Human Anti-VP6 Recombinant Antibody (clone 6-26) (CAT#: PABL-323)

Figure 1 The equilibrium binding affinity of the wild-type or mutant Fabs determined by ELISA binding to viral double-layered particles.

Kallewaard, N. L., McKinney, B. A., Gu, Y., Chen, A., Prasad, B. V., & Crowe, J. E. (2008). Functional maturation of the human antibody response to rotavirus. The Journal of Immunology, 180(6), 3980-3989.

Figure 2 Representative surface plasmon resonance binding curves showing the interaction between VP6 and the mutant RV-specific Fabs that were decreased in overall affinity in comparison to the wild-type Fab. Binding curves of RV6-26 wild-type and mutants at a concentration of 12.5 nM.

Kallewaard, N. L., McKinney, B. A., Gu, Y., Chen, A., Prasad, B. V., & Crowe, J. E. (2008). Functional maturation of the human antibody response to rotavirus. The Journal of Immunology, 180(6), 3980-3989.

Figure 3 RRV DLP were coated on microplates and differing concentrations of Fab, IgG or IgA forms of RV6-26 or 2D1 control human antibody (specific for the HA protein of 1918 influenza) normalized for binding sites (Fab = 1; IgG = 2 and IgA = 4) were allowed to bind to DLP. Bound antibodies were detected using peroxidase-conjugated anti-human κ chain antibodies, and the absorbance values are shown.

Aiyegbo, M. S., Sapparapu, G., Spiller, B. W., Eli, I. M., Williams, D. R., Kim, R.,... & Nannemann, D. P. (2013). Human rotavirus VP6-specific antibodies mediate intracellular neutralization by binding to a quaternary structure in the transcriptional pore. PloS one, 8(5), e61101.

Figure 4 Anti-rotaviral activity of RV6-26.

(A) Inhibition of in vitro transcription: EDTA-activated DLP were incubated with 200 nM combining sites of different antibodies and mRNA was synthesized in vitro using selected components of the Riboprobe SP6 system (transcription was mediated by the viral RNA-dependent RNA polymerase not the SP6 DNA-dependent RNA polymerase). First-strand cDNA was synthesized by reverse transcription using a VP6-specific primer. Amplification of cDNA with VP6-specific primers was monitored in a real-time PCR using SYBR Green; the concentrations of RNA estimated from a standard curve constructed using reference RNA extracted from RRV are plotted. (B) Inhibition of rotavirus replication by IgA: polarized monolayers of Caco-2 cells grown on Transwell inserts were treated with polymeric IgA in the basal compartment and inoculated apically with trypsin-activated RRV (MOI = 5) at ambient temperature for 1 h and then cultured for 16 in medium containing trypsin. Amount of rotavirus in the inserts was titrated by inoculating MA104 cells and culturing for 16 h, followed by acetone-fixation and staining with anti-rotavirus polyclonal antibodies conjugated to either Alexa568 or IRDye 800. Detection was done either by scanning on Licor or by counting fluorescent foci.

Aiyegbo, M. S., Sapparapu, G., Spiller, B. W., Eli, I. M., Williams, D. R., Kim, R.,... & Nannemann, D. P. (2013). Human rotavirus VP6-specific antibodies mediate intracellular neutralization by binding to a quaternary structure in the transcriptional pore. PloS one, 8(5), e61101.