Related Experiment Videos
Enumeration of IgE secreting B cells. A filter spot-ELISA
C L King1, G Thyphronitis, T B Nutman
1Laboratory of Parasitic Diseases, National Institutes of Health, Bethesda, MD 20892.
Insights
A new filter immunoplaque assay accurately detects IgE-producing B lymphocytes in human blood. This sensitive method quantifies immunoglobulin E (IgE) secreting cells, aiding in allergy and immunology research.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Assessing immunoglobulin E (IgE) producing cells is crucial for understanding allergic diseases and immune responses.
- Existing methods for quantifying IgE secretion from individual human B lymphocytes can be complex or lack sensitivity.
Purpose of the Study:
- To develop and validate a sensitive and specific filter immunoplaque assay for detecting IgE secretion from individual B lymphocytes in human peripheral blood mononuclear cells (PBMC).
Main Methods:
- Peripheral blood mononuclear cells (PBMC) were incubated on microfilter plates coated with anti-human IgE antibodies.
- IgE production by single cells was detected using a monoclonal antibody and enzymatic development, visualized as blue plaques.
- Assay sensitivity and specificity were confirmed using an IgE-producing myeloma cell line (U266) and Epstein-Barr Virus (EBV)-transformed B cells producing IgG or IgM.
Main Results:
- The assay demonstrated a strong correlation (near 1:1) between cell counts and plaque formation for U266 cells.
- No detectable plaques were observed for EBV-transformed cells producing only IgG or IgM, confirming specificity.
- IgE levels in culture supernatants correlated highly with IgE-producing cell counts (r=0.93) and intracytoplasmic IgE staining (r=0.94).
Conclusions:
- The developed filter immunoplaque assay is a simple, direct, sensitive, and specific method for assessing the frequency of IgE-producing lymphocytes in humans.
- This assay facilitates research into IgE-mediated immune responses and allergic conditions.
Abstract:
To assess the frequency of IgE producing cells in humans a filter immunoplaque assay has been developed to detect IgE secretion from individual B lymphocytes in unfractionated peripheral blood mononuclear cells (PBMC). PBMC were incubated in microfilter plates containing nitrocellulose membranes coated with polyclonal anti-human IgE antibody, and the IgE production by a single cell was detected using a specific anti-human IgE monoclonal antibody followed by enzymatic development. The products of the enzymatic reaction were visualized as blue plaques on the membranes. The assay was both sensitive and specific as determined by: (1) a near 1:1 correlation between direct cell counts of an IgE producing myeloma cell line (U266) and the number of plaques in the filter immunoassay; and (2) the absence of detectable plaques generated by human B cells transformed by Epstein-Barr Virus (EBV) and producing only IgG or IgM. The presence of other cell types in PBMC did not affect the ability to detect IgE secreting cells. Replicate cultures of highly purified B lymphocytes, first transformed with EBV and then stimulated with recombinant human interleukin-4, produced IgE levels in culture supernatants that correlated closely with the number of IgE producing cells (r = 0.93; P less than 0.001). Furthermore, using the same transformed cells, the number of IgE secreting cells assessed by the immunoplaque assay was significantly correlated (r = 0.94; P = 0.002) with the number of IgE producing cells assessed by immunofluorescence staining of intracytoplasmic IgE. This assay provides a simple and direct method to assess the frequency of IgE producing lymphocytes in humans.