Replacement of murine sera by allergen-specific monoclonal IgE antibodies: a new approach for the characterisation of allergen extracts.

Allergen characterisation that is based on patients' sera or monoclonal allergen-specific IgG antibodies has several disadvantages. Current methods such as immunoblotting or allergen-specific EUSA are non-functional assays and cannot be used to evaluate the biological allergenic activity of allergen products. We have established an in vitro assay based on polyclonal murine IgE and allergen-dependent mediator release of rat basophilic leukaemia (RBL) cells as an alternative to passive cutaneous anaphylaxis (PCA), an animal model of IgE-mediated allergic reactions. The RBL assay is a functional in vitro test which enables the measurement of biological potency of allergen extracts to be made. Up to now, allergen-specific IgE-containing murine sera were used for sensitisation of RBL cells. Sensitisation with allergen-specific IgE monoclonal antibodies (IgE mAbs) would reduce the number of animals necessary for the production of allergen-specific IgE. In addition, IgE mAbs are better defined and will offer more exact determination of allergens. Since allergen-specific IgE mAbs were not available, the aim of this study was to produce such antibodies. As a new strategy to select IgE-producing hybridomas the RBL mediator release assay was used: the cells were incubated with hybridoma supernatant and stimulated with allergen and crosslinking allergen-specific polyclonal IgG antibodies. By this technology IgE mAbs specific for the birch pollen allergens Bet v 1 and Bet v 6 were produced. In conclusion, this novel strategy enables the production of panels of allergen-specific IgE mAbs by immunisation of a limited number of mice to be made. These IgE mAbs in combination with the RBL mediator release assay may serve as new tools for the evaluation of diagnostic and therapeutic allergen extracts.