Serum samples from adult age-matched, sex-matched mice were taken before (n = 3 per genotype) or 14 hours after (n = 5 per genotype) a 100 ug intraperitoneal injection of polyinosine-polycytidylic acid (high molecular weight polyI:C; InvivoGen). IFNα and IFNβ production were measured using commercial ELISA kits according to manufacturer’s protocol (Mouse IFNα Platinum ELISA, eBioscience; VeriKine Mouse IFNβ ELISA kit, PBL interferon source). Serum samples were run in duplicate and mean OD levels were used to calculate interferon concentration. Interferon levels below the assay detection limit (IFNα 31.3 pg/ml, IFNβ 15.6 pg/ml) were recorded as not detected. Significant differences between WT and RdRP interferon concentrations were assessed using the Student’s two-tailed unpaired t test (Graphpad Prism). P values < 0.05 were considered significant.
Mouse ifnα platinum elisa
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The Mouse IFNα Platinum ELISA is a laboratory equipment product designed for the quantitative measurement of mouse interferon-alpha (IFNα) levels in various sample types. It utilizes an enzyme-linked immunosorbent assay (ELISA) technique to detect and quantify the target analyte.
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4 protocols using mouse ifnα platinum elisa
1
Serum Interferon Levels in Mice
2
Interferon-α Antibody Administration in Mice
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Interferon-α monoclonal antibodies were purchased from BioXcell and 200–500 μg/dose were administered twice weekly in the peritoneum of C57Bl/6 mice. IFN-α ELISA was performed using a mouse IFN-α Platinum ELISA (eBioscience) per manufacturer's instructions.
3
Murine Pharmacokinetics and Cytokine Induction
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MHV370 in MC/Tween (0.5% methylcellulose/0.5% Tween-80) or vehicle alone was administered p.o. to 129/Sv mice (5 per group). At 1 h, mice were injected i.v. with 20 μg R0006 or 20 mg CpG1585, each pre-complexed with 140 μg DOTAP in Hanks Balanced Salt solution (HBSS). At 3 h, blood was withdrawn into EDTA Microvettes (Sarstedt) and plasma TNF and IFNα quantified using a mouse IFNα Platinum ELISA (Invitrogen) and a DuoSet TNF ELISA (R&D Systems), as single technical replicate per mouse. MHV370 serum concentrations were quantified by liquid chromatography coupled to mass spectrometry (LC-MS/MS).
4
Quantification of Proinflammatory Cytokines
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The concentration of proinflammatory cytokine TNF-α, IL-1β and IFN-α was measured from the peripheral blood samples collected 6 hours following the first Aldara treatment by ELISA method (Mouse TNF-α or IL-1β ELISA Set, catalogue number: 555268 or 559603, respectively, BD Biosciences Eastern Europe, Heidelberg, Germany; Mouse IFN-α Platinum ELISA, catalogue number: BMS6027, Invitrogen, USA). The principal of the assay is to coat capture antibodies specific for the protein of interest onto the wells of a microplate. Standards and unknown samples are added into the plate. During the first incubation step, the antigen binds to the capture antibody. Following incubation, unbound proteins are removed by washing and a biotinylated detection antibody is added to the wells to form capture antibody-antigen-detection antibody complex. After the removal of unbound detection antibodies, streptavidin-HRP conjugate is added and binds to the biotinylated detection antibody. Following the last incubation and washing cycle to remove the unbound HRP conjugate, a substrate solution is added and is converted by the HRP enzyme to a detectable color product read by spectrophotometry. The color intensity is directly proportional to the concentration of antigen present in the unknown sample.
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