Development of Allergen ELISA Kits for Dust Mites, Pollen, and Pet Dander

<Background>Allergic diseases such as asthma, atopic dermatitis and allergic rhinitis have remarkably increased, and they are caused mainly by allergens such as dust mites, pollen and pet dander. For the measures to prevent and control allergic diseases, it is important to measure allergens in the environment.

<Objective>We established a highly sensitive enzyme-linked immunosorbent assay (ELISA) to quantify specifically Dermatophagoides mite allergens (Der p 1, Der f 1 and Der 2), Japanese cedar pollen allergens (Cry j 1 and Cry j 2) and cat allergen (Fel d 1).

<Methods>Allergen samples were incubated in the wells coated with a primary antibody for 2 hours at 37^oC. After washing, a biotinylated secondary antibody against the allergen was incubated for 1 hour at 37^oC. The allergens were detected using HRP-conjugated streptavidin and its substrate at 450 nm.

<Result>The working range was 500 to 32,000 pg/mL for Der p 1 and Der f 1, 100 to 6,400 pg/mL for Der 2, 250 to 16,000 pg/mL for Cry j 1, 2,500 to 160,000 pg/mL for Cry j 2, and 100 to 6,400 pg/mL for Fel d 1. In all systems, the intra- and inter-assay coefficient of variation was less than 5%. Furthermore, each assay showed no significant cross-reactivity with other allergens.

<Conclusion>Our established ELISA systems for these allergens are useful for not only the qualification of allergens in the environment but also the quality control of allergenic products for sublingual immunotherapy and the evaluation of the allergen inactivation effect in industrial products