Pla 1 1 and Ole e 1 pollen allergens share common epitopes and similar ultrastructural localization.

BACKGROUND: English plantain (Plantago lanceolata L.) and olive (Olea europaea L.) pollens are important causes of pollinosis in large areas of North America, Australia, and the Mediterranean basin. The major pollen allergens of both plants, Pla I 1 and Ole e 1, share 38.7% of their amino acid sequences. OBJECTIVE: To analyze putative cross-reactivity between these 2 proteins. METHODS: Several antibodies and patients' sera were used in immunoblot and immunocytochemistry experiments. RESULTS: Two anti-Pla I 1 antibodies were able to bind to 3 polypeptides from olive pollen protein extracts, which correspond to the 3 glycosylation isoforms of Ole e 1 (18-22 kDa) previously described. Moreover, Pla I 1 protein was found in the cytoplasm of both the vegetative and the generative cells of P lanceolata mature pollen. On olive pollen sections, these anti-Pla I 1 antibodies displayed significant labeling in the cytoplasm of the vegetative cell and in both the exine and the material adhering to this outer layer of the pollen wall. In addition, the anti-Ole e 1 antibody 10H1 was found to cross-react with proteins of similar masses (16-20 kDa) to Pla I 1 variants. In Plantago pollen sections, the 10H1 antibody recognized proteins located in the cytoplasm of both the vegetative and generative cells. Cross-reaction was confirmed using sera from patients allergic to either plant pollen. CONCLUSION: Both allergens share common epitopes, which can be cross-recognized by different antibodies and sera from different patients, although this antigenic similarity seems to have little clinical relevance.

Monoclonal antibody-based ELISA to quantify the major allergen of Cynodon dactylon (Bermuda grass) pollen, Cyn d 1.

BACKGROUND: Pollen of Bermuda grass (Cynodon dactylon) is an important cause of pollinosis in many areas of the world. Most patients show sensitivity to the major allergen Cyn d 1, a glycoprotein composed of a number of isoforms with a molecular mass of 31-32 kDa. The aim of this work was to develop a monoclonal antibody (mAb)-based ELISA to quantify Cyn d 1, and to assess the correlation of the allergen content with the biological activity of C. dactylon pollen extracts. METHODS: After fusion of myeloma cells with spleen cells from a BALB/c mouse immunized with C. dactylon pollen extract, Cyn d 1-specific mAbs secreting hybridomas were selected, and the antibodies characterized. One of them (4.4.1) was used as the capture antibody in an ELISA method for Cyn d 1 quantitation. An anti-Cyn d 1 rabbit serum was used as the second antibody. Cyn d 1 was purified by immunoaffinity chromatography with mAb 4.4.1, characterized, and used as the standard in the assay. RESULTS: The identity, purity and isoallergen composition of affinity-purified Cyn d 1 was confirmed by N-terminal amino acid sequencing, SDS-PAGE, Western blot and 2D electrophoresis. The Cyn d 1 ELISA is highly specific and sensitive, with a detection limit of 0.24 ng/ml and a linear range of 1.1-9.2 ng/ml. An excellent correlation was found when the content of Cyn d 1, measured in 16 different extracts, was compared with the allergenic activity of the same extracts determined by RAST inhibition. CONCLUSIONS: The results prove the usefulness of the Cyn d 1 ELISA for the standardization of C. dactylon-allergen products on the basis of major allergen content.

Studies on the carbohydrate moiety of Pla l 1 allergen. identification of a major N-glycan and significance for the immunoglobulin E-binding activity.

BACKGROUND: Pla l 1, the major allergen of Plantago lanceolata pollen, is a glycoprotein that contains an N-glycosylation site. Carbohydrate moieties of many allergenic glycoproteins have been reported to be IgE-binding determinants responsible for cross-reactivity among different species. OBJECTIVE: To identify the kind of linkages and the type of glycans present in Pla l 1 and to investigate their contribution to the allergic response to this allergen. METHODS: Pla l 1 was deglycosylated by N-glycosidase A and the IgE-binding ability of the unglycosylated protein was evaluated by dot-blot. Identification of beta1 --> 2 xylose and/or alpha1 --> 3 fucose residues in Pla l 1 N-glycan was carried out by incubation with specific antibodies from rabbit antiserum against HRP (anti-HRP). The contribution of this N-glycan to total IgE reactivity was analysed quantitatively by pre-incubation of Pla l 1 with anti-HRP prior to incubation with sera. The role of the carbohydrate moiety of Pla l 1 in cross-reactivity was studied by RAST using unrelated glycoproteins with known sugar composition and structure. RESULTS: The effectiveness of N-glycosidase A to deglycosylate Pla l 1 and the ineffectiveness of the treatment with PNGase F indicate that Pla l 1 carries a complex N-glycan with an alpha1 --> 3 fucose residue in its structure. Furthermore, the presence of beta1 --> 2 xylose and/or alpha1 --> 3 fucose residues was identified in this N-glycan by means of an ELISA. Pre-incubation of Pla l 1 with an anti-HRP antibody caused a weak but significant reduction in IgE reactivity. Some sera from P. lanceolata-allergic patients reacted positively with four glycoproteins that bear N-glycans of complex type but not with fetuine. CONCLUSIONS: Pla l 1 is a glycoprotein that carries at least a complex, major N-linked glycan, with a alpha1 --> 3 fucose residue in its structure and probably also a beta1 --> 2 xylose. This glycan moiety does not seem to constitute a relevant allergenic epitope of Pla l 1.

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. CONCLUSION: The results prove the usefulness of the Pla l 1-ELISA for the standardization of extracts of P. lanceolata pollen intended for clinical use.

Purification and characterization of the main allergen of Plantago lanceolata pollen, Pla l 1.

English plantain (Plantago lanceolata) pollen is an important cause of pollinosis in the temperate regions of North America, Australia and Europe. However, very little is known about its allergen composition. The aim of this study was to identify plantain allergens, and to isolate and characterize a major allergen. Allergens were identified by immunoblotting with individual allergic patients' sera. Isolation of the major allergen was achieved by sequential reverse-phase and size-exclusion HPLC. Allergenic characterization was performed by ELISA and immunoblotting after SDS-PAGE with sera from plantain-allergic patients. N-terminal amino acid sequence was established by Edman degradation. Allergograms showed that 13 out of the 14 sera assayed had IgE to a group of proteins with a molecular weight in the range of 16-20 kd, that turned out to be different isoforms or variants of the major allergen Pla l l. Eighteen amino acid residues from the N-terminal end of one of the isoforms, and 10 of three others, were sequenced, and a partial sequence identity with Ole e 1 was found. Prevalence of specific IgE to purified Pla l 1 in plantain allergic patients was 86%, and represents about 80% of the total IgE-binding capacity of the plantain extract. The most relevant allergen from P.lanceolata pollen, Pla l 1, has been purified and characterized. This contributes to a greater knowledge of the allergen composition of this important weed, and clears the way for the standardization of plantain allergen products in terms of major allergen content.

Monoclonal antibody-based method to quantify Gly m 1. Its application to assess environmental exposure to soybean dust.

BACKGROUND: The demonstration that some asthma epidemics have been caused by allergens of soybean-hull dust prompted us to develop a two-site ELISA, suitable for the quantification of the major allergen (Gly m 1), to be used for the prevention of new episodes. METHODS: BALB/c mice were injected with Gly m 1 purified from soybean hulls. After fusion and screening, 10 monoclonal antibodies (mAbs) were obtained that were shown to be specific for Gly m 1 Two of them (6G1 as the capture antibody; 1G10 as the tracer) were selected to develop a quantitative two-site ELISA for the indoor and outdoor determination of Gly m 1. RESULTS: The two-site ELISA developed is very sensitive, with a detection limit of less than 0.2 ng/ml and a practical working range of 0.4-10 ng/ml. The assay is also highly reproducible with an intra-assay coefficient of variation of 3.5% and an interassay coefficient of variation of 12.5%. The method was applied to measure the concentration of Gly m 1 in air-sampler filters and in house-dust samples. Our results illustrate that there is a good correlation between the content of Gly m 1 in a number of samples and the allergenic activity as measured by ELISA inhibition. CONCLUSIONS: A specific and sensitive method is presented that can be used for the quantification of Gly m 1. The application of this method may allow the establishment of risk limits for soybean dust, and thus may contribute to the control of environmental contamination and to the prevention of new asthma epidemics.