Description
The B27 monoclonal antibody specifically binds to human interferon-γ (IFN-γ), a 14-18 kDa glycoprotein containing 143 amino acid residues. IFN-γ is a potent multifunctional cytokine produced by several activated cell types including NK, NKT, CD4+TCRαβ+, CD8+TCRαβ+, and TCRγδ+ T cells. IFN-γ exerts its biological effects through specific binding to the high-affinity IFN-γ receptor complex comprised of IFN-γRα (CD119) and IFN-γRβ subunits. In addition to its antiviral effects, IFN-γ upregulates a number of lymphoid cell functions including the antimicrobial and anti-tumor responses of macrophages, NK cells, and neutrophils. In addition, IFN-γ influences the regulation of proliferation, differentiation, and effector responses of B cell and T cell subsets. These influences can involve IFN-γ's capacity to boost MHC class I and II expression by antigen-presenting cells as well as direct effects on B cells and T cells themselves. B27 is a neutralizing antibody. The use of B27 antibody for epitope mapping of human IFN-γ has been described. The B27 antibody has been reported not to bind to denatured IFN-γ.
Immunofluorescent Staining and Flow Cytometric Analysis: The APC-conjugated B27 antibody (Cat. No. 554702) is useful for multicolor immunofluorescent staining and flow cytometric analysis to identify and enumerate IFN-γ producing cells within mixed cell populations (see image). For optimal immunofluorescent staining for flow cytometric analysis, this anti-cytokine antibody should be titrated (≤ 0.5 µg mAb/1X10^6 cells). For specific methodology, please visit the protocols section under "Intracellular Flow" on our web site, http://www.bdbiosciences.com/us/s/resources. A useful control for demonstrating specificity of staining is the following: pre-block the paraformaldehyde-fixed/saponin-permeabilized cells with unlabeled B27 antibody (Cat. No. 554699/550011) prior to staining. The intracellular cytokine staining technique and blocking controls are described in detail by C. Prussin and D. Metcalfe. A suitable mouse IgG1 isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized human cells is APC-MOPC-21 (Cat. No. 554681); use at comparable concentrations to antibody of interest (e.g., ≤ 0.5 µg mAb/1X10^6 cells). Neutralization: The NA/LE™ B27 antibody (Cat. No. 554698) is useful for neutralization of human IFN-γ bioactivity. A suitable NA/LE mouse IgG1 isotype control to match the NA/LE B27 antibody is the 107.3 antibody, (Cat. No. 554698). IP/WB: The B27 antibody has been reported to be useful for immunoprecipitation studies. The B27 antibody has been reported not to bind to denatured IFN-γ. Please note that this application is not routinely tested.
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to APC under optimum conditions, and unconjugated antibody and free APC were 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. This APC-conjugated reagent can be used in any flow cytometer equipped with a dye, HeNe, or red diode laser.
6. Cy is a trademark of Amersham Biosciences Limited.
7. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
8. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
