Detection of Parechovirus A1 with Monoclonal Antibody against Capsid Protein VP0

Figures

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  Figure 1. Generation procedures of PeV-A1 VP0 hybridoma. (A,B) Nucleotide and amino acid sequence similarity of VP0 from PeV-A1 and PeV-A3. Red and blue bands indicate identical and nonidentical amino acid residues, respectively. (C) Schematic for hybridoma generation.

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  Figure 2. Evaluation of mouse antiserum against PeV-A1 VP0. (A) A549 cells (1 × 105) were infected with mock or PeV-A1 (MOI = 5). Cell lysates were harvested at postinfection 24 h for VP0 detection by antiserum (dilution factor of 1:5000) from four immunized mice. Arrow: PeV-A VP1. (B) Immunofluorescence assay was conducted in PeV-A1-infected A549 cells (1 × 105) for 24 h. Both PeV-A VP0 polyclonal antibody (left panels) and Mouse #1 antiserum (right panels) were used (dilution factor of 1:500). The fluorescence-merged images of PeV-A1 VP0 (red) and DAPI (nuclear, blue) are shown. Scale bar, 100 μm.

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  Figure 3. Identification of anti-PeV-A1 VP0 monoclonal antibody (mAb)-producing hybridoma clones. (A) A549 cells were infected with mock and PeV-A1 (MOI = 5) for 24 h; cell lysates were separated in SDS-PAGE for Western blot with hybridoma mAb supernatant, polyclonal VP0 antibody, or mouse antiserum. The arrow indicates the detected PeV-A1 VP0 protein. (B) ELISA assay was conducted with hybridoma mAb supernatant, polyclonal PeV-A VP0 antibody, or Aichi-VP1 mAb. The dilution factor of each antibody is indicated. The absorbance value of O.D.450 (-blank) is shown. (C) The representative image of hybridoma growth is shown. Scale bar, 100 μm.

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  Figure 4. Detection of PeV-A1 by anti-PeV-A1 VP0 mAb. Mock-, PeV-A1-, and PeV-A3-infected A549 cells, after 24 h, were subjected to immunofluorescence analysis with hybridoma mAb supernatant (dilution factor = 1:10) and polyclonal VP0 antibody (dilution factor = 1:500). The polyclonal VP0 antibody detected PeV-A1 and PeV-A3 (red). PeV-A1 VP0 mAb detected PeV-A1- but not PeV-A3-infected cells. DAPI showed nuclear staining (blue). Scale bar, 100 μm.

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  Figure 5. Anti-PeV-A1 VP0 mAb detects PeV-A1 but not PeV-A3. Immunoblotting assay was conducted in mock-, PeV-A1-, and PeV-A3-infected A549 cells for 24 h. Both hybridoma mAb supernatant and polyclonal VP0 antibody were employed to recognize PeV-A VP0 (upper panel). The protein-loading control (Ponceau S. staining) is shown in the lower panels.