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Published on: May 4, 2012
Human monoclonal antibody-based quantification of group 2 grass pollen allergens
Katharina Marth1, Margarete Focke, Sabine Flicker
1Department of Pathophysiology, Vienna General Hospital, Medical University of Vienna, Austria.
Background:
Grasses belong to the most potent allergen sources worldwide. Group 2 grass pollen allergens are recognized by more than 100 million allergic patients.
Objective:
The aim was to develop an assay for the specific detection and quantification of group 2 grass pollen allergens.
Methods:
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 rPh1 p 2 to develop a competitive ELISA for the specific measurement of group 2 allergens. ELISA plate-bound purified recombinant human Ph1 p 2-specific IgG1 is incubated with a constant amount of biotinylated rPh1 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 rPh1 p 2, which can be detected with peroxidase-labeled streptavidin.
Results:
The competition-ELISA measured rPh1 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.
Conclusions:
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.
