Supplementary file 1_Monoclonal antibody 1B9 against the African swine fever virus viroporin pB169L: molecular characterization and epitope mapping.pdf

Introduction<p>The African swine fever virus (ASFV) viroporin pB169L is a conserved membrane protein, but well-defined reagents for its detection and molecular characterization remain limited.</p>Methods<p>A soluble recombinant antigen comprising the non-transmembrane regions of pB169L was used to generate a mouse monoclonal antibody (mAb), designated 1B9. The antibody specificity was characterized by immunoblotting and immunofluorescence assays.

The expression and subcellular localization of pB169L were examined in ASFV-infected WSL-R4 cells and primary porcine alveolar macrophages. The variable regions of 1B9 were sequenced, and its epitope was mapped using truncated antigenic fragments.</p>Results<p>The 1B9 antibody belonged to the IgG1 kappa subclass and recognized pB169L by immunoblotting and immunofluorescence at a working dilution of 1:2,000. In ASFV-infected cells, pB169L was detectable by immunofluorescence from 6 h post-infection and by Western blot from 12 h post-infection, with progressive accumulation in cytoplasmic viral factories together with p54.