Grasses belong to the most potent allergen sources worldwide. Group 2 grass pollen allergens are recognized by more than 100 million allergic patients.

The aim was to develop an assay for the specific detection and quantification of group 2 grass pollen allergens.

We have isolated a monoclonal human IgE Fab specific for group 2 grass pollen allergens by combinatorial cloning from lymphocytes of a grass pollen–allergic patient. This Fab was converted into a complete human IgG₁ antibody and used together with rPhl p 2 to develop a competitive ELISA for the specific measurement of group 2 allergens. ELISA plate-bound purified recombinant human Phl p 2–specific IgG₁ is incubated with a constant amount of biotinylated rPhl p 2 competing with increasing concentrations of group 2 allergens to be determined. Defined concentrations of purified rPhl p 2 are used to establish a standard curve. The concentration of unlabeled group 2 allergens can thus be deduced from the displacement of biotinylated rPhl p 2, which can be detected with peroxidase-labeled streptavidin.

The competition-ELISA measured rPhl p 2 concentrations ranging from 10 ng/mL to 500 ng/mL and allowed to quantify group 2 allergens from 9 different grass families. The results were in good agreement with immunoblot data.

The described assay can be used for standardization of diagnostic and therapeutic vaccines as well as for the quantification of group 2 allergens in environmental samples.