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Updated: Aug 8, 2026
Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function
Published on: February 16, 2017
Isolation and characterization of allergen-binding cells from normal and allergic donors
J Irsch1, N Hunzelmann, H Tesch
1Institute for Genetics, University of Cologne, Germany.
Insights
This study introduces antigen-specific high gradient magnetic cell sorting to isolate rare immune cells from blood. This method effectively enriches allergen-specific cells for allergy diagnosis and research.
Area of Science:
- Immunology
- Cell Biology
- Allergy Research
Background:
- Traditional flow cytometry is limited in analyzing rare immune cells.
- Identifying antigen-specific cells is crucial for understanding immune responses.
- High gradient magnetic cell sorting offers a novel enrichment method.
Purpose of the Study:
- Develop efficient technology for staining and isolating antigen-binding cells from human peripheral blood.
- Analyze and compare allergen-specific cells from normal and allergic donors.
- Establish a cellular diagnosis for allergy.
Main Methods:
- Utilized high-gradient magnetic cell sorting (MACS) for rare cell isolation.
- Employed haptenized phospholipase A2 (PLA2) and ParoI as specific allergens.
- Characterized cells phenotypically using immunofluorescence and immortalized B-cells via EBV transformation.
Main Results:
- Achieved up to 75% purity in enriching allergen-specific cells from both normal and allergic donors.
- Confirmed specificity through established allergen-specific EBV-transformed B-cell lines.
- Identified distinct cellular compositions in allergen-binding cells between normal and allergic donors.
Conclusions:
- High gradient magnetic cell sorting enables effective enrichment of allergen-specific immune cells.
- Normal donors primarily show allergen-binding B-cells (CD19+, CD21+).
- Allergic donors exhibit allergen-binding B-cells and IgE+ basophilic granulocytes (CD38+, CD9+, CD25+).
Background:
Flow cytometry of the immune system so far has been limited to the analysis of subpopulations according to lineage markers. The cells involved in a particular immune response could not be assayed due to their low frequency. Here we show the potential of antigen-specific high gradient magnetic cell sorting to enrich cells for visualisation in multiparameter cytometry, functional studies and immortalization.
Objectives:
The aim of this study was the development of an efficient technology for staining and isolation of antigen-binding cells from human peripheral blood. In particular, allergen-specific cells from normal and allergic donors should be analysed and compared to develop a cellular diagnosis of allergy.
Study Design:
The rare antigen-specific cells were sorted by high-gradient magnetic cell sorting with MACS. Haptenized phospholipase A2 (PLA2), the major allergen of bee venom, or haptenized ParoI, the major allergenic component of Parietaria officinalis, were used as antigens. The cells from normal and allergic donors, binding to the allergen were characterized phenotypically by immuno-fluorescence. Allergen-specific B-cells were immortalized by EBV transformation.
Results And Conclusion:
Allergen-specific cells can be enriched from blood of both allergic and normal donors to purities of up to 75%, by high gradient magnetic cell sorting. The specificity of labelling with allergen was confirmed by establishing allergen-specific EBV-transformed B-cell lines from the sorted cells. Clear differences exist in the cellular composition of allergen-binding cells from normal compared to allergic donors. In normal donors the allergen-binding cells are B-cells expressing CD19 and CD21. In allergic donors, in addition to allergen-binding B-cells, occurring in about equal absolute numbers as in normal donors, basophilic granulocytes are labeled by allergen. These cells express CD38, CD9 and CD25 on their surface, and stain for IgE.
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