Recombinant Human Anti-EV71 VP1 Antibody (D5) (CAT#: PABL-088)

Figure 1 Characterization of D5, H7 and C4.

(A) Binding specificity of MAbs. Three forms of EV71 particles (EV71-F, EV71-E and EV71 VLP) and two inactivated virus (CA16 and CA10) were examined for reactivity to MAbs by ELISA assay. (B) Binding affinity of the MAbs to EV71 F-particle determined by bio-layer interferometry analysis. (C) Neutralization abilities of D5, H7 and C4 against EV71/G082 strain. The neutralization IC50 for each MAb was shown.

Ku, Z., Ye, X., Shi, J., Wang, X., Liu, Q., & Huang, Z. (2015). Single neutralizing monoclonal antibodies targeting the VP1 GH-loop of enterovirus 71 inhibit both virus attachment and internalization during viral entry. Journal of virology, JVI-02189.

Figure 2 D5 and C4 inhibit EV71 infection at post-attachment stage.

(A) Neutralization efficacies of D5 at pre- and post-attachment stages. (B) Neutralization efficacies of C4 at pre-and post-attachment stages. (C-D) The neutralization efficacies of (C) D5 and (D) C4 when added into virus-attached RD cells at different time points post-attachment. The virus-attached cells were incubated without MAb at 37°C for periods indicated, and then treated with MAbs. The cell viability was measured, and the percent of neutralization for each treatment was calculated. The dashed line indicates 50% neutralization.

Ku, Z., Ye, X., Shi, J., Wang, X., Liu, Q., & Huang, Z. (2015). Single neutralizing monoclonal antibodies targeting the VP1 GH-loop of enterovirus 71 inhibit both virus attachment and internalization during viral entry. Journal of virology, JVI-02189.

Figure 3 D5 inhibits virus internalization, uncoating and RNA release at post-attachment stage.

A) Localization of EV71 and MAb revealed by confocal microscopy. Vero cells were pre-attached by EV71 F-particles and then treated by D5 or 3F5. The cells were then incubated at 37°C for 0, 30, 120 or 300 min as indicated. Then, the cells were subjected to immunostaining and confocal microscopy as described in Materials and Methods. Green signal indicates D5 or the control 3F5 mouse MAbs; red signal indicates the presence of EV71 virus which was recognized by a rabbit anti-VLP polyclonal antibody; blue signal indicates DAPI staining. Merge 1 represents the merge of green, red and blue channels. Merge 2 represents the merge of green, red, blue and white field channels.

Ku, Z., Ye, X., Shi, J., Wang, X., Liu, Q., & Huang, Z. (2015). Single neutralizing monoclonal antibodies targeting the VP1 GH-loop of enterovirus 71 inhibit both virus attachment and internalization during viral entry. Journal of virology, JVI-02189.

Figure 4 D5 or C4 treatment interferes with EV71 binding to SCARB2.

Different amounts of D5 or C4 were mixed with EV71/G082 virus and then incubated at 37°C for 1 hr. Subsequently, the antibody/virus mixtures were incubated with the immobile (A-B) SCARB2 on agarose beads. After washing to remove unbound virus, the agarose beads were precipitated by centrifugation, and the binding of virus was detected by Western blotting with a virus-specific antibody or an anti-Fc antibody.

Ku, Z., Ye, X., Shi, J., Wang, X., Liu, Q., & Huang, Z. (2015). Single neutralizing monoclonal antibodies targeting the VP1 GH-loop of enterovirus 71 inhibit both virus attachment and internalization during viral entry. Journal of virology, JVI-02189.

Figure 5 In vivo protective efficacy of antibody D5.

Groups of mice were administered i.p. with PBS or the indicated mAbs, and one day later, inoculated i.p. with 5 × 105 TCID50 of EV71/MAV-W. The mice were monitored daily for (A) survival and (B) clinical signs for a period of 14 days. Clinical scores were graded as follows: 0, healthy; 1, reduced mobility; 2, limb weakness; 3, paralysis; 4, death. The data shown are survival rates and mean clinical scores for each group at the indicated time points. The logrank test was used to compare the survival rate between each mAb group and the PBS control group.

Ye, X., Fan, C., Ku, Z., Zuo, T., Kong, L., Zhang, C.,... & Jiang, W. (2016). Structural basis for recognition of human enterovirus 71 by a bivalent broadly neutralizing monoclonal antibody. PLoS pathogens, 12(3), e1005454.

Figure 6 A yeast library expressing a combinatorial library of the entire polyprotein of EV71 on the surface was screened for D5 binding by fluorescence activating cell sorter (FACS) analysis. D5-positive yeast clones were isolated, and 34 of them were sequenced for the insertion. This panel shows the representative results of two rounds of sorting.

Ye, X., Fan, C., Ku, Z., Zuo, T., Kong, L., Zhang, C.,... & Jiang, W. (2016). Structural basis for recognition of human enterovirus 71 by a bivalent broadly neutralizing monoclonal antibody. PLoS pathogens, 12(3), e1005454.