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FITC Rat Anti-Human IL-6
FITC Rat Anti-Human IL-6
Expression of IL-6 by stimulated CD14+ human monocytes. Human PBMC were stimulated for 6 hours with LPS (100 ng/ml final concentration) in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724). The PBMC were harvested, stained with PE-mouse anti-human CD14 monoclonal antibody (PE-M5E2, Cat. No. 555398), fixed, permeabilized, and subsequently stained with 0.25 µg of FITC-rat anti-human IL-6 antibody (Cat. No. 554544), following Pharmingen's staining protocol (left panel). The data reflect gating on monocytes, based on forward and side scattered light signals. To demonstrate specificity of staining, the binding by FITC-MQ2-13A5 antibody was blocked by each of the following: 1) preincubation of the fluorochrome-conjugated antibody with molar excess of recombinant human IL-6 (0.5 µg, Cat. No. 550071; (middle panel) and by 2) preincubation of the fixed/permeabilized cells with excess unlabeled MQ2-13A5 antibody (e.g., 5 µg; Cat. No. 554543; right panel) prior to staining with the FITC-MQ2-13A5 antibody. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabeled antibody blocking specificity controls.
Expression of IL-6 by stimulated CD14+ human monocytes. Human PBMC were stimulated for 6 hours with LPS (100 ng/ml final concentration) in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724). The PBMC were harvested, stained with PE-mouse anti-human CD14 monoclonal antibody (PE-M5E2, Cat. No. 555398), fixed, permeabilized, and subsequently stained with 0.25 µg of FITC-rat anti-human IL-6 antibody (Cat. No. 554544), following Pharmingen's staining protocol (left panel). The data reflect gating on monocytes, based on forward and side scattered light signals. To demonstrate specificity of staining, the binding by FITC-MQ2-13A5 antibody was blocked by each of the following: 1) preincubation of the fluorochrome-conjugated antibody with molar excess of recombinant human IL-6 (0.5 µg, Cat. No. 550071; (middle panel) and by 2) preincubation of the fixed/permeabilized cells with excess unlabeled MQ2-13A5 antibody (e.g., 5 µg; Cat. No. 554543; right panel) prior to staining with the FITC-MQ2-13A5 antibody. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabeled antibody blocking specificity controls.
Product Details
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BD Pharmingen™
Human (QC Testing)
Rat IgG1
Human IL-6 Recombinant Protein
Intracellular staining (flow cytometry) (Routinely Tested)
0.5 mg/ml
AB_395468
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO


Preparation And Storage

The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with FITC under optimum conditions, and unreacted FITC was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.

Recommended Assay Procedures

Immunofluorescent Staining and Flow Cytometric Analysis: The PE conjugated MQ2-13A5 antibody is useful for immunofluorescent staining and flow cytometric analysis to identify and enumerate IL-6 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/million cells)  For specific methodology, please visit our web site, www.bdbiosciences.com, and go to the protocols section or the chapter on intracellular staining in the Immune Function Handbook.

A useful control for demonstrating specificity of staining is either of the following: 1) pre-block the FITC-MQ2-13A5 antibody with excess ligand (e.g., recombinant human IL-6; Cat No. 550071) prior to staining, or 2) pre-block the fixed/permeabilized cells with unlabelled MQ2-13A5 antibody (Cat. No. 554543) prior to staining. A suitable rat IgG1 isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized human cells is FITC-R3-34 (Cat. No. 554684); use at comparable concentrations to antibody of interest (e.g., ≤ 0.5 µg mAb/1 million cells).

OTHER APPLICATIONS

ELISA Capture: The purified MQ2-13A5 antibody (Cat. No. 554543) is useful as a capture antibody for a sandwich ELISA for measuring human IL-6 protein levels.  Purified MQ2-13A5 antibody can be paired with the biotinylated MQ2-39C3 antibody (Cat. No. 554546) as the detecting antibody, with recombinant human IL-6 protein (Cat. No. 550071) as the standard. For specific methodology, please visit our web site, www.bdbiosciences.com, and go to the protocols section or the chapter on ELISA in the Immune Function Handbook. For testing IL-6 in serum or plasma, our OptEIA™ Human IL-6 ELISA Set (Cat.No. 550799) or OptEIA Human IL-6 ELISA Kit (Cat. No. 550799) is recommended.

Neutralization: The no azide/low endotoxin formulation of the MQ2-13A5 antibody (Cat. No. 554541) is useful for neutralization of human IL-6 bioactivity.

Western blot: The MQ2-13A5 antibody (Cat. No. 554543) has been found useful for Western blot. Please note that this application is not routinely tested at BD Biosciences Pharmingen.

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  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.
554544 Rev. 1
Antibody Details
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MQ2-13A5

The MQ2-13A5 monoclonal antibody specifically binds to human interleukin-6 (IL-6). IL-6 is a multifunctional cytokine that plays a central role in host defense mechanisms, including hematopoiesis, immune responses (eg, T and B cell activation and differentiation) and acute phase reactions. IL-6 can be expressed by a variety of cells including monocytes/macrophages, eosinophils, fibroblasts, vascular endothelial cells, bone marrow stromal cells, mesangial cells, hepatocytes, keratinocytes, astrocytes, T lymphocytes, B lymphocytes, and various tumor cells. IL-6 production is upregulated by cells in response to bacterial products such as lipopolysaccharide, viruses and other pro-inflammatory cytokines such as IL-1, TNF , and IFN-γ. IL-6 transcription is downregulated by cells in response to IL-4 and IL-10. The functional IL-6 Receptor (IL-6R) complex consists of two transmembrane glycoproteins, an 80-kDa low-affinity ligand-binding receptor subunit (IL-6Rα/CD126) and a 130 kDa (gp130/CD130) subunit that binds to IL-6-IL-6Rα to form the high-affinity signal transducing complex. Abnormal expression of IL-6 is related to the pathogenesis of many diseases including neoplastic (eg, multiple myeloma) and autoimmune diseases (eg, rheumatoid arthritis). The immunogen used to generate this hybridoma was COS-7 -expressed recombinant human IL-6.

This antibody is routinely tested by flow cytometric analysis. Other applications were tested at BD Biosciences Pharmingen during antibody development only or reported in the literature.

554544 Rev. 1
Format Details
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FITC
Fluorescein (FITC) is part of the BD blue family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 494-nm and an emission maximum (Em Max) at 518-nm. FITC is designed to be excited by the Blue laser (488-nm) and detected using an optical filter centered near 520 nm (e.g., a 530/30-nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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FITC
Blue 488 nm
494 nm
518 nm
554544 Rev.1
Citations & References
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Development References (4)

  1. Abrams J. Immunoenzymetric assay of mouse and human cytokines using NIP-labeled anti-cytokine antibodies. Curr Protoc Immunol. 2001; 1:6.20-6.21. (Clone-specific: ELISA, Neutralization). View Reference
  2. Abrams JS, Roncarolo MG, Yssel H, Andersson U, Gleich GJ, Silver JE. Strategies of anti-cytokine monoclonal antibody development: immunoassay of IL-10 and IL-5 in clinical samples. Immunol Rev. 1992; 127:5-24. (Clone-specific: ELISA, Neutralization). View Reference
  3. Gaines Das RE, Poole S. The international standard for interleukin-6. Evaluation in an international collaborative study. J Immunol Methods. 1993; 160(2):147-153. (Clone-specific: ELISA, Neutralization). View Reference
  4. Prussin C, Metcalfe DD. Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies. J Immunol Methods. 1995; 188(1):117-128. (Methodology: IC/FCM Block). View Reference
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554544 Rev. 1

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For Research Use Only. Not for use in diagnostic or therapeutic procedures.