Preparation And Storage
Recommended Assay Procedures
Immunofluorescent Staining and Flow Cytometric Analysis: The BVD3-1F9 antibody is useful for immunofluorescent staining and flow cytometric analysis to identify and enumerate IL-3 producing cells within mixed cell populations. The PE-conjugated BVD3-1F9 antibody (Cat. No. 554676) is especially suitable for these studies. For optimal immunofluorescent staining and flow cytometric analysis, this anti-cytokine antibody should be titrated ( ≤ 0.5 µg mAb/million cells). 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.
A suitable rat IgG1 isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized human cells is R3-34 (Cat. No. 554685); use at comparable concentrations to antibody of interest (e.g., ≤ 0.5 µg mAb/1 million cells). A useful control for demonstrating specificity of staining is either of the following: 1) pre-block the conjugated BVD3-1F9 antibody with ligand (e.g., recombinant human IL-3; Cat No. 554604) prior to staining, or 2) pre-block the fixed/permeabilized cells with unlabeled BVD3-1F9 antibody prior to staining. The staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe.
ELISA: The biotinylated BVD3-1F9 antibody (Cat. No. 554674) is useful as a detection antibody for a sandwich ELISA for measuring human IL-3 protein levels. Biotinylated BVD3-1F9 antibody can be paired with the purified BVD8-3G11 antibody (Cat. No. 554672) as the capture antibody, with recombinant human IL-3 protein (Cat. No. 554604) as the standard.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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.
The BVD3-1F9 antibody reacts with human interleukin-3 (IL-3). The immunogen used to generate the BVD3-1F9 hybridoma was recombinant human IL-3. This is a weakly neutralizing antibody.
Development References (5)
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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). View Reference
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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). View Reference
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Abrams JS, Silver JE, Van Dyke RE, Gleich GI. Eosinophil-active cytokines in human disease: development and use of monoclonal antibodies to IL-3, IL-5 and GMCSF. In: Gleich GJ and Kay AB, ed. Eosinophils in Allergy and Inflammation. New York: Dekker; 1994:133-157.
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Kaushansky K, Shoemaker SG, Broudy VC. Structure-function relationships of interleukin-3. An analysis based on the function and binding characteristics of a series of interspecies chimera of gibbon and murine interleukin-3. J Clin Invest. 1992; 90(5):1879-1888. (Clone-specific). View Reference
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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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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.