Mouse Anti-WNV E Recombinant Antibody (clone E-11) (CAT#: FAMAB-0734WJ)

Figure 1 Binding of scFvs to rWNV-E as measured by ELISA.

ELISA plates were coated with rWNV-E (100 ng/well) overnight and incubated with serial dilutions of antibodies. Anti-His-HRP (1:4,000) was used as a secondary antibody, and the plates were developed and read at 450 nm (OD450).

Gould, L. H., Sui, J., Foellmer, H., Oliphant, T., Wang, T., Ledizet, M., ... & Fikrig, E. (2005). Protective and therapeutic capacity of human single-chain Fv-Fc fusion proteins against West Nile virus. Journal of virology, 79(23), 14606-14613.

Figure 2 Binding of scFvs to rWNV-E.

rWNV-E (10 μg/gel) was run on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel under reducing (in the presence of 2-mercaptoethanol) or nonreducing conditions and blotted to nitrocellulose. Blots were incubated with scFvs (1 μg/ml), and binding was detected by incubation with anti-His-HRP (1:4,000). Lane Ab refers to control for background anti-His-HRP antibody binding to rWNV-E.

Gould, L. H., Sui, J., Foellmer, H., Oliphant, T., Wang, T., Ledizet, M., ... & Fikrig, E. (2005). Protective and therapeutic capacity of human single-chain Fv-Fc fusion proteins against West Nile virus. Journal of virology, 79(23), 14606-14613.

Figure 3 Survival of mice passively immunized with scFvs-Fcs.

(a) Groups of 5 to 10 mice were injected subcutaneously with 100 μg of scFv-Fcs 1 day prior to intraperitoneal inoculation with 100 PFU WNV. The number of mice surviving was recorded daily. One hundred percent of mice treated with scFv-Fcs 11, 15, 73, and 95 survived. (b) Groups of 5 to 10 mice were injected subcutaneously with 100 μg of scFv-Fcs 1 and 4 days after intraperitoneal inoculation with 100 PFU WNV. The number of mice surviving was recorded daily. Eighty percent of mice treated with scFv-Fc 11 or 15 survived, and 60% of mice treated with scFv-Fc 71 or 73 survived.

Gould, L. H., Sui, J., Foellmer, H., Oliphant, T., Wang, T., Ledizet, M., ... & Fikrig, E. (2005). Protective and therapeutic capacity of human single-chain Fv-Fc fusion proteins against West Nile virus. Journal of virology, 79(23), 14606-14613.

Figure 4 Binding of scFv-Fcs to WNV E protein ectodomain, DI/DII, and DIII.

The binding of scFv-Fcs to yeast displaying either the E protein ectodomain, DI/DII, or DII was measured by fluorescence-activated cell sorting. Yeast cells displaying the indicated fusions were incubated with scFv-Fcs, followed by incubation with the appropriate secondary antibody conjugated to Alexa Fluor 647, and the binding was measured by flow cytometry. The percentage of cells binding to each region was determined after subtracting background binding to control yeast cells expressing only the pYD1 vector. Mouse MAbs E24 and E53 served as positive controls for binding to DIII and DI/DII, respectively.

Gould, L. H., Sui, J., Foellmer, H., Oliphant, T., Wang, T., Ledizet, M., ... & Fikrig, E. (2005). Protective and therapeutic capacity of human single-chain Fv-Fc fusion proteins against West Nile virus. Journal of virology, 79(23), 14606-14613.

Figure 5 Partial epitope mapping of therapeutic recombinant antibodies previously selected by phage display.

To map linear peptide sequences of scFv-Fcs 11, 71, 73, 79, and 95, we measured scFv-Fc binding to a set of overlapping 20-mer peptides spanning the entire sequence of West Nile virus sE by ELISA. scFv-Fcs 11, 71, and 73 bind a peptide consisting of West Nile virus residues 281 to 300. All three scFv-Fcs also recognize the homologous E peptide from dengue virus type 2 (DEN2) but not a peptide composed of the same amino acids in a randomized sequence. Furthermore, scFv-Fc binding was critically dependent on positively charged side chains (Lys or Arg) at positions 287 and 291 of West Nile virus E.

Kanai, R., Kar, K., Anthony, K., Gould, L. H., Ledizet, M., Fikrig, E., ... & Modis, Y. (2006). Crystal structure of West Nile virus envelope glycoprotein reveals viral surface epitopes. Journal of virology, 80(22), 11000-11008.