Human monoclonal antibody-based quantification of group 2 grass pollen allergens.
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.
Department of Pathophysiology, Vienna General Hospital, Medical University of Vienna, Austria.
Katharina
K
Marth
Margarete
M
Focke
Sabine
S
Flicker
Rudolf
R
Valenta
D000485:Allergens / Q000032:analysis* / Q000145:classification; D000911:Antibodies, Monoclonal; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000706:statistics & numerical data; D006801:Humans; D006109:Poaceae / Q000276:immunology*; D011058:Pollen / Q000276:immunology*; D012680:Sensitivity and Specificity; D013045:Species Specificity
D000485:Allergens; D000911:Antibodies, Monoclonal