Mouse Anti-EDA Recombinant Antibody (clone EctoD2) (CAT#: FAMAB-0124CQ)

Figure 1 Anti-EDA antibodies recognize epitopes on native EDA1

A, SDS-PAGE analysis and Coomassie Blue staining of 10 μg/lane of the indicated purified mouse IgG1 monoclonal antibodies under reducing conditions. Migration positions of molecular mass standards (in kDa) are shown. B, Fc-EDA1 (200 or 50 ng) and Fc-hAPRIL (Fc-A, 500 ng) were resolved by Western blotting (WB) under reducing conditions and revealed with anti-human immunoglobulin (anti-Fc), anti-EDA (Renzo2, EctoD1, EctoD2, or EctoD3), or anti-APRIL (Aprily2) antibodies.

Kowalczyk-Quintas, C., Willen, L., Dang, A. T., Sarrasin, H., Tardivel, A., Hermes, K., ... & Headon, D. J. (2014). Generation and characterization of function-blocking anti-ectodysplasin A (EDA) monoclonal antibodies that induce ectodermal dysplasia. Journal of Biological Chemistry, 289(7), 4273-4285.

Figure 2 Anti-EDA antibodies recognize epitopes on native EDA1

Purified Fc-EDA1 and Fc-APRIL proteins were coated directly in an ELISA plate. Fc-EDA1 was also captured via the Fc portion using an anti-human antibody. Coated proteins were revealed with the indicated antibodies at the indicated concentrations.

Kowalczyk-Quintas, C., Willen, L., Dang, A. T., Sarrasin, H., Tardivel, A., Hermes, K., ... & Headon, D. J. (2014). Generation and characterization of function-blocking anti-ectodysplasin A (EDA) monoclonal antibodies that induce ectodermal dysplasia. Journal of Biological Chemistry, 289(7), 4273-4285.

Figure 3 Sensitive detection of Fc-EDA1 in serum by sandwich ELISA

A, sandwich ELISAs for Fc-EDA1 were performed using anti-EDA antibodies (Renzo2, EctoD2, and EctoD3) at capture and biotinylated forms of the same antibodies for detection. Biotinylated antibodies were revealed with horseradish-coupled streptavidin. B, comparison of sandwich ELISAs for Fc-EDA1 using EctoD2, EctoD3, EDAR-Fc, or anti-human immunoglobulin antibodies at capture and biotinylated EcotD2, EctoD3, or horseradish-coupled goat anti-human (hu) immunoglobulin for revelation. C, comparison of sandwich ELISA (EctoD2 and biotinylated EctoD3) for Fc-EDA1 in buffer or in the presence of 50% mouse or human serum.

Kowalczyk-Quintas, C., Willen, L., Dang, A. T., Sarrasin, H., Tardivel, A., Hermes, K., ... & Headon, D. J. (2014). Generation and characterization of function-blocking anti-ectodysplasin A (EDA) monoclonal antibodies that induce ectodermal dysplasia. Journal of Biological Chemistry, 289(7), 4273-4285.

Figure 4 Anti-EDA antibodies EctoD2 and EctoD3 recognize epitopes overlapping the EDA receptor-binding site.

Fc-EDA1 WT or containing the indicated point mutations, or Fc-BAFF as a control, were coated onto ELISA plates and revealed with the indicated antibodies at the indicated concentration.

Kowalczyk-Quintas, C., Willen, L., Dang, A. T., Sarrasin, H., Tardivel, A., Hermes, K., ... & Headon, D. J. (2014). Generation and characterization of function-blocking anti-ectodysplasin A (EDA) monoclonal antibodies that induce ectodermal dysplasia. Journal of Biological Chemistry, 289(7), 4273-4285.

Figure 5 Anti-EDA monoclonal antibodies EctoD2 and EctoD3 inhibit mammalian and avian EDA1 and EDA2.

Receptors (human or chicken EDAR or XEDAR) fused to the GPI anchor of TRAILR3 were expressed in 293T cells together with an EGFP tracer (x axis). Cells were stained with or without cell supernatants containing Fc-EDA1 or Fc-EDA2 of human/mouse (human) or chicken origin (y axis). The interactions of Fc-EDAs with GPI-anchored receptors were challenged by preincubation of the ligand with anti-EDA antibodies (EctoD1, EctoD2, and EctoD3) or with an irrelevant antibody (anti-APRIL). Both scattergram axes show fluorescence intensity on a logarithmic scale (10⁰–10⁴).

Kowalczyk-Quintas, C., Willen, L., Dang, A. T., Sarrasin, H., Tardivel, A., Hermes, K., ... & Headon, D. J. (2014). Generation and characterization of function-blocking anti-ectodysplasin A (EDA) monoclonal antibodies that induce ectodermal dysplasia. Journal of Biological Chemistry, 289(7), 4273-4285.

Figure 6 Anti-EDA monoclonal antibodies EctoD2 and EctoD3 inhibit mammalian and avian EDA1 and EDA2.

FLAG-EDA1 was diluted at the indicated concentrations and preincubated with or without 125 ng/ml concentration of anti-EDA (Renzo2, EctoD1, EctoD2, or EctoD3) or anti-APRIL (Aprily2) monoclonal antibodies. The binding of FLAG-EDA1 to coated EDAR-Fc protein was then probed by an ELISA-like assay.

Kowalczyk-Quintas, C., Willen, L., Dang, A. T., Sarrasin, H., Tardivel, A., Hermes, K., ... & Headon, D. J. (2014). Generation and characterization of function-blocking anti-ectodysplasin A (EDA) monoclonal antibodies that induce ectodermal dysplasia. Journal of Biological Chemistry, 289(7), 4273-4285.

Figure 7 Anti-EDA monoclonal antibodies EctoD2 and EctoD3 inhibit mammalian and avian EDA1 and EDA2.

EDA1-sensitized Jurkat JOM2 EDAR-Fas reporter cells were incubated in the presence of the indicated concentrations of Fc-EDA1, in the presence or absence of the indicated concentrations of anti-EDA monoclonal antibodies EctoD1, EctoD2, or EctoD3. Cell viability was determined by colorimetry (phenazine methosulfate/(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay) and expressed as percentage of maximal survival.

Kowalczyk-Quintas, C., Willen, L., Dang, A. T., Sarrasin, H., Tardivel, A., Hermes, K., ... & Headon, D. J. (2014). Generation and characterization of function-blocking anti-ectodysplasin A (EDA) monoclonal antibodies that induce ectodermal dysplasia. Journal of Biological Chemistry, 289(7), 4273-4285.

Figure 8 Anti-EDA monoclonal antibodies EctoD2 and EctoD3 induce ectodermal dysplasia when administered during embryonic development in wild type mice.

Kowalczyk-Quintas, C., Willen, L., Dang, A. T., Sarrasin, H., Tardivel, A., Hermes, K., ... & Headon, D. J. (2014). Generation and characterization of function-blocking anti-ectodysplasin A (EDA) monoclonal antibodies that induce ectodermal dysplasia. Journal of Biological Chemistry, 289(7), 4273-4285.