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Monoclonal antibody‐based ELISA to quantify the major allergen of Artemisia vulgaris pollen, Art v 1
Author(s) -
Jimeno L.,
Duffort O.,
Serrano C.,
Barber D.,
Polo F.
Publication year - 2004
Publication title -
allergy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.363
H-Index - 173
eISSN - 1398-9995
pISSN - 0105-4538
DOI - 10.1111/j.1398-9995.2004.00464.x
Subject(s) - mugwort , allergen , affinity chromatography , monoclonal antibody , artemisia , radioallergosorbent test , immunoglobulin e , chemistry , antibody , chromatography , allergy , medicine , immunology , biochemistry , traditional medicine , enzyme , alternative medicine , pathology
Background: Pollen of Artemisia vulgaris (mugwort) is a relevant cause of pollinosis in temperate and humid regions. Recently, the major allergen of this pollen, Art v 1, has been characterized. Objective: To develop a monoclonal antibody (mAb)‐based enzyme‐linked immunosorbent assay (ELISA) to quantify Art v 1, and to assess the correlation of Art v 1 content with the biological activity of mugwort pollen extracts. Methods: Art v 1‐specific mAbs were obtained from a BALB/c mouse immunized with high‐performance liquid chromatography (HPLC)‐purified Art v 1. One of these antibodies (Av 3.7), which recognizes the N‐terminal defensin‐like domain of Art v 1, was used as the capture antibody in an ELISA method for allergen quantitation. An anti‐ A. vulgaris rabbit serum was used as the second antibody. Art v 1 was purified by immunoaffinity chromatography and used as the standard in the assay. Results: The purity and identity of the affinity‐purified Art v 1 was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), mass spectrometry, amino acid composition, and N‐terminal amino acid sequencing. The prevalence of specific IgE against Art v 1, determined by radioallergosorbent test (RAST) in a population of 44 mugwort‐allergic patients, was 79%. The Art v 1‐ELISA developed displays a detection limit of 0.1 ng/ml, and a practical working range of 0.2–10 ng/ml. The concentration of Art v 1 was measured in 10 A. vulgaris pollen extracts, and a good correlation was observed between the Art v 1 content and the allergenic activity of the extracts. Conclusions: The results prove the usefulness of the Art v 1‐ELISA for the standardization of A. vulgaris pollen extracts intended for clinical use.