BD Horizon™ BV480 Biosimilar Anti-Human TNF
Clone Adalimumab297.rMAb (also known as Adalimumab N297A Biosimilar.rMAb) (RUO)
Multiparameter flow cytometric analysis of TNF (Adalimumab 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 permeabilized cells were then stained with either BD OptiBuild™ BV480 Human IgG1 (N297A), κ Isotype Control (Cat. No. 756244; Left Plot) or BD Horizon™ BV480 Biosimilar Anti-Human TNF antibody (Cat. No. 571507/571557; Right Plot) in BD Perm/Wash™ Buffer. The bivariate pseudocolor density plot showing the correlated expression of TNF (or Ig Isotype control staining) versus forward light scatter (FSC-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 FACSymphony™ A5 SE Cell Analyzer System and FlowJo™ Software.
Multiparameter flow cytometric analysis of TNF (Adalimumab 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 permeabilized cells were then stained with either BD OptiBuild™ BV480 Human IgG1 (N297A), κ Isotype Control (Cat. No. 756244; Left Plot) or BD Horizon™ BV480 Biosimilar Anti-Human TNF antibody (Cat. No. 571507/571557; Right Plot) in BD Perm/Wash™ Buffer. The bivariate pseudocolor density plot showing the correlated expression of TNF (or Ig Isotype control staining) versus forward light scatter (FSC-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 FACSymphony™ A5 SE 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.
For optimal and reproducible results, BD Horizon Brilliant™ Stain Buffer should be used anytime BD Horizon Brilliant dyes are used in a multicolor flow cytometry panel. Fluorescent dye interactions may cause staining artifacts which may affect data interpretation. The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions. When BD Horizon Brilliant Stain Buffer is used in in the multicolor panel, it should also be used in the corresponding compensation controls for all dyes to achieve the most accurate compensation. For the most accurate compensation, compensation controls created with either cells or beads should be exposed to BD Horizon Brilliant Stain Buffer for the same length of time as the corresponding multicolor panel. More information can be found in the Technical Data Sheet of the BD Horizon Brilliant Stain Buffer (Cat. No. 563794/566349) or the BD Horizon Brilliant Stain Buffer Plus (Cat. No. 566385).
Product Notices
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- For U.S. patents that may apply, see bd.com/patents.
- 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.
- 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.
Companion Products
The Adalimumab297.rMAb (also known as, Adalimumab N297A Biosimilar.rMAb) is a research grade recombinant Human IgG1, kappa monoclonal antibody that specifically recognizes Human Tumor Necrosis Factor (TNF) similarly to therapeutic Adalimumab. Adalimumab297.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. Adalimumab is a recombinant Human IgG1 monoclonal anti-Human TNF antibody that is used in the treatment of a wide variety of inflammatory conditions such as rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, septic shock, and cancer. Therapeutic Adalimumab specifically binds to TNF and neutralizes its biological activity by blocking its binding to its cell surface receptors. Adalimumab does not bind to or inactivate lymphotoxin (Tumor necrosis factor-beta; TNF-β). TNF, also known as TNF alpha (TNF-α) 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 is expressed as a noncovalently bound homotrimer that is 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 Adalimumab297.rMAb is intended for research use only. It is not intended for use in therapeutic or diagnostic procedures for humans or animals.
Development References (4)
-
Grewal IS. Overview of TNF superfamily: a chest full of potential therapeutic targets. Adv Exp Med Biol. 2009; 647:1-7. (Biology). View Reference
-
Ingelman-Sundberg HM, Laestadius Å, Chrapkowska C, et al. Diverse effects on vaccine-specific serum IgG titres and memory B cells upon methotrexate and anti-TNF-α therapy in children with rheumatic diseases: A cross-sectional study.. Vaccine. 2016; 34(10):1304-11. (Biology). View Reference
-
Meager A. Assays for cytotoxicity.. Methods Mol Biol. 2004; 249:135-52. (Methodology). View Reference
-
Ward-Kavanagh LK, Lin WW, Šedý JR, Ware CF. The TNF Receptor Superfamily in Co-stimulating and Co-inhibitory Responses.. Immunity. 2016; 44(5):1005-19. (Biology). View Reference
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.