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Supplementary file 1_Monoclonal antibody 1B9 against the African swine fever virus viroporin pB169L: molecular characterization and epitope mapping.pdf Zhiying Cui (12986093) Jianing Feng (9456153) Miaomiao Ye (1836151) Qin Wen (3505655) Peng Gao (35465) Qiongqiong Zhou (92521) Yongning Zhang (9511486) Lei Zhou (19808) Xinna Ge (573347) Xin Guo (123593) Jun Han (231645) Hanchun Yang (67762) Microbiology African swine fever virus monoclonal antibody pB169L viral factory viroporin 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. Two immunoreactive species of approximately 18 and 21 kDa were consistently observed. Variable-region sequencing defined the VH/VL gene usage and complementarity-determining regions of 1B9. Epitope mapping identified a linear epitope at residues ¹⁴⁷SKPLVPLQE¹⁵⁵, which was broadly conserved across several ASFV genotypes and predicted to reside in a hydrophilic, flexible, surface-exposed C-terminal region.</p>Discussion<p>Thus, 1B9 provides a useful reagent for studying the expression, localization, and antigenic properties of pB169L.</p> 2026-09-22T05:40:45Z Dataset Dataset 10.3389/fmicb.2026.1942750.s001 https://figshare.com/articles/dataset/Supplementary_file_1_Monoclonal_antibody_1B9_against_the_African_swine_fever_virus_viroporin_pB169L_molecular_characterization_and_epitope_mapping_pdf/33962257 CC BY 4.0