MDA5, IRF3, and IRF7 expression in N2a cells at various time points (0–48 h) post-PHEV infection. ( F ) WB analysis of RIG-I, IRF7, MAVS, and viral N protein levels in N2a cells at indicated times (0–48 h) after PHEV infection. ( G ) Detection of PHEV, RIG-I, IRF3, IRF7, and IFNB1 in brain tissues from mice at 5 days post-PHEV infection. Data represent mean ± SD (** P < 0.01 and *** P < 0.001 by unpaired two-tailed Student’s t-test).

Article Snippet: The IFN-β recombinant protein was purchased from MCE.

Techniques: Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Two Tailed Test

Journal: Journal of Virology

Article Title: Porcine hemagglutinating encephalomyelitis virus nucleocapsid protein targets RIG-I and IRF3 to evade IFN immunity

doi: 10.1128/jvi.02112-25

Figure Lengend Snippet: IRF3 initiates the interferon response to suppress viral replication. ( A ) Inhibition of PHEV replication by recombinant IFN-β. WB analysis of viral protein levels to evaluate the suppression of PHEV replication by recombinant IFN-β (0.5 or 1 μg/mL). ( B ) QRT-PCR analysis of IRF3 mRNA in N2a cells overexpressing Myc-tagged IRF3 (1, 2, or 4 μg) for 24 h, followed by 24 h PHEV infection. ( C ) QRT-PCR analysis of IRF7 mRNA in N2a cells overexpressing Myc-tagged IRF7 (1, 2, or 4 μg) for 24 h, followed by 24 h PHEV infection. ( D ) QRT-PCR analysis of PHEV mRNA expression as described in panels B and C . ( E ) WB analysis of PHEV N protein as described in panels B and C . ( F ) QRT-PCR analysis of IFNB1 mRNA expression as described in panels B and C . Data represent mean ± SD (** P < 0.01 and *** P < 0.001 by unpaired two-tailed Student’s t-test). ns, not significant.

Article Snippet: The IFN-β recombinant protein was purchased from MCE.

Techniques: Inhibition, Recombinant, Quantitative RT-PCR, Infection, Expressing, Two Tailed Test

Journal: Journal of Virology

Article Title: Porcine hemagglutinating encephalomyelitis virus nucleocapsid protein targets RIG-I and IRF3 to evade IFN immunity

doi: 10.1128/jvi.02112-25

Figure Lengend Snippet: PHEV N protein targets IRF3 to suppress IFN production. ( A ) Schematic illustration of the experimental design for the dual-luciferase assay using HT1080 cells with a stably integrated IFN-β promoter reporter. ( B ) PHEV N protein inhibits the IFN-β promoter. HT1080 cells were transfected with PHEV proteins, cultured for 24 h, and then stimulated with Poly(I:C) (20 μM) for another 24 h to induce IFN-β promoter activity. IFN-β-Luc reporter activity is normalized to that of Renilla luciferase and shown. Detection of viral protein expression by WB. ( C ) WB analysis demonstrated that the protein levels of RIG-I and IRF3 in N2a cells were unaltered by transfection with a gradient of N protein (0.5–4 μg). ( D ) Nuclear-cytoplasmic fractionation. N2a cells were transfected with 2 μg GFP-N recombinant plasmid for 24 h, and then treated with Poly(I:C) (20 μM) for 24 h or infected with VSV (MOI = 1) for 12 h. Cells were collected for cytoplasmic and nuclear isolation. WB analysis of IRF3 nuclear translocation using cytoplasmic and nuclear fractions prepared from harvested cells. ( E ) N2a cells were infected with PHEV for 24 h (with Poly(I:C) treatment (20 μM, 24 h) as a positive control), followed by immunostaining with anti-N (red) and anti-IRF3 (green) antibodies. Nuclei were counterstained with Hoechst (blue). Scale bar, 10 μm. Data represent mean ± SD (** P < 0.01 and *** P < 0.001 by unpaired two-tailed Student’s t-test).

Article Snippet: The IFN-β recombinant protein was purchased from MCE.

Techniques: Luciferase, Stable Transfection, Transfection, Cell Culture, Activity Assay, Expressing, Fractionation, Recombinant, Plasmid Preparation, Infection, Isolation, Translocation Assay, Positive Control, Immunostaining, Two Tailed Test