PMC4285236_01
WU polyomavirus antigen in bronchoalveolar lavage specimens from lungs transplanted into a recipient (28-year-old woman) with Job syndrome. Immunohistochemical analysis of 293T cells transfected with pDEST26-WU-virus protein 1 and stained as follows. A) WU virus protein 1 monoclonal antibody (NN-Ab06). B) Isotype control. C) Mock transfected 293T cells stained with NN-Ab06. D) Bronchoalveolar lavage specimen stained with NN-Ab06 showing prominent dark staining of cells with enlarged nuclei and a ground glass appearance characteristic of viral cytopathic changes (arrows). E) Isotype control. Original magnifications x400 in panels A-C and x600 in panels D and E.
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A). A serial section of the same cell block stained with an isotype-matched antibody (#557351, mouse IgG2b: BD Biosciences, San Jose, CA, USA) (Figure 1, panel B) and mock transfected cells stained with NN-Ab06 (Figure 1, panel C) showed negative results. Prominent dark staining of cells with enlarged nuclei and a ground glass appearance characteristic of viral cytopathic changes were observed (Figure 1, panel D). Staining was not seen in serial sections stained with the isotype antibody (Figure 1, panel E) or with no antibodies.
WU polyomavirus antigen in respiratory epithelial cells from lungs transplanted into a recipient (28-year-old woman) with Job syndrome. Immunofluorescence of 293T cells transfected with pDEST26-WU-virus protein 1 and stained with A) WU virus protein 1 polyclonal antibody (NN-Ab01) or B) preimmune serum. C) Double immunofluorescence with NN-Ab01 (red) and a monoclonal antibody against cytokeratin (green) showing a double-positive cell from the bronchoalveolar lavage specimen. D) Bronchoalveolar lavage specimen with multiple WU virus protein 1/cytokeratin double-positive cells. Original magnifications x100 in panels A and B, x600 in panel C, and x400 in panel D.
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Scientific, Waltham, MA, USA). To validate the assay, we performed immunofluorescence analysis with NN-Ab01 on positive control 293T cells expressing WU-VP1. The nucleus was counterstained with Hoechst (#H21491; Life Technologies). Several WU-VP1-positive cells were observed (Figure 2, panel A); a serial section stained with preimmune serum at the same dilution showed a negative result (Figure 2, panel B). We observed cells positive for WU-VP1 and cytokeratin (Figure 2, panels C, D), which identified these cells as epithelial cells.