Molecular basis of IgE-recognition of Lol p 5, a major allergen of rye-grass pollen.

Grass pollen, especially of rye-grass (Lolium perenne). represents an important cause of type I allergy. Identification of IgE-binding (allergenic) epitopes of major grass pollen allergens is essential for understanding the molecular basis of interaction between allergens and human IgE antibodies and therefore facilitates the devising of safer and more effective diagnostic and immunotherapy reagents. The aim of this study was to identify the allergenic epitopes of Lol p 5, a major allergen of rye-grass pollen, immunodissect these epitopes further so that the amino acid residues critical for antibody binding can be determined and investigate the conservation and nature of these epitopes within the context of the natural grass pollen allergens. Peptides, 12-13 amino acid residues long and overlapping each other by 4 amino acid residues, based on the entire deduced amino acid sequence of the coding region of Lol p 5, were synthesised and assayed for IgE-binding. Two strong IgE-binding epitopes (Lol p 5 (49-60) and (265-276), referred to as peptides 7 and 34, respectively) were identified. These epitopes were further resolved by truncated peptides and amino acid replacement studies and the amino acid residues critical for IgE-binding determined (Lol p 5 (49-60) residue Lys57 and (265-276) residue Lys275). Sequences of these epitopes were conserved in related allergens and may form the conserved allergenic domains responsible for the cross-reactivity observed between pollen allergens of taxonomically related grasses. Furthermore, due to its strong IgE-reactivity, synthetic peptide Lol p 5 (265-276) was used to affinity-purify specific IgE antibodies which recognised proteins of other clinically important grass pollens. further indicating presence of allergenic cross-reactivity at the level of allergenic epitope. Moreover, Lol p 5 (265 276) demonstrated a strong capacity to inhibit IgE-binding to natural rye-grass pollen proteins highlighting the antibody accessibility to these sequences within the context of the natural allergens. Strong IgE-binding epitopes of Lol p 5 have been identified down to single critical amino acid residues and are shown to occur as linear or continuous domains in the natural conformation of natural Lol p 5 and other group 5 grass pollen allergens. The fact that such an allergenic synthetic epitope has the capacity to strongly inhibit IgE-binding to natural allergens highlight its potential for use as a candidate in future therapeutics to treat pollen-associated allergies.

A novel grass pollen allergen mimotope identified by phage display peptide library inhibits allergen-human IgE antibody interaction.

The aim of this study was to investigate the molecular basis of human IgE-allergen interaction by screening a phage-displayed peptide library with an allergen-specific human IgE-mimicking monoclonal antibody (mAb). A mAb that reacted with major grass pollen allergens was successfully identified and shown to inhibit human IgE-allergen interaction. Biopanning of a phage-displayed random peptide library with this mAb yielded a 12 amino acid long mimotope. A synthetic peptide based on this 12-mer mimotope inhibited mAb and human IgE binding to grass pollen extracts. Our results indicate that such synthetic peptide mimotopes of allergens have potential as novel therapeutic agents.

Molecular cloning, expression and immunological characterisation of Lol p 5C, a novel allergen isoform of rye grass pollen demonstrating high IgE reactivity.

A novel isoform of a major rye grass pollen allergen Lol p 5 was isolated from a cDNA expression library. The new isoform, Lol p 5C, shares 95% amino acid sequence identity with Lol p 5A. Both isoforms demonstrated shared antigenic activity but different allergenic activities. Recombinant Lol p 5C demonstrated 100% IgE reactivity in 22 rye grass pollen sensitive patients. In comparison, recombinant Lol p 5A showed IgE reactivity in less than 64% of the patients. Therefore, Lol p 5C represents a novel and highly IgE-reactive isoform allergen of rye grass pollen.

Comparison of commercially available ELISA kits with human sera-based detection methods for peanut allergens in foods.

Undeclared peanut allergens as contaminants in foodstuffs represent a major health problem for sensitized persons. Various immunochemical techniques are employed to detect and quantify peanut allergens. There is an urgent need to compare and standardize those test systems to enable comparable allergen analyses of foodstuffs, comparable studies, and consequent and consistent measures against the presence of hidden peanut allergens. The present study compared commercially available peanut ELISA kits with human sera-based immunoassay techniques (dot blotting and Western blotting), enabling semiquantitative and quantitative detection, and identification of peanut contaminants in foodstuffs. Additionally, the effect of conventional roasting conditions on the detection and quantification of peanut with the selected methods was investigated.

Direct determination of allergens in ambient aerosols: methodological aspects.

In Switzerland the birch tree (Betula verrucosa) major allergen Bet v 1 and the grass (Phleum pratense) pollen major allergen Phl p 5 are of particular relevance for inducing pollinosis. In this study, the protein and major allergen contents of aerosols of different aerodynamic diameters were determined. The aerosols were sampled by Andersen-Impactors and submitted to protein assays and allergen assays (ELISA) specific for Bet v 1 and Phl p 5. The total protein, Bet v 1 and Phl p 5 concentrations were correlated with the corresponding pollen counts. The presence of Bet v 1 in smaller aerosol fractions was demonstrated before and after birch pollen was counted, especially in the lower particle size ranges.

Concentrations of the major birch tree allergen Bet v 1 in pollen and respirable fine particles in the atmosphere.

BACKGROUND: Birch tree pollen allergens are an important cause of early spring hay fever and allergic asthma. Pollen counts provide a guide for individuals with birch pollen allergy. However, birch pollen, because of its size, has a low probability of entering the lower airways to trigger asthma. Yet birch pollen allergens are known to be associated with respirable particles present in the atmosphere. OBJECTIVE: We sought to determine the concentration of major allergen Bet v 1 in birch pollen and respirable particles in the atmosphere during the birch pollen season. METHODS: We used a two-site monoclonal antibody-based assay (ELISA) to quantitate Bet v 1 in pollen extracts and high-volume air sampler filters collecting particles larger and smaller than 7.2 microm. RESULTS: Bet v 1 (0.006 ng) is detectable per birch pollen grain, of which 0.004 ng is present in aqueous extracts (13.9% of soluble proteins). Atmospheric Bet v 1 concentrations are correlated with birch pollen counts. Heavy rainfall tended to wash out pollen and particles, indicated by a mean daily Bet v 1 concentration of 0.12 ng/m3 (20 pollen equivalents), but light rainfall produced a dramatic increase in allergen-loaded respirable particles with Bet v 1 concentrations of 1.2 ng/m3 (200 pollen equivalents). CONCLUSION: These results highlight the different environmental risk factors for hay fever and allergic asthma in patients sensitized to Bet v 1. Light rainfall causes an increase in respirable particles; hence, this is an important risk factor for asthma.

Immunologic significance of respirable atmospheric starch granules containing major birch allergen Bet v 1.

BACKGROUND: Birch-pollen allergens are an important cause of early spring hay fever and allergic asthma. Recently, we reported a mechanism for the release of respirable allergenic particles from birch pollen containing the major allergen Bet v 1. In this study, we aimed to assess the immunologic significance of the released Bet v 1-containing starch granules in the environment. METHODS: A two-site monoclonal antibody-based assay (ELISA) was employed to quantitate Bet v 1 in high-volume air sampler filter extracts, and immunogold-labelling was used on sections of these extracts to localize Bet v 1. Immunoblot analyses were performed with pooled sera from patients sensitive to birch pollen. RESULTS: Atmospheric starch granules contained Bet v 1, and the concentration increased upon light rainfall. Sera from patients allergic to birch allergens recognized extracts from isolated starch granules. CONCLUSIONS: The clinical implications of these findings are that starch granules released from birch pollen are potentially able to trigger allergic asthmatic reactions to Bet v 1, since the allergen occurs in respirable particles. Thus, clinicians can advise asthma patients to remain indoors on days of light rainfall during the birch-pollen season to avoid high levels of allergen exposure.

Hidden peanut allergens detected in various foods: findings and legal measures.

BACKGROUND: Undeclared allergens in foodstuffs represent a major health problem for sensitized persons. Until recently, most food control authorities were not in the position to monitor hidden allergens and to take legal measures against their presence in foodstuffs. METHODS: In this study, we employed human sera-based immunoassay techniques, enabling semiquantitative detection and identification of peanut allergens in a variety of foodstuffs. RESULTS: This study showed the presence of undeclared allergens in products belonging to various food categories, such as cereals, cookies, cakes, and snacks. The detection limit for peanut contamination was in most instances less than 50 mg peanut material per kg, i.e., less than about 5 mg peanut allergens per kg. We legally objected to products with more than one part per thousand or 1000 mg/kg of peanut contamination. CONCLUSIONS: In most cases, food producers, confronted with our results, were able to detect and eliminate the sources of the contamination. They implemented measures to prevent the presence of hidden peanut allergens in their products, increasing food safety for sensitized persons and overall food quality.

Concentrations of major grass group 5 allergens in pollen grains and atmospheric particles: implications for hay fever and allergic asthma sufferers sensitized to grass pollen allergens.

BACKGROUND: Grass pollen allergens are the most important cause of hay fever and allergic asthma during summer in cool temperate climates. Pollen counts provide a guide to hay fever sufferers. However, grass pollen, because of its size, has a low probability of entering the lower airways to trigger asthma. Yet, grass pollen allergens are known to be associated with atmospheric respirable particles. OBJECTIVE: We aimed (1) to determine the concentration of group 5 major allergens in (a) pollen grains of clinically important grass species and (b) atmospheric particles (respirable and nonrespirable) and (2) to compare the atmospheric allergen load with clinical data to assess different risk factors for asthma and hay fever. METHODS: We have performed a continuous 24 h sampling of atmospheric particles greater and lower than 7.2 microm in diameter during the grass pollen season of 1996 and 1997 (17 October 1996-16 January 1997) by means of a high volume cascade impactor at a height of about 15 m above ground in Melbourne. Using Western analysis, we assessed the reactivity of major timothy grass allergen Phl p 5 specific monoclonal antibody (MoAb) against selected pollen extracts. A MoAb-based ELISA was then employed to quantify Phl p 5 and cross-reactive allergens in pollen extracts and atmospheric particles larger and smaller than 7.2 microm. RESULTS: Phl p 5-specific MoAb detected group 5 allergens in tested grass pollen extracts, indicating that the ELISA employed here determines total group 5 allergen concentrations. On average, 0.05 ng of group 5 allergens were detectable per grass pollen grain. Atmospheric group 5 allergen concentrations in particles > 7.2 microm were significantly correlated with grass pollen counts (rs = 0.842, P < 0. 001). On dry days, 37% of the total group 5 allergen load, whereas upon rainfall, 57% of the total load was detected in respirable particles. After rainfall, the number of starch granule equivalents increased up to 10-fold; starch granule equivalent is defined as a hypothetical potential number of airborne starch granules based on known pollen count data. This indicates that rainfall tended to wash out large particles and contributed to an increase in respirable particles containing group 5 allergens by bursting of pollen grains. Four day running means of group 5 allergens in respirable particles and of asthma attendances (delayed by 2 days) were shown to be significantly correlated (P < 0.001). CONCLUSION: Here we present, for the first time, an estimation of the total group 5 allergen content in respirable and nonrespirable particles in the atmosphere of Melbourne. These results highlight the different environmental risk factors for hay fever and allergic asthma in patients, as on days of rainfall following high grass pollen count, the risk for asthma sufferers is far greater than on days of high pollen count with no associated rainfall. Moreover, rainfall may also contribute to the release of allergens from fungal spores and, along with the release of free allergen molecules from pollen grains, may be able to interact with other particles such as pollutants (i.e. diesel exhaust carbon particles) to trigger allergic asthma.