Jackson Immuno Research Inc.
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Whole IgG Affinity-Purified Secondary Antibodies

"I have used a wide variety of secondaries and Jackson ImmunoResearch has consistently been the best. The fluorophores are bright and stable and their selective (x reactivity removed) secondaries have always shown species specificity in multiple labeling."

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Anti-Human IgE

Anti-Human IgE

Introduction

Jackson ImmunoResearch Mouse Monoclonal Anti-Human IgE antibodies are the latest addition to our suite of products suitable for diagnostics research and development. Specific for Human IgE antibodies, they complement our existing Anti-Human IgG, IgM, and IgA antibodies. Anti-Human IgE is available conjugated to a select range of reporter molecules, including Alkaline Phosphatase and Biotin enabling excellent sensitivity.


About Jackson ImmunoResearch Anti-Human IgE

Jackson ImmunoResearch Anti-Human IgE is a mouse-derived monoclonal antibody with reactivity to human E class immunoglobulins and is epsilon (ε) chain specific.

Jackson ImmunoResearch Mouse Anti-Human IgE (clone number ME.114) (209-002-241) is available conjugated to a range of reporter molecules and is suitable to detect human IgE by a variety of techniques, including ELISA (Enzyme-linked immunosorbent assay), Lateral flow immunoassay (LFIA), flow cytometry, western blot, and CLIA (Chemiluminescent immunoassay).


Specificity

Jackson ImmunoResearch Anti-Human IgE epsilon (ε) chain specific and has been tested by ELISA and shows minimal cross-reactivity to human IgG, IgM, or IgA. Figure 1 is a Western blot that demonstrates the specificity of Jackson ImmunoResearch Anti-Human IgE showing no detection of other immunoglobulin classes. We have not tested if this antibody will detect immunoglobulins from other species.

Western blot showing the specificity of Jackson ImmunoResearch Anti-Human IgE.
Figure 1: Western blot showing the specificity of Jackson ImmunoResearch Anti-Human IgE antibody for Human IgE. 1μg of purified human immunoglobulin (IgE, IgG, IgA or IgM as indicated) was loaded into individual wells. SDS-PAGE was performed under non-reducing conditions and the proteins blotted onto nitrocellulose membrane. Blots were blocked with 5% BSA in PBS/T and probed with Peroxidase Anti-Human IgE (209-032-241) at 1:10K dilution. The blot was visualized using ECL.
Western blot showing detection of purified human IgE by Jackson ImmunoResearch Anti-Human IgE in reducing and non-reducing conditions.
Figure 2: Western blot showing detection of 100ng of purified human IgE by Anti-Human IgE in reducing and non-reducing conditions. Please note that this antibody will give a weaker signal when detecting reduced protein and therefore is not recommended for applications where detection of reduced IgE is required.

Sensitivity

Jackson ImmunoResearch Anti-Human IgE can be used to detect human IgE from various sources with excellent sensitivity. Figure 3 demonstrates the sensitivity of Jackson ImmunoResearch Anti-Human IgE when used in an indirect ELISA, showing its ability to detect IgE coating the wells at two different concentrations. Figure 4 demonstrates the sensitivity of Jackson ImmunoResearch Anti-Human IgE when used in an indirect ELISA to detect IgE purified from different myeloma and plasma sources.

Detection of purified IgE at different coating concentrations

Detection of IgE by indirect ELISA with Jackson ImmunoResearch Anti-Human IgE.
Figure 3: Detection of IgE by indirect ELISA. Plate coated with 100μl/well purified IgE from human plasma at 2 or 0.2μg/ml in carbonate coating buffer overnight at 4oC. The plate was blocked with 300μl/well of 1% BSA (IgG-Free, Protease-Free Bovine Serum Albumin 001-000-161) in PBS/T for 2 hr. The primary antibody, Anti-Human IgE (209-002-241), was serially diluted in PBS/T at 1:3 across the plate and incubated for 2 hr at room temperature. Secondary antibody, Peroxidase AffiniPure Goat Anti-Mouse IgG, Fcγ fragment specific (min X Hu, Bov, Hrs Sr Prot) (115-035-071), diluted 1:20,000 in PBS/T was incubated on the plate for 1.5 hr. TMB was applied and read at 620nm after 30 min.

Detection of IgE from Plasma and Myeloma

Detection of human IgE from different sources by indirect ELISA with Jackson ImmunoResearch Anti-Human IgE.
Figure 4: Detection of human IgE from different sources by indirect ELISA. Plate coated with 100μl/well purified human IgE from the indicated sources at 2μg/ml in carbonate coating buffer overnight at 4oC. The plate was blocked with 300μl/well of 1% Bovine serum albumin (BSA) (001-000-161) in PBS/T for 2 hr at room temperature. The primary antibody, Anti-Human IgE (209-002-241), was serially diluted in PBS/T at 1:3 across the plate and incubated for 1 hr at room temperature. Secondary antibody, Peroxidase AffiniPure Goat Anti-Mouse IgG, Fcγ fragment specific (min X Hu, Bov, Hrs Sr Prot) (115-035-071), diluted 1:20,000 was incubated on the plate for 1.5 hr. TMB was applied and read at 620nm after 30 min.

Detection of IgE from plasma

Jackson ImmunoResearch Anti-Human IgE offers excellent sensitivity when used in a sandwich ELISA with its capture partner antibody. We recommend using two different monoclonal antibodies for capture and detection. In this format, use an unconjugated Anti-Human IgE as your coating antibody before blocking with Bovine Serum Albumin (IgG-Free, Protease-Free 001-000-161). Following washing, incubate with your diluted sample. For the detection step, use a conjugated Anti-Human IgE antibody, such as Alkaline Phosphatase Anti-Human IgE (209-052-241) or Peroxidase Anti-Human IgE (209-032-241), followed by the appropriate substrate or visualization technique. Figure 5 illustrates the application of the direct sandwich assay to validate the quantification of total IgE from patient samples using unconjugated Anti-Human IgE in partnership with an alkaline phosphatase conjugated detection antibody. Total IgE (kIU/l) in patient samples used in this experiment were previously measured by the Roche Cobas Total IgE assay. These results are displayed on the x-axis of Figure 5.

Total IgE in Patient Samples

Detection of total IgE from human serum and plasma samples by direct sandwich ELISA with Jackson ImmunoResearch Anti-Human IgE.
Figure 5: Detection of total IgE from human serum and plasma samples by direct sandwich ELISA. Plate was coated with unconjugated Anti-Human IgE (clone 10A10) antibody at 100μl/well at 2μg/ml in coating buffer overnight at 4oC. The plate was blocked with 300μl/well of 1% BSA (001-000-161) in Phosphate buffered saline, 0.05% Tween® (PBS/T) for 1.5 hrs. Incubate with 100μl/well of plasma/serum samples diluted 1:80 in PBS/T for 1hr. Detection antibody, Jackson ImmunoResearch Alkaline Phosphatase Anti-Human IgE (209-055-241), was diluted at 1:10,000 in PBS/T and incubated at 100μl/well for 1 hr. 100μl/well of freshly prepared DEA substrate was incubated on the plate for 30 min and read at 405nm.

Suggested Dilution Factors

The dilution factors suggested in the following table are presented as ranges because the optimal dilution is a function of many factors, such as antigen density, permeability, etc. The optimal working dilution should be determined empirically for each application.


  Show Dilution Table  
Application
Conjugate ELISA Western Blots* Flow Cytometry
Unconjugated 10-20 µg/ml
FITC 1:50-1:200
R-PE 1:50-1:200
Horseradish Peroxidase 1:5,000-1:100,000 1:5,000-1:100,000 (non-ECL)
1:10,000-1:200,000 (ECL)
Alkaline Phosphatase 1:5,000-1:50,000 1:5,000-1:50,000
Biotin-SP (using Fluorophore-Conjugated Streptavidin) 1:200-1:1,000
Biotin-SP (using enzyme-Conjugated Streptavidin) 1:20,000-1:400,000 1:20,000-1:400,000

* Please note that this antibody will give a weaker signal when detecting reduced protein and therefore is not recommended for applications where detection of reduced IgE is required by western blot.


References

  • Cox, L., Larenas-Linnemann, D., Lockey, R. and Passalacqua, G., 2010. Speaking the same language: The World Allergy Organization Subcutaneous Immunotherapy Systemic Reaction Grading System. Journal of Allergy and Clinical Immunology, 125(3), pp.569-574.e7.
  • Pawankar, R., Canonica, G., Holgate, S., and Lockey, R., 2011. World Allergy Organization (WAO) white book on allergy. United Kingdom: WAO.
  • Sánchez-Borges, M., Ansotegui, I. & Cox, L. World Allergy Organization Grading System for Systemic Allergic Reactions: it Is Time to Speak the Same Language When it Comes to Allergic Reactions. Curr Treat Options Allergy 6, 388–395 (2019). https://doi.org/10.1007/s40521-019-00229-8
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