title: Development and in-house validation of an allergen-specific ELISA for quantification of Bet v 4 in diagnostic and therapeutic birch allergen products.
abstract: Birch (Betula) pollen is a major cause of allergy in northern and central Europe. The allergenic potency of products for diagnosis and therapy of birch pollen allergy is adjusted nearly exclusively to the major birch pollen allergen Bet v 1. Although every fifth patient is additionally sensitized to Bet v 4, both content and variability of this minor allergen in birch allergen products remain unclear due to a lack of simple and cost-effective quantitative methods. This study aimed to develop and in-house validate the first Bet v 4-specific sandwich enzyme-linked immunosorbent assay (ELISA). Based on a murine monoclonal antibody in combination with a polyclonal rabbit antiserum, the ELISA proved to be highly sensitive, with a lower limit of quantification of 30 pg/ml Bet v 4. After confirmation of satisfactory accuracy, reproducibility, and robustness, the ELISA was utilized to quantify Bet v 4 in 30 authorized birch allergen products. The allergen was detected in all samples tested, ranging from 0.2 to 4.4 μg/ml. No significant correlation of Bet v 4 was found with the respective amount of Bet v 1. In contrast to Bet v 1, also no correlation of Bet v 4 with total protein content or total allergenic activity could be observed. Thus, it seems presently unfeasible to base birch allergen product standardization additionally on Bet v 4. In light of these results, the continuous monitoring of Bet v 4 in birch allergen products with the presented ELISA will provide a basis for the understanding of the clinical relevance of minor allergens.
authors.affiliation: Division of Allergology, Paul-Ehrlich-Institut, Paul-Ehrlich-Straße 51-59, 63225, Langen, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Dehus
authors.forename: Julia
authors.initials: J
authors.lastname: Zimmer
authors.forename: Sascha
authors.initials: S
authors.lastname: Döring
authors.forename: Frank
authors.initials: F
authors.lastname: Führer
authors.forename: Kay-Martin
authors.initials: KM
authors.lastname: Hanschmann
authors.forename: Thomas
authors.initials: T
authors.lastname: Holzhauser
authors.forename: Florian
authors.initials: F
authors.lastname: Neske
authors.forename: Daniel
authors.initials: D
authors.lastname: Strecker
authors.forename: Jan-Hendrik
authors.initials: JH
authors.lastname: Trösemeier
authors.forename: Stefan
authors.initials: S
authors.lastname: Vieths
authors.forename: Susanne
authors.initials: S
authors.lastname: Kaul
mesh_terms: D000818:Animals; D052179:Antigens, Plant / Q000032:analysis*; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D010940:Plant Proteins / Q000032:analysis*; D011058:Pollen / Q000737:chemistry*; D015203:Reproducibility of Results
chemical_list: D052179:Antigens, Plant; C108867:BETV4 protein, Betula pendula; D002135:Calcium-Binding Proteins; D010940:Plant Proteins