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.
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.
Calabozo
B
B
Research and Development Department, ALK-Abelló, Madrid, Spain.
Duffort
O
O
Carpizo
J A
JA
Barber
D
D
Polo
F
F
D000485:Allergens / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002846:Chromatography, Affinity / Q000379:methods* / Q000592:standards; D003429:Cross Reactions; D019662:Cucurbitaceae; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D006801:Humans; D006967:Hypersensitivity / Q000175:diagnosis* / Q000453:epidemiology / Q000209:etiology*; D015151:Immunoblotting; D051379:Mice; D008807:Mice, Inbred BALB C; D010943:Plantago; D010946:Plants, Medicinal; D011058:Pollen / Q000276:immunology*; D012680:Sensitivity and Specificity
D000485:Allergens; D000911:Antibodies, Monoclonal