title: Detection of specific IgE-secreting cells with an enzyme-linked immunospot assay.
abstract: BACKGROUND
  An in vitro assay system for monitoring the induction of human allergen-specific IgE synthesis with an enzyme-linked immunospot assay is described.
  
  
  METHODS
  IgE-secreting cells, responding specifically to short ragweed pollen allergens. Amb a V and Amb a VI, were directly enumerated after the coculture of high-density, resting B cells with either autologous T cells or allergen-specific T-cell clones from the peripheral blood of two ragweed-allergic individuals. Both T-cell clones were type 2-like T helper cells (TH2) as determined by reverse transcription polymerase chain reaction. The IgE-producing cells were detected on a nitrocellulose-based paper disc coated with antigen after treatment with biotinylated, anti-human IgE and avidin-peroxidase conjugate.
  
  
  RESULTS
  We demonstrated the induction of Amb a V- or Amb a VI-specific IgE synthesis from either peripheral blood B cells or purified resting B cells. Specific IgE-secreting B cells could be detected only when T cells were present in the culture, providing that they were not separated by a membrane. The addition of interleukin-4 had an enhancing effect on the overall numbers of IgE-secreting cells and allergen-specific T cells and a synergistic effect in the coculture of B cells and an autoreactive T-cell line.
  
  
  CONCLUSIONS
  These results suggest that cognate interaction T and B cells is required for the de novo induction of IgE responses. This in vitro system could thus provide a useful model for analysis of the molecular and cellular mechanisms that regulate the allergen-specific IgE responses in human beings.
authors.affiliation: Johns Hopkins Asthma and Allergy Center, Johns Hopkins University School of Medicine, Baltimore, Md. 21224.
authors.forename: N
authors.initials: N
authors.lastname: Shinomiya
authors.forename: M
authors.initials: M
authors.lastname: Kumai
authors.forename: D G
authors.initials: DG
authors.lastname: Marsh
authors.forename: S K
authors.initials: SK
authors.lastname: Huang
mesh_terms: D001402:B-Lymphocytes / Q000276:immunology*; D001483:Base Sequence; D002478:Cells, Cultured; D007124:Immunoenzyme Techniques; D007073:Immunoglobulin E / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012313:RNA / Q000302:isolation & purification; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D007073:Immunoglobulin E; D012313:RNA