The scarcity of monoclonal human
IgE antibodies with specificity for defined
allergens is a bottleneck for the molecular characterisation of
allergens and their
epitopes. Insights into the characteristics of such
antibodies may allow for analyses of the molecular basis underlying allergenicity and cross-reactivity, standardisation of
allergens as well as improvement of
allergy diagnostics and
therapeutics. Here we report the generation and application of the first set of authentic human
IgG,
IgE and
IgA antibodies. On the basis of a Phl p 5a specific
antibody fragment, a lambda light chain and the
IgG1,
IgG4,
IgE,
IgA1, and
IgA2 heavy chains, the corresponding human
immunoglobulins were constructed and produced in mammalian cells. In parallel, a murine hybridoma line with specificity for Phl p 5a was established, recloned and produced as human chimeric
IgE. After purification, immunoreactivity of the
antibodies with the
allergen was assessed. Applicability in
allergy diagnostics was confirmed by establishment of artificial human sera. Functionality of both
antibodies was further demonstrated in receptor binding studies and mediator release assays using humanised rat basophil leukaemia cells (RBL-SX38) suggesting the presence of spatially separate
epitopes. By using Phl p 5 fusion
proteins and recombinant
IgE in immunoblotting and mediator release assays we assigned the
epitope of the authentic
IgE to a looped stretch exclusively present in Phl p 5a. In summary, the Phl p 5-specific
antibodies are the first full set of
allergy-related antibody isotypes of their kind and represent valuable tools for studies of fundamental mechanisms and structure/function relationships in
allergy.