Description
The TNFR1-B1 monoclonal antibody specifically recognizes the extracellular domain of CD120a. CD120a is encoded by TNFRSF1A (Tumor necrosis factor receptor superfamily member 1A). CD120a is also known as the 55 kDa receptor for the human cytokines, tumor necrosis factor (TNF) and lymphotoxin-alpha (LT-a). This receptor is referred to as the p55 or Type I Tumor Necrosis Factor Receptor (TNFRI). TNFRI are expressed by a variety of cell lines, tumor cells, and normal cell types including T cells, monocytes, macrophages, neutrophils, endothelial cells, hepatocytes, chondrocytes, and fibroblasts. Naive B cells express very low or undetectable levels of TNFRI whereas mature erythrocytes and platelets are uniformly negative for TNFRI expression. MABTNFR1-B1 specifically binds to natural and recombinant truncated forms of TNFRI.
Immunofluorescent Staining and Flow Cytometric Analysis: Biotin Mouse Anti-Human CD120a (Cat. No. 550900) can be used for immunofluorescent staining (≤ 1 µg antibody/10×6 cells) and flow cytometric analysis of nucleated human cells to measure their expressed levels of surface TNFRI. A two-layer staining protocol is recommended for maximizing the detection of TNFRI expressed by cells as detailed in the figure legend. As a demonstration of specificity, the binding of MABTNFR1-B1 to TNFRI is inhibited when human TNFRI+ target cells are preincubated with saturating levels of recombinant human TNF at 4°C, i.e., when the TNFRI are bound with ligand. Based on our data, a recombinant human TNF level above 50 ng/10×6 cells is sufficient to completely inhibit the binding of MABTNFR1-B1 (0.06 µg/10×6 cells). Please note also that as a consequence of in vivo or in vitro activation, cell surface TNFRI can either be shed by cells or transiently expressed at higher levels. As a result, cellular activation can affect the cell's overall expressed level of surface TNFRI. ELISA Detection: Biotin Mouse Anti-Human CD120a serves as the detection antibody in a sandwich ELISA for measuring human soluble TNFRI protein levels. Biotin Mouse Anti-Human CD120a antibody can be paired with the Purified Mouse Anti-Human CD120a (Cat No. 552535) using recombinant human soluble TNFRI as the standard. This detection antibody should be titrated between 0.5 - 1 µg/ml to determine its optimal concentration for ELISA detection. To obtain linear standard curves, doubling dilutions of recombinant soluble human TNFRI ranging from 200 to 15 pg/ml are recommended for inclusion in each ELISA plate. For specific methodology, please visit the protocols section under "ELISA and ELISPOT" at our website, http://www.bdbiosciences.com/us/s/resources.
Store undiluted at 4°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with biotin under optimum conditions, and unreacted biotin was removed.
1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2. An isotype control should be used at the same concentration as the antibody of interest.
3. 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.
4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
5. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
6. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
