BD Horizon™ RB780 Biosimilar Anti-Human TNF
Clone Infliximab297.rMAb (RUO)
Two-color flow cytometric analysis of TNF (Infliximab Biosimilar) expression by stimulated Human peripheral blood lymphocytes. Human peripheral blood mononuclear cells (PBMC) were stimulated for 5 hours with Phorbol 12-Myristate 13-Acetate (Sigma P-8139; 50 ng/ml final concentration) and Ionomycin (Sigma I-0634; 1 μg/ml final concentration) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724). The cells were harvested, washed with BD Pharmingen™ Stain Buffer (FBS) [Cat. No. 554656] and fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655) followed by washing and permeabilization using BD Perm/Wash™ Buffer (Cat. No. 554723).
The fixed and permeabilized cells were then stained in BD Perm/Wash™ Buffer with either BD OptiBuild™ RB780 Human IgG1 (N297A), κ Isotype Control (Cat. No. 756229; Left Plot) or BD Horizon™ RB780 Biosimilar Anti-Human TNF antibody (Cat. No. 570958/571029; Right Plot). The bivariate pseudocolor density plot showing the correlated expression of TNF (or Ig Isotype control staining) versus side light-scatter (SSC-A) signals was derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ Cell Analyzer System and FlowJo™ Software.
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Two-color flow cytometric analysis of TNF (Infliximab Biosimilar) expression by stimulated Human peripheral blood lymphocytes. Human peripheral blood mononuclear cells (PBMC) were stimulated for 5 hours with Phorbol 12-Myristate 13-Acetate (Sigma P-8139; 50 ng/ml final concentration) and Ionomycin (Sigma I-0634; 1 μg/ml final concentration) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724). The cells were harvested, washed with BD Pharmingen™ Stain Buffer (FBS) [Cat. No. 554656] and fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655) followed by washing and permeabilization using BD Perm/Wash™ Buffer (Cat. No. 554723).
The fixed and permeabilized cells were then stained in BD Perm/Wash™ Buffer with either BD OptiBuild™ RB780 Human IgG1 (N297A), κ Isotype Control (Cat. No. 756229; Left Plot) or BD Horizon™ RB780 Biosimilar Anti-Human TNF antibody (Cat. No. 570958/571029; Right Plot). The bivariate pseudocolor density plot showing the correlated expression of TNF (or Ig Isotype control staining) versus side light-scatter (SSC-A) signals was derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ Cell Analyzer System and FlowJo™ Software.
Preparation And Storage
Recommended Assay Procedures
BD® CompBeads can be used as surrogates to assess fluorescence spillover (compensation). When fluorochrome conjugated antibodies are bound to BD® CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and BD® CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.
Product Notices
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
- Human donor specific background has been observed in relation to the presence of anti-polyethylene glycol (PEG) antibodies, developed as a result of certain vaccines containing PEG, including some COVID-19 vaccines. We recommend use of BD Horizon Brilliant™ Stain Buffer in your experiments to help mitigate potential background. For more information visit https://www.bdbiosciences.com/en-us/support/product-notices.
- When using high concentrations of antibody, background binding of this dye to erythroid fragments produced by ammonium chloride-based lysis, such as with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899), has been observed when the antibody conjugate was present during the lysis procedure. This may cause nonspecific staining of target cells, such as leukocytes, which have bound the resulting erythroid fragments. This background can be mitigated by any of the following: titrating the antibody conjugate to a lower concentration, fixing samples with formaldehyde, or removing erythrocytes before staining (eg, gradient centrifugation or pre-lysis with wash). This background has not been observed when cells were lysed with BD FACS™ Lysing Solution (Cat. No. 349202) after staining.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- An isotype control should be used at the same concentration as the antibody of interest.
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- Please observe the following precautions: We recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to protect exposure of conjugated reagents, including cells stained with those reagents, to any room illumination. Absorption of visible light can significantly affect the emission spectra and quantum yield of tandem fluorochrome conjugates.
- Tandem fluorochromes contain both an energy donor and an energy acceptor. Although every effort is made to minimize the lot-to-lot variation in the efficiency of the fluorochrome energy transfer, differences in the residual emission from the donor may be observed. Additionally, multi-laser cytometers may directly excite both the donor and acceptor fluorochromes. Therefore, we recommend for every tandem conjugate, a matched individual single-stain control be acquired for generating a compensation or spectral unmixing matrix.
The Infliximab N297A Biosimilar.rMAb is a research grade recombinant Human IgG1, kappa monoclonal antibody that specifically recognizes Human Tumor Necrosis Factor (TNF) similarly to therapeutic Infliximab. Infliximab N297A Biosimilar.rMAb was engineered to code for a replacement of asparagine with alanine at position 297 (N297A) of the Human IgG1 heavy chain to reduce potential nonspecific staining caused by Fc receptor binding. Infliximab is a chimeric recombinant Human IgG1 Anti-Human TNF monoclonal antibody that is used to treat a wide variety of inflammatory conditions such as rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, Bechet's disease, psoriasis, septic shock, cachexia, and cancer. Therapeutic Infliximab specifically binds to soluble and transmembrane TNF and neutralizes its biological activity by blocking the binding to its cell surface receptors. It may also bind to transmembrane TNF on cells that produce it and mediate both antibody-dependent (ADCC) and complement-dependent (CDC) cytotoxicity. Infliximab does not bind to or inactivate lymphotoxin (Tumor necrosis factor-beta; TNF-ß). TNF, also known as TNF alpha (TNF-a) or Cachectin, is an ~26 kDa type II transmembrane protein that is encoded by TNF. TNF is a multifunctional cytokine that plays roles in inflammation, immune system development, innate and adaptive immune responses, apoptosis, lipid metabolism, and necrosis of some tumor cells. TNF is variably produced by a wide variety of activated leucocytes, epithelial cells, endothelial cells, and tumor cells. This mediator forms noncovalently bound homotrimers that are expressed on the cell surface. Membrane TNF is enzymatically cleaved from the cell surface by tumor necrosis factor alpha converting enzyme (TACE), also known as ADAM17 and CD156b, into a soluble biologically active trimer of the TNF extracellular domains. TNF mediates its biological activities through binding to cell surface TNF Receptor Type 1 (TNFR1, CD120a) and Type 2 (TNFR2, CD120b), which are widely expressed on normal hemopoietic and nonhemopoietic cells as well as tumor cells.
The Infliximab N297A Biosimilar.rMAb is intended for research use only. It is not intended for use in therapeutic or diagnostic procedures for humans or animals.
Development References (3)
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Knight DM, Trinh H, Le J, et al. Construction and initial characterization of a mouse-human chimeric anti-TNF antibody.. Mol Immunol. 1993; 30(16):1443-53. (Immunogen: Blocking, ELISA, Radioimmunoassay). View Reference
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Mitoma H, Horiuchi T, Tsukamoto H, Ueda N. Molecular mechanisms of action of anti-TNF-α agents - Comparison among therapeutic TNF-α antagonists.. Cytokine. 2018; 101:56-63. (Biology). View Reference
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Scallon BJ, Moore MA, Trinh H, Knight DM, Ghrayeb J. Chimeric anti-TNF-alpha monoclonal antibody cA2 binds recombinant transmembrane TNF-alpha and activates immune effector functions.. Cytokine. 1995; 7(3):251-9. (Clone-specific: Blocking, Cytotoxicity, Flow cytometry, Inhibition, Radioimmunoassay). View Reference
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.