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Monoclonal antibodies against the major allergen of Plantago lanceolata pollen, Pla l 1: affinity chromatography purification of the allergen and development of an ELISA method for Pla l 1 measurement
Author(s) -
Calabozo B.,
Duffort O.,
Carpizo J. A.,
Barber D.,
Polo F.
Publication year - 2001
Publication title -
allergy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.363
H-Index - 173
eISSN - 1398-9995
pISSN - 0105-4538
DOI - 10.1034/j.1398-9995.2001.056005429.x
Subject(s) - allergen , monoclonal antibody , pollen , immunoglobulin e , affinity chromatography , antibody , plantago , airborne allergen , microbiology and biotechnology , chromatography , immunology , chemistry , biology , allergy , biochemistry , botany , enzyme
Background: Plantago lanceolata (English plantain) pollen is a relevant cause of pollinosis in temperate regions. The major allergen of this pollen, Pla l 1, is recognized by the specific IgE from more than 80% of plantain‐sensitive patients. It displays significant sequence homology with the major olive‐pollen allergen Ole e 1. The objective was to develop a monoclonal antibody‐based ELISA to quantify Pla l 1, and to assess the correlation of Pla l 1 content with the biologic activity of plantain pollen extracts. We also aimed to establish the specificity of the monoclonal antibodies against the potentially cross‐reactive allergen Ole e 1, and to investigate the presence of Pla l 1‐like proteins in psyllium and melon that have been reported to cross‐react with P. lanceolata pollen. Methods: After fusion of myeloma cells with spleen cells from a BALB/c mouse, two Pla l 1‐specific monoclonal antibodies secreting hybridomas were selected, and the antibodies characterized. One of them (2A10) was used as the capture antibody in an ELISA for Pla l 1 quantitation. An anti‐ P. lanceolata rabbit serum was used as the second antibody. Pla l 1 was purified by immunoaffinity chromatography and used as the standard in the assay. Results: The ELISA developed was highly reproducible and sensitive, with a detection limit of 0.1 ng/ml, and a practical working range of 0.4–12 ng/ml. The specificity was demonstrated against a large battery of allergens, including Ole e 1. The concentration of Pla l 1 was measured in 19 extracts of P. lanceolata pollen, and a good correlation was observed between the Pla l 1 content and the allergenic activity of the extracts. Pla l 1 was not detected in psyllium or melon extracts. Conclusions: The results prove the usefulness of the Pla l 1‐ELISA for the standardization of extracts of P. lanceolata pollen intended for clinical use.