Detection of B-lymphocytes secreting antibodies to Dermatophagoides antigens

An enzyme-linked immunospot assay (ELI-spot assay) has been established to count individual cells secreting antibodies to Dermatophagoides spp. allergens. Initial optimization of the assay was performed using Der p I-specific murine hybridoma cell lines. Inhibition with soluble purified allergen demonstrated that spot formation was specific. Addition of cycloheximide showed that spot formation was due to antibodies actively produced and secreted and not caused by preformed surface-associated Ig. An analogous method was used to count immunoglobulin-secreting cells (IgSC) of any specificity, and the number of circulating IgSC was measured in young healthy subjects. The results (means) were 331 IgM-SC, 532 IgG-SC, 850 IgA-SC and less than 1 IgE-SC per 10(6) mononuclear cells (MNC). Initial experiments performed with blood from patients undergoing immunotherapy with Dermatophagoides pteronyssinus Alutard, showed that 1 week after injection, circulating allergen-specific antibody-secreting cells (AbSC) were detected with this assay. The ranges were between 1 and 13 IgM-, 2 and 20 IgG- and 20 and 55 IgA allergen-specific AbSC per 10(6) MNC. It is expected that this assay will help to understand more thoroughly the basic immunological mechanisms behind the efficacy of immunotherapy.