Monoclonal immunoassay for major dust mite ( Dermatophageides) allergens, Derp I and Derf I, and quantitative analysis of the allergen content of mite and house dust extracts

Abstract

Monoclonal, two-site radioimmunoassays (RIAs) were developed to measure allergen Der p I of Dermatophagoides pteronyssinus or Der f I of D. farinae. Microtiter plates coated with monoclonal antibody (Mab) were incubated with mite extract, and bound allergen was detected with a second 125I-labeled Mab of different epitope specificity. The Mab RIAs were very sensitive (nanogram range) and highly specific. D. pteronyssinus extracts with different concentrations of Der p I demonstrated parallel binding curves, whereas a potent D. farinae extract demonstrated <5% of the Der p I binding in the same assay. Similar parallel curves were obtained with several D. farinae extracts in the Der f I assay, whereas D. pteronyssinus extract demonstrated little or no binding. The Mab RIAs were compared with an inhibition RIA that measured cross-reacting determinants on both Der p I and Der f I (antigen P 1 equivalent [AgP 1Eq].) The results demonstrated good quantitative agreement between these assays in commercial mite and house dust extracts (mean difference 1.57 ± 0.5-fold). Thirty house dust samples with known mite counts, Der p I, and AgP 1Eq content were also compared. The summed Mab RIA values for Der p I and Der f I demonstrated a very good correlation with AgP 1Eq values (r = 0.86; p < 0.001) and with assessments of total mite-allergen content by RAST inhibition (n = 21, r = 0.77; p < 0.001). Furthermore, in samples with more than 10 mites per 100 mg of dust, the Der p I: Der f I ratio closely correlated with the ratio of the two mites counted by microscopy ( n = 15, r = 0.89; p < 0.001). The Mab RIAs can measure allergen levels in mite or dust extracts without the need for purified allergen or affinity-purified antibodies and can readily be standardized. These assays will be useful in epidemiologic studies of allergic asthma, to assess patients' exposure to mite allergens, and the effects of avoidance regimens. Because of the long-term stability and reproducibility of the reagents, Mab-based assays for specific allergens will also play an important role in the standardization of mite and other allergen extracts.