Mice (n = 8) were immunized intraperitoneally (i.p.) by injection of 100 μg ganglidiximab in combination with 400 μg of adjuvant aluminum hydroxide (Invivogen, Toulouse, France). aluminum hydroxide is a clinically approved highly effective adjuvant. It forms a long-lasting depot maintaining local antigen concentrations and promotes antigen uptake by antigen-presenting cells. Furthermore, aluminum hydroxide mainly induces a polarized TH2 cell (T helper cell subtype 2) response to nearly all protein antigens [29 (link)]. Control groups received Al(OH)3 or 0.9% NaCl. Immunization was repeated six times at 2-week intervals and serum samples were taken before and after the last immunization step.
Next, serum of immunized mice was analyzed for Abs directed against the GD2-mimicking paratopes of ganglidiximab based on our optimized and recently reported ELISA method [17 (link)]. In this case, 96-well plates were coated with ganglidiximab. For quantification, seven standard samples containing known concentration of murine anti-GD2 Ab 14G2a were prepared. Binding was detected using HRP-conjugated goat anti-mouse IgG (Fc-specific) (# A0168-1ML, Sigma Aldrich, Steinheim, Germany) as secondary mAb. To avoid quantification of Abs directed against IgG1 human constant regions, data were normalized to signals obtained using chimeric isotype rituximab as a capture Ab.
Next, serum of immunized mice was analyzed for Abs directed against the GD2-mimicking paratopes of ganglidiximab based on our optimized and recently reported ELISA method [17 (link)]. In this case, 96-well plates were coated with ganglidiximab. For quantification, seven standard samples containing known concentration of murine anti-GD2 Ab 14G2a were prepared. Binding was detected using HRP-conjugated goat anti-mouse IgG (Fc-specific) (# A0168-1ML, Sigma Aldrich, Steinheim, Germany) as secondary mAb. To avoid quantification of Abs directed against IgG1 human constant regions, data were normalized to signals obtained using chimeric isotype rituximab as a capture Ab.