Recombinant Mouse Anti-VACV A13 Antibody (11F7) (CAT#: FAMAB-0327CQ)

Figure 1 Identification and Characterization of an anti-A13 mAb 11F7

HeLa cells were infected with VACV WR at a MOI of 10 and metabolically labeled with 35S-methionine and -cysteine from 8 to 16 hpi. The cells were lyzed and immunoprecipitated with either the culture supernatant from 11F7 hybridoma cells or DMEM medium only. The precipitated proteins were analyzed by SDS-PAGE, and the autoradiograph is shown.

Xu, C., Meng, X., Yan, B., Crotty, S., Deng, J., & Xiang, Y. (2011). An epitope conserved in orthopoxvirus A13 envelope protein is the target of neutralizing and protective antibodies. Virology, 418(1), 67-73.

Figure 2 Identification and Characterization of an anti-A13 mAb 11F7

B). Proteins from uninfected (-) cells or cells infected with VACV WR (+) were analyzed by Western blot using the culture supernatant from 11F7. The same membranes were also blotted with anti-HSP70 antibody as a loading control.

Xu, C., Meng, X., Yan, B., Crotty, S., Deng, J., & Xiang, Y. (2011). An epitope conserved in orthopoxvirus A13 envelope protein is the target of neutralizing and protective antibodies. Virology, 418(1), 67-73.

Figure 3 Identification and Characterization of an anti-A13 mAb 11F7

C). The same amount of purified recombinant MBP or MBP fused with VACV A13 were used to coat ELISA plates, and ELISA were performed with the culture supernatant from 11F7. Average OD from triplicate experiments is shown.

Xu, C., Meng, X., Yan, B., Crotty, S., Deng, J., & Xiang, Y. (2011). An epitope conserved in orthopoxvirus A13 envelope protein is the target of neutralizing and protective antibodies. Virology, 418(1), 67-73.

Figure 4 Identification and Characterization of an anti-A13 mAb 11F7

BHK cells grown on cover-slips were infected with VACV WR at a MOI of 0.5 PFU/cell for 8 hrs and then analyzed by immunofluorescence with 11F7 supernatant. The primary antibody was stained with a Cy3-conjugated goat anti-mouse secondary antibody (red), and the DNA was stained with DAPI (blue).

Xu, C., Meng, X., Yan, B., Crotty, S., Deng, J., & Xiang, Y. (2011). An epitope conserved in orthopoxvirus A13 envelope protein is the target of neutralizing and protective antibodies. Virology, 418(1), 67-73.

Figure 5 Neutralization of VACV MV by 11F7

Sucrose-gradient purified VACV mature virions were incubated with the indicated amount of purified anti-A13 mAb 11F7, anti-H3 mAb #41, or anti-WR148 mAb HE7 in the presence (+) or absence (-) of rabbit complement for 1 hr at 4°C. The mixture was then added to monolayers of BS-C-1 cells, and the inoculum was removed after one hour. The number of plaques that appeared after 2 days was enumerated. The amount of viruses used for inoculation in (B) was three times of that in (A). The average number of plaques from untreated inoculum is 55 for (A) and 184 for (B). The number of plaques obtained under the indicated condition as the percentage to the number of plaques from untreated inoculums is shown. The average and standard deviation are from three independent inoculums.

Xu, C., Meng, X., Yan, B., Crotty, S., Deng, J., & Xiang, Y. (2011). An epitope conserved in orthopoxvirus A13 envelope protein is the target of neutralizing and protective antibodies. Virology, 418(1), 67-73.

Figure 6 Protection of mice from intranasal VACV infection by 11F7

(A and B). Groups of 5 BALB/c mice were given by the intraperitoneal route with either PBS or 2 mg of the indicated antibodies (anti-A10, anti-A13 11F7, anti-H3 #41) on day -1 and challenged with 1 × 104 PFU of VACV WR by the intranasal route on day 0. The average body-weight changes with SEM (A) and survivals (B) of each group are shown. (C and D). The experiment was done as in (A and B) except with different antibodies.

Xu, C., Meng, X., Yan, B., Crotty, S., Deng, J., & Xiang, Y. (2011). An epitope conserved in orthopoxvirus A13 envelope protein is the target of neutralizing and protective antibodies. Virology, 418(1), 67-73.

Figure 7 Identification of epitope for 11F7

A). E. coli strains were either not induced (-) or induced with IPTG (+) to express fusion of MBP with the indicated A13 fragments. Proteins from the whole cell lysates were resolved by SDS-PAGE and analyzed by either Coomassie staining or by Western blot with 11F7. B). The multiple sequence alignment of A13 orthologues from vaccinia virus (VACV), monkeypox virus (MPXV) and variola virus (VARV). The box indicates the identified 11F7 epitope.

Xu, C., Meng, X., Yan, B., Crotty, S., Deng, J., & Xiang, Y. (2011). An epitope conserved in orthopoxvirus A13 envelope protein is the target of neutralizing and protective antibodies. Virology, 418(1), 67-73.

Figure 8 Binding affinity of 11F7 to recombinant A13 proteins

A). Recombinant MBP, MBP fusion with A13(23-70) or MBP fusion with A13(59-69) were expressed in E. coli BL21 strains and purified with metal affinity chromatograph. Coomassie stain of the purified recombinant proteins is shown. B). 11F7 was immobilized on a BIAcore CM5 sensor chip, and its binding with MBP-A13(23-70) or A13(59-69) was then monitored with a BIAcore 3000 sensor. The lines are the responses obtained with the indicated concentrations of recombinant proteins. The response curves were globally fitted to a 1:1 binding model, and obtained affinity constants are shown.

Xu, C., Meng, X., Yan, B., Crotty, S., Deng, J., & Xiang, Y. (2011). An epitope conserved in orthopoxvirus A13 envelope protein is the target of neutralizing and protective antibodies. Virology, 418(1), 67-73.