Monoclonal antibody-based ELISA to quantify the major allergen of Artemisia vulgaris pollen, Art v 1.
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.
Research and Development Department, ALK-Abelló, Madrid, Spain.
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Jimeno
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Duffort
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Serrano
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Barber
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Polo
D000485:Allergens / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D052179:Antigens, Plant; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D010940:Plant Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D011058:Pollen / Q000276:immunology*
D000485:Allergens; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; C102724:Art v 1 protein, mugwort; D010940:Plant Proteins