The multispecies double antigen ELISA (IDVet, Grabels, France) was applied according to the manufacturer’s instructions. An in-house ELISA based on recombinant N protein and a species-adapted protocol for the commercial human Vector-Best IgG ELISA (Vector-Best, Novosibirsk, Russia) and an immunofluorescence assay (Euroimmun, Luebeck, Germany) were used as described before [28 (link)].
Western blot analysis was conducted according to standard procedures. For the immune detection, sera were diluted 1/20 in 5% skim milk and incubated for 1 h. Membranes were washed with PBS-Tween20 and the conjugate (Peroxidase-conjugated AffiniPure Goat Anti-Bovine IgG or Peroxidase-conjugated AffiniPure Donkey Anti-Sheep IgG, Jackson ImmunoResearch, Cambridgeshire, UK) was diluted to 1/3000 in 5% skim milk and added to the membrane. After 1 h, membranes were washed again and the substrate (SuperSignal West Pico PLUS Chemiluminescent Substrate, Thermo Scientific, Braunschweig, Germany) was applied. Chemiluminescence was measured with ChemiDocTM Touch Imaging System (Bio-Rad Laboratories, Hercules, CA, USA) and blots were analyzed with Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA).
Western blot analysis was conducted according to standard procedures. For the immune detection, sera were diluted 1/20 in 5% skim milk and incubated for 1 h. Membranes were washed with PBS-Tween20 and the conjugate (Peroxidase-conjugated AffiniPure Goat Anti-Bovine IgG or Peroxidase-conjugated AffiniPure Donkey Anti-Sheep IgG, Jackson ImmunoResearch, Cambridgeshire, UK) was diluted to 1/3000 in 5% skim milk and added to the membrane. After 1 h, membranes were washed again and the substrate (SuperSignal West Pico PLUS Chemiluminescent Substrate, Thermo Scientific, Braunschweig, Germany) was applied. Chemiluminescence was measured with ChemiDocTM Touch Imaging System (Bio-Rad Laboratories, Hercules, CA, USA) and blots were analyzed with Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA).