Table 1.
The viruses used in this study.
Fig 1.
SDS-PAGE analysis of the purified Fabs.
Purified Fabs 2A6 and 4E2 were preheated and then individually resolved by SDS-PAGE under denaturing and reducing conditions. The heterodimer was dissociated into the light chain (28 kDa) and the Fd fragment (32 kDa). Lane M represents the protein marker.
Fig 2.
SDS-PAGE analysis of the purified knob proteins.
The purified knob proteins of HAdV-B3, E4, and C5 and the mutant knob protein with and without preheating (lanes 1 and 2, respectively) were individually analyzed by SDS-PAGE under denaturing and reducing conditions. Coomassie Brilliant Blue staining of the purified knob proteins of HAdV-B3, E4 and C5 and the mutant knob protein with preheating revealed bands corresponding to the expected molecular weight of the monomeric proteins (23 kDa, 18 kDa, 20 kDa and 20 kDa, respectively). The purified knob proteins of HAdV-E4 and C5 without preheating were revealed in polymeric form, while the purified knob protein of HAdV-B3 and the mutant knob protein without preheating were revealed in monomeric form. Lane M represents the protein marker.
Fig 3.
Measurement of virus-binding capacity of Fabs 2A6 and 4E2 using CLEIA.
The virus-binding capacities of Fabs 2A6 and 4E2 were measured by separately binding to several types of HAdV, IFV, RSV, CVA16, and EV71. PBS was used as a blank control. The photons of light emitted were measured as RLU. Each virus was tested in duplicate, and the results represent the mean ± SD of three measurements. The average RLU of Fab 2A6 that reacted with HAdV-B35 was higher than those of the Fab that reacted with IFV, RSV, CVA16, and EV71. A similar result was found with Fab 4E2.
Fig 4.
Immunofluorescence analysis of Fab 2A6 with different types of HAdV-infected cells.
HAdV-B3, B7, B11, B14, B35, B55, C1, C2, C5, E4, F40, and F41 were individually used to examine Fab 2A6 with HAdV-infected cells using immunofluorescence. Cell cultures with obvious CPE were visualized. The cells were observed by a fluorescence microscope (100×).
Fig 5.
Immunofluorescence analysis of Fab 4E2 with different types of HAdV-infected cells.
HAdV-B3, B7, B11, B14, B35, B55, C1, C2, C5, E4, F40, and F41 were individually used to examine Fab 4E2 with HAdV-infected cells using immunofluorescence. Cell cultures with obvious CPE were visualized. The cells were observed by a fluorescence microscope (100×).
Fig 6.
Measurement of knob protein-binding capacity of Fabs 2A6 and 4E2 using CLEIA.
The knob protein-binding capacities of Fabs 2A6 and 4E2 were measured by binding to the knob proteins of HAdV-B3, E4, and C5 (negative control). PBS was used as a blank control. The photons of light emitted were measured as RLU. Each knob protein was tested in duplicate, and the result represents the mean ± SD of three measurements.
Fig 7.
Multiple alignment of amino acid sequences of the knob proteins of HAdV-B3, E4, and C5 and mutant knob protein.
The multiple alignment results showed that, compared with the sequences outside of the boxed region, the amino acid sequence (residues 71–91) of HAdV-E4 was found to be more similar to that of HAdV-B3 (residues 69–89) than to that of HAdV-C5 (residues 70–90) within the boxed region. The amino acid fragment of the HvdA-E4 knob protein within the boxed region was predicted to be the linear epitope. The mutant knob protein was designed by replacing the amino acid sequence of the HAdV-C5 knob protein within the boxed region with the amino acid sequence of the predicted linear epitope.
Fig 8.
3D structure of the HAdV-E4 knob protein and the predicted linear epitope.
The predicted linear epitope region on the HAdV-E4 knob protein is located in the neck region on the side of the 3D model. The predicted linear epitope region is highlighted in red on the model in both top (A) and side (B) views. The amino acid sequence of the predicted linear epitope is highlighted in red in the sequence of HAdV-E4 knob protein.
Fig 9.
Western blot analysis of Fabs 2A6 and 4E2 binding to knob proteins under denaturing and reducing conditions.
Note that Fabs 2A6 (A) and 4E2 (B) bind to the knob proteins of HAdV-B3 and E4 with molecular masses of about 23 kDa and 18 kDa, respectively, with preheating. However, both Fabs did not recognize the HAdV-C5 knob protein with preheating. In addition, a band around 20 kDa, which is the predicted molecular weight of the mutant knob (mknob) protein with preheating, was detected with both Fabs. Two non-specific bands appeared below the knob proteins of HAdV-B3 and E4, but these were not found with SDS-PAGE, suggesting that the concentrations were low. These non-specific proteins could be purified by Ni-NTA resin and recognized by Fab, suggesting that they contained the 6×His tag and the linear epitope. We inferred that these non-specific proteins were produced as partially expressed knob proteins that were degraded in E.coli.