Anti- Rituximab (Anti-Idiotype) Antibody

Detection of Rituximab by Western Blot. Western blot shows Cetuximab (negative control), Rituximab, Brentuximab (negative control), and Gemtuzumab (negative control) biosimilars. PVDF membrane was probed with 0.5 µg/mL of Anti-Rituximab (Anti-Idiotype) Monoclonal Antibody (Catalog # MAB9630) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (HAF008). A specific band was detected for Rituximab at approximately 240 kDa (as indicated). This experiment was conducted under non-reducing conditions and using Immunoblot Buffer Group 1.

Anti-MS4A1/CD20 (Rituximab) binding to MS4A1/CD20-transfected HEK293 Human Cell Line is Blocked by Anti-Rituximab (Anti-Idiotype) Antibody. In a functional flow cytometry test, Biotinylated Anti-MS4A1/CD20 (Rituximab) (Catalog # FAB9575B) binds to HEK293 human embryonic kidney cell line transfected with human MS4A1/CD20 (black dotted line). Binding is completely blocked (orange histogram) by 0.25 µg/mL of Anti-Rituximab (Anti-idiotype) Monoclonal Antibody (Catalog # MAB9630), but not by Normal Rabbit IgG (MAB1050) (blue line). Cells were stained with APC-conjugated Streptavidin (F0050).

Detection of Anti-MS4A1/CD20 (Rituximab) on Human MS4A1/CD20 transfectants by flow cytometry. HEK293 human cell line transfected with (A) human MS4A1/CD20 or (B) irrelevant transfectants and eGFP were stained with Human anti-Human MS4A1/CD20 (Rituximab) Monoclonal Antibody (MAB9575), then Anti-Rituximab (Anti-Idiotype) (Catalog # MAB9630) followed by APC-conjugated anti-rabbit IgG secondary antibody (F0111).

Specificity of Anti-Rituximab (Anti-Idiotype) Antibody in ELISA Capture Assay. Biotinylated biosimilars were serially diluted and incubated with Anti-Rituximab (Anti-Idiotype) Antibody (Catalog # MAB9630, green line) coated at 1 μg/mL on a 96-well polystyrene plate. No cross-reactivity with Brentuximab (black line) or biotinylated IgG control (orange line) was observed.

Specificity of Anti-Rituximab (Anti-Idiotype) Antibody in Direct ELISA. A panel of biosimilars was coated at 1 μg/mL in 96-well polystyrene plates. Anti-Rituximab (Anti-Idiotype) Antibody (Catalog # MAB9630, 333 ng/mL) detected Rituximab. 100% cross-reactivity with Tositumomab and Ibritumomab (95% or higher sequence identity with Rituximab) and no cross-reactivity with Cetuximab, Trastuzumab, Brentuximab, Gemtuzumab, Adalimumab, Solanezumab, Teprotumumab, and Aducanumab was observed.

Anti-Rituximab Bridging Assay. The anti-idiotype antibody (catalog number MAB9630) directed against Rituximab was coated at 4 μg/mL in a 96-well polystyrene plate to capture the biosimilar Rituximab. Biotinylated anti-Rituximab (Anti-Idiotype) Antibody was used at 0.5 μg/mL as the detection antibody before incubation with Streptavidin-HRP. Other biosimilars did not bridge the anti-idiotype antibody, showing specificity of the anti-Rituximab (Anti-Idiotype) antibody.

NKs cells are potent killers in ADCC assays with Raji cells. 200x105 human NK cells were seeded from PBMCs and cultured for 13 days in plates/flasks using plate-bound Anti-Human NKp46 Monoclonal Antibody in ExCellerate Human NK Cell Expansion Media (Xeno-Free) plus cytokines (see expansion protocol, CCM032). Prior to setting up the ADCC assay, CD56+CD16+ levels were ascertained to be >30% on NKs using Human Fc gamma RIII (CD16) PerCP-conjugated Antibody (FAB2546C). Raji target cells were labeled with Mito Mark Green and Effectors/NKs were labeled with Janelia Fluor 646 (see FMC032 for details). Raji cells were preincubated with 10 ug/mL anti-CD20/Rituximab (Catalog # MAB9630) for 30 minutes at room temperature prior to setting up the ADCC assay with the NKs. 2:1 E:T ratios were used for the Raji/Rituximab model, and NKs and Raji cells were co-incubated for 2.5 hours at 37°C. The cells were then harvested, stained with antibodies for Human CD3 Alexa Fluor 405 and Human CD56 Alexa Fluor 700, and analyzed by flow cytometry. Importantly, non-NKs (JF 646-CD56- cells) are shown in the Mito Mark dye graph that depict killing. These results are representative of 3+ experiments.