Development of a Sandwich-Type ELISA for Measuring Pla a 1, the Major Allergen of Platanus acerifolia Pollen

Abstract

Background:Platanus acerifolia is an important cause of pollinosis in Western European cities. Pla a 1, a nonglycosylated 18-kDa protein with a prevalence of 80%, is a major allergen in P. acerifolia pollen extracts. Our aim was to develop a Pla a 1-specific ELISA to quantify this protein in allergenic extracts and preparations for clinical use. Methods: Pla a 1 was purified by cation exchange at pH 7.0, gel filtration, and anion exchange chromatography at pH 10.0. Monoclonal (mAb) and polyclonal antibodies were obtained by immunizing mice and rabbits with nPla a 1. One (5C1) of the 13 mAb obtained was used as capture antibody at 5 µg/ml and biotin-labeled specific polyclonal antiserum at 0.63 µg/ml served for detection. Results: The prevalence of Pla a 1-specific IgE to purified Pla a 1 among 47 P. acerifolia-allergic patients was 79%. The Pla a 1-ELISA developed has a linear range of 3–25 ng/ml, high sensitivity with a detection limit of 0.5 ng/ml and is highly specific as none of the 24 pollen, mite, mold, and plant food extracts tested gave positive results. The assay could quantify Pla a 1-like proteins in other planetree pollen extracts. A good correlation was obtained between Pla a 1 content of 11 P. acerifolia pollen extracts (average content 0.69% of the total protein) and their IgE-binding activity. Conclusions: The described two-site sandwich ELISA to measure Pla a 1 is useful for standardization of planetree pollen extracts intended for clinical use.