Verikine mouse ifn β enzyme linked immunosorbent assay elisa kit

RNA samples obtained from infected cells were treated with DNase I (Roche, Germany) for 60 min in 37°C. For total RNA from RRV-T48- or RRV-T48_A534V-infected cells (multiplicity of infection [MOI] of 10; 6 h postinfection [p.i.]), 100-µg aliquots were also incubated with biotinylated oligo(dT) probe, and the bound poly(A)⁺ fraction was removed using streptavidin MagneSphere paramagnetic particles (PolyATtract mRNA isolation systems kit; Promega, USA), resulting in poly(A)⁻ RNA samples. All samples were repurified with RNeasy kit (Qiagen, Germany) and eluted with 20 µl water. Prior to transfection of Cop5 cells, the infectious viral RNAs were UV inactivated for 5 min in a Hoefer UV crosslinker UVC5000. One microgram of total or poly(A)⁻ RNAs were used to transfect confluent cultures of Cop5 cells in 12-well plates using Lipofectamine 2000 reagent. Transfected cells were incubated at 37°C. At 24 h posttransfection (p.t.), the supernatants were collected and centrifuged at 3,000 × g for 5 min. The amount of IFN-β produced by the transfected cells was determined using VeriKine mouse IFN-β enzyme-linked immunosorbent assay (ELISA) kit (PBL Assay Science, USA).

For measuring IFN-β production, cells were seeded onto 96-well plates (Falcon) at a density of 20,000 cells per well and infected with RSV or IAV as described above. The culture medium (total of 200 μL) from infected cells was collected at the designated time points and stored at −20°C until further analysis. IFN-β levels were estimated using the VeriKine mouse IFN-β enzyme-linked immunosorbent assay (ELISA) kit (PBL Assay Science). Standards and diluted samples in duplicates or triplicates were added to a precoated plate included in the kit and incubated for 1 h. After subsequent washing, an antibody solution was prepared and added to wells for another 1 h, followed by another wash and 1 h of incubation with an HRP solution. Finally, a TMB substrate solution was added to the wells, and the developing color reaction was stopped after 15 min with the addition of a stop solution. The optical densities of the samples after the resulting color development were determined using a Multiskan Go plate reader (Thermo Fisher Scientific) set to 450 nm, with wavelength correction at 570 nm. IFN-β concentrations were obtained based on the standard curve (4-parameter logistic function fitted to 7 data points from the standards).