Peroxidase conjugated species specific secondary antibodies

CEFs were infected with rNDV and rNDV_H_Kur at an moi of 5 and harvested 48 h p.i. Lysates of VDS cells infected with PPRV Kurdistan/11 at an moi of 1, which were harvested 3 and 4 days p.i., were provided by the German National Reference Laboratory for PPR. All cell lysates were denatured at 95 °C for 10 min, proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and subsequently transferred to nitrocellulose membranes. Viral proteins were detected by incubation with α PPRV-H, α NDV-HN, or α NDV-F. After primary antibody incubation, binding of peroxidase-conjugated species-specific secondary antibodies (Dianova) was detected by chemiluminescence substrate (Thermo Scientific, Rockford, IL, USA) and visualized by ChemiDoc XRS+ (BioRad, Hercules, CA, USA).

DF-1, MDBK or BHK-21 cells, seeded in 6 well plates, were infected with rNDV and rNDV_G_RABV at an moi of 5, harvested 24 h and 48 h p. i. and lysed in 1x Roti-Load buffer (Roth, Karlsruhe, Germany). Purified virion solutions were mixed 1:1 with 1x Roti-Load buffer. All lysates were denatured at 95° for 10 min, proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and subsequently transferred to nitrocellulose membranes. Viral proteins were detected by incubation with α RABV-G, α NDV-HN, or α NDV-F. After primary antibody incubation, binding of peroxidase-conjugated species-specific secondary antibodies (Dianova) was detected by chemiluminescence substrate (Thermo Scientific, Rockford, IL, USA) and visualized by ChemiDoc XRS+ (BioRad, Hercules, CA, USA).