Frontiers in microbiology · 2026
Monoclonal antibody 1B9 against the African swine fever virus viroporin pB169L: molecular characterization and epitope mapping.
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Cui Zhiying, Feng Jianing, Ye Miaomiao, Wen Qin, Gao Peng, Zhou Qiongqiong, Zhang Yongning, Zhou Lei, Ge Xinna, Guo Xin, Han Jun, Yang Hanchun
Аннотация
INTRODUCTION: 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.
METHODS: 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.
RESULTS: 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.
DISCUSSION: Thus, 1B9 provides a useful reagent for studying the expression, localization, and antigenic properties of pB169L.
METHODS: 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.
RESULTS: 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.
DISCUSSION: Thus, 1B9 provides a useful reagent for studying the expression, localization, and antigenic properties of pB169L.