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Purified Rat Anti-Human IL-4
Purified Rat Anti-Human IL-4
Expression of IL-4 by stimulated human peripheral blood mononuclear cells (PBMC). Human PBMC were stimulated with soluble anti-human CD3 antibody (1 µg/ml; Cat. No. 555329), recombinant human IL-2 (10 ng/ml, Cat. No. 554603) and IL-4 (20 ng/ml; Cat. No. 554605) for 2 days. The cells were subsequently cultured in medium containing recombinant human IL-2 and recombinant human IL-4 for 3 days. Finally, the cells were harvested and stimulated for 6 h with PMA (Sigma, Cat. No. P-8139) and calcium ionophore A23187 (Sigma) in the presence of 2 µM GolgiStop™ (Cat. No. 554724). The cells were harvested, stained with PE-Cy™5 -anti CD4 (Cat. No. 555348), fixed, permeabilized, and subsequently stained with 0.05 µg of PE-rat anti-human IL-4 antibody (PE-MP4-25D2, Cat. No. 554485) by using Pharmingen's staining protocol (left panel). To demonstrate specificity of staining, the binding of PE-MP4-25D2 antibody was blocked by preincubation of the fixed/permeabilized cells with unlabeled MP4-25D2 antibody (2.5 µg, Cat. No. 554482; right panel) prior to staining. The quadrant markers for the bivariate dot plot were set based on the autofluorescence controls and verified using unlabeled antibody blocking controls.
Expression of IL-4 by stimulated human peripheral blood mononuclear cells (PBMC). Human PBMC were stimulated with soluble anti-human CD3 antibody (1 µg/ml; Cat. No. 555329), recombinant human IL-2 (10 ng/ml, Cat. No. 554603) and IL-4 (20 ng/ml; Cat. No. 554605) for 2 days. The cells were subsequently cultured in medium containing recombinant human IL-2 and recombinant human IL-4 for 3 days. Finally, the cells were harvested and stimulated for 6 h with PMA (Sigma, Cat. No. P-8139) and calcium ionophore A23187 (Sigma) in the presence of 2 µM GolgiStop™ (Cat. No. 554724). The cells were harvested, stained with PE-Cy™5 -anti CD4 (Cat. No. 555348), fixed, permeabilized, and subsequently stained with 0.05 µg of PE-rat anti-human IL-4 antibody (PE-MP4-25D2, Cat. No. 554485) by using Pharmingen's staining protocol (left panel). To demonstrate specificity of staining, the binding of PE-MP4-25D2 antibody was blocked by preincubation of the fixed/permeabilized cells with unlabeled MP4-25D2 antibody (2.5 µg, Cat. No. 554482; right panel) prior to staining. The quadrant markers for the bivariate dot plot were set based on the autofluorescence controls and verified using unlabeled antibody blocking controls.
Product Details
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BD Pharmingen™
Human (QC Testing)
Rat IgG1
Purified Recombinant Human IL-4
Intracellular block/flow cytometry (Routinely Tested), Western blot (Reported)
0.5 mg/ml
AB_398561
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. Store undiluted at 4°C.

Recommended Assay Procedures

Recommended Assay Procedure:

1. Blocking Control for Intracellular Staining: The unlabeled MP4-25D2 antibody can be used as a blocking control to demonstrate specificity of IL-4 staining by conjugated MP4-25D2 antibody (Cat. No. 554486, 554485, No. 554484). To perform this control, the fixed/permeabilized cells (~ 1 million) can be incubated with 1-10 µg of unlabeled MP4-25D2 antibody (Cat. No. 554482) for 20 minutes at 4°C, prior to staining with conjugated MP4-25D2 antibody.  The intracellular cytokine staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe. For specific methodology, please visit the protocols section or chapter on intracellular staining in the Immune Function Handbook, both of which are posted on our web site, www.bdbiosciences.com.

2. Neutralization: The NA/LE™ MP4-25D2 antibody (Cat. No. 554481) is useful for neutralization of human IL-4 bioactivity. A suitable NA/LE™ rat IgG1 isotype control to match the MP4-25D2 antibody is the R3-34 antibody, Cat. No. 554682.

3. WB: The purified MP4-25D2 antibody has been reported to be useful for Western blotting. 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.
554482 Rev. 1
Antibody Details
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MP4-25D2

The MP4-25D2 monoclonal antibody specifically binds to Interleukin-4 (IL-4). IL-4 is also known as Lymphocyte stimulatory factor 1, B cell stimulatory factor 1 (BSF-1), or B cell growth factor 1 (BCGF-1). IL-4 is produced by activated T cells, basophils, and mast cells. IL-4 is a multifunctional cytokine and growth factor that affects a variety of different target cell types. IL-4 can costimulate T cell proliferation and survival, as well as help drive Th2-like cell differentiation and effector functions. It costimulates B cell proliferation and survival, and can help B cells differentiate into IgG4- or IgE-producing cells. IL-4 can also diminish the proinflammatory functions of monocytes and macrophages. IL-4 exerts its biological effects by binding with high affinity to cell surface CD124 (IL-4Rα chain). CD124 forms signaling IL-4 receptor complexes with either CD132/common γ chain or CD213a1/IL-13Rα1 to form either Type I or Type II IL-4 Receptor complexes, respectively. The immunogen used to generate the MP4-25D2 hybridoma was purified recombinant human IL-4. This is a neutralizing antibody. The MP4-25D2 antibody has been reported to crossreact with IL-4 from rhesus monkeys. The use of the MP4-25D2 antibody for epitope mapping of human IL-4 has been described.

This antibody is routinely tested as a blocking control for intracellular staining. Other applications were tested at BD Biosciences Pharmingen during antibody development only or reported in the literature.

554482 Rev. 1
Format Details
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Purified
Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
Purified
554482 Rev.1
Citations & References
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Development References (5)

  1. 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
  2. Chretien I, Van Kimmenade A, Pearce MK, Banchereau J, Abrams JS. Development of polyclonal and monoclonal antibodies for immunoassay and neutralization of human interleukin-4. J Immunol Methods. 1989; 117(1):67-71. (Clone-specific: ELISA, Neutralization). View Reference
  3. Jung T, Schauer U, Rieger C, et al. Interleukin-4 and interleukin-5 are rarely co-expressed by human T cells. Eur J Immunol. 1995; 25(8):2413-2416. (Clone-specific: Flow cytometry). 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
  5. Ramanathan L, Ingram R, Sullivan L, et al. Immunochemical mapping of domains in human interleukin 4 recognized by neutralizing monoclonal antibodies. Biochemistry. 1993; 32(14):3549-3556. (Clone-specific: Neutralization). View Reference
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554482 Rev. 1

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