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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

Authors


Florentino Polo, Departamento de Investigación y Desarrollo
ALK-Abelló
C/Miguel Fleta, 19
E-28037 Madrid
Spain

Abstract

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.

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