Anti- Rituximab (Anti-Idiotype) Antibody
Anti- Rituximab (Anti-Idiotype) Antibody Summary
Please Note: Optimal dilutions should be determined by each laboratory for each application. General Protocols are available in the Technical Information section on our website.
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.
Preparation and Storage
- 12 months from date of receipt, -20 to -70 °C as supplied.
- 1 month, 2 to 8 °C under sterile conditions after reconstitution.
- 6 months, -20 to -70 °C under sterile conditions after reconstitution.
The anti-Rituximab antibody is an anti-Idiotype antibody raised
against and selected for the antigenic determinants of the
variable region of Rituximab. The antibody has been tested against a
broad range of other biosimilars to ensure specificity. This highly
specialized anti-idiotypic antibody
can be used in a variety of pharmacokinetic (PK) assay formats. In addition to Rituximab, the anti-Idiotype antibody also detects derivatives of Rituximab (Ibritumumab and Tositumumab).
What is an anti-idiotype antibody?
An anti-idiotype antibody is specific for the unique variable (antigen binding) region of another antibody. We have anti-idiotype antibodies to commecially available therapeutic antibodies or biosimilars.
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