Ferret ifn γ elisa development kit alp

Heparinised whole blood was diluted 1:10 with serum free RPMI 1640 medium and incubated with either A/California/07/2009 (H1N1) or A/Perth/16/2009 (H3N2), corresponding with the virus of infection. Phytohemagglutinin PHA-M (PHA) (Sigma-Aldrich, Dorset, United Kingdom) was used as a positive control and egg allantoic fluid was used as a negative control as viruses were egg grown. Blood was stimulated for 4 days at 37°C, after which plasma supernatants were collected and cryopreserved at -80°C. The Ferret IFN-γ ELISA Development Kit (ALP) (Mab Tech, Nacka. Sweden) was used to determine the quantity of IFNɣ secreted by cells in the blood in responses to influenza-specific stimulations. ELISA plates were read using the VersaMax ELISA Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) with SoftMax^TM PRO software (Molecular Devices, Sunnyvale, CA, USA).

Ferret lung and spleen cells were pelleted by centrifugation at 1,500 × g_max for 5 min and washed in PBS. Cells (1.25 × 10⁴/96-well) were cultured with or without live H7N9 virus for 48 h at 37°C/5% CO₂. The Ferret IFN-γ ELISA Development Kit (ALP) (Mabtech, Stockholm, Sweden) was used to determine the quantity of IFN- γ secreted by cells ex vivo at endpoint and after restimulation with live H7N9 virus (MOI 0.1). ELISA plates were measured using a Multiskan Ascent Plate Reader with Ascent Software Version 2.6 (ThermoFisher, Waltham, MA, USA). IFN-γ-producing cells were detected using a ferret IFN-γ ELISpot assay, as per the manufacturer's instructions (Mabtech, Stockholm, Sweden).

Heparinised whole blood was diluted 1:10 with serum-free RPMI 1640 medium and incubated with either A/California/07/2009 (H1N1) or A/Perth/16/2009 (H3N2) allantoic fluids (1.6 × 10⁶ PFU). Phytohemagglutinin PHA-M (Sigma-Aldrich, Dorset, UK) was used as a positive control and egg allantoic fluid was used as a negative control. Blood was stimulated for 4 days at 37 °C, after which plasma supernatants were collected and cryopreserved at −80 °C. The Ferret IFN-γ ELISA Development Kit (ALP) (Mabtech, Nacka, Sweden) was used to determine the quantity of IFN-ɣ secreted by cells in the blood as described^{18 (link)}.