DNA was extracted from the EDTA-anticoagulated blood samples using a MagMAX system (Thermo Fisher) and a Magvet universal isolation kit (Life Technologies). qPCR was carried out following a modified protocol described previously (15 (link), 64 (link)). Infectious virus in blood was titrated by endpoint dilution in PBM cells. Infected cells were detected by immunofluorescence using a monoclonal antibody against the ASFV p30/CP204L protein.
Serum samples were assayed using a commercial competition ELISA kit for the detection of ASFV-specific antibodies against VP72 (Ingezim PPA3 Compac; Ingenasa, Madrid, Spain) and by commercial ELISA kits for the detection of porcine immunoregulatory cytokines (IFN-γ and IL-10; R&D Systems, Abingdon, UK) according to the manufacturers’ instructions. For IFN-α, an in-house ELISA using antibodies purchased from PBL Interferon Source were used as previously described (65 (link)).
Serum samples were assayed using a commercial competition ELISA kit for the detection of ASFV-specific antibodies against VP72 (Ingezim PPA3 Compac; Ingenasa, Madrid, Spain) and by commercial ELISA kits for the detection of porcine immunoregulatory cytokines (IFN-γ and IL-10; R&D Systems, Abingdon, UK) according to the manufacturers’ instructions. For IFN-α, an in-house ELISA using antibodies purchased from PBL Interferon Source were used as previously described (65 (link)).
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