Detection of specific IgE-secreting cells with an enzyme-linked immunospot assay

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 (T_H2) 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.