Human ifn β duoset elisa kit

The type I IFN concentrations in cell culture supernatants and murine sera were quantified by a bioluminescent mouse IFN-β ELISA kit 2.0 (InvivoGen, Cat# luex-mifnbv2), a human IFN-β DuoSet ELISA kit (R&D, Cat# DY814-05) or a mouse IFN-α ELISA Kit (Invitrogen, Cat# BMS6027). A LEGENDplex™ bead-based multiplex ELISA kit (Biolegend, San Diego, CA, USA) was employed to quantify multiple serum cytokine concentrations. The procedures were exactly the same as in the product manuals. For the LEGENDplex assay, briefly, (25 µL) of samples or standards were mixed with antibody-coated premix microbeads in a filter-bottom microplate and incubated at room temperature for 2 h with vigorous shaking at 500 × g. After removal of unbound analytes and two washes, 25 µL of detection antibody were added to each well, and the plate was incubated at room temperature for 1 h with vigorous shaking at 500 × g. 25 µL of SA-PE reagent was then added directly to each well, and the plate was incubated at room temperature for 30 min with vigorous shaking at 500 × g. The beads were washed twice with wash buffer, re-suspended in 1× wash buffer, then transferred to a 96-well microplate. The beads were run through a BIORAD ZE5 and the concentration of each analyte was calculated with the standards using the LEGENDPlex data analysis software.

The antibodies used in this study were as follows: HRP-conjugated anti-HA (12013819001) and HRP-conjugated anti-Flag (A8592) antibodies (Sigma, Dallas, TX, USA); anti-GFP (11814460001), TBK1 (3013S) antibodies (Cell Signaling Technology, Danvers, MA, USA); anti-NS2 (GTX125953) antibody (Genetex, Irvine, CA, USA); anti-IRF7 (ab238137), anti-GAPDH (ab181602), and HRP-conjugated mouse anti-rabbit IgG (light-chain specific) (93702) antibodies (Abcam, Boston, USA); Alexa Fluor 488-conjugated anti-mouse IgG (A0428), Cy3-labeled goat anti-rabbit IgG (A0516) (Beyotime, Shanghai, China).
Reagents used in the study included: anti-Flag agarose affinity beads (A2220) and protein A/G agarose affinity beads (P6486/E3403) (Sigma, Dallas, TX, USA); human IFN-β DuoSet ELISA kit (DY814-05) (R&D, Minneapolis, MN, USA); NP-40 (ST366), DAPI (C1002), and Passive Lysis 5× Buffer (E1941) (Promega, Madison, WI, USA). The scrambled negative control RNA (NC), and IRF7-specific short interfering RNA were purchased from RiboBio Co. (Guangzhou, China). Lipofectamine 2000, RNAi MAX, and TRIzol were obtained from Invitrogen (Carlsbad, CA, USA). SYBR Green I Master Mix was purchased from Roche (Darmstadt, Germany).