Cellular localization of water soluble, allergenic proteins in rye-grass (Lolium perenne) pollen using monoclonal and specific IgE antibodies with immunogold probes.

A postembedding method has been developed for localizing water soluble allergens in rye-grass pollen. This uses dry fixation in glutaraldehyde vapour, followed by 2,2-dimethoxypropane, prior to a 100% ethanol series leading into embedment in LR Gold. This has allowed the attachment of specific monoclonal antibodies to the allergen, which are themselves probed with specific immunogold labels to the antibodies. Wall and cytoplasmic sites have been identified, representing an improvement of fixation and localization of allergens over previous studies employing polyclonal, broad spectrum antibodies. Rye-grass allergens are labelled in mature pollen grains in the exine (tectum, nexine and central chamber), and in the electron opaque areas of the cytoplasm, especially mitochondria. The allergens are absent from the intine, polysaccharide (P) particles, amyloplasts, Golgi bodies and endoplasmic reticulum. IgE antibodies derived from humans allergic to rye-grass pollen, bind to similar sites in the cytoplasm but only to the outer surface of the pollen grain wall. This method now provides a valuable tool for further developmental studies on the pollen grains, in order to establish the site/s of synthesis of the allergens.

Localization of the two major allergens in rye-grass pollen using specific monoclonal antibodies and quantitative analysis of immunogold labelling.

The intracellular localization of the two major allergens, Lol p I and Lol p IX, in rye-grass anthers was examined using monoclonal antibodies FMCA1 (specific for Lol p I) and FMCA7 (specific for Lol p IX) with immunocytochemical techniques and quantitative analysis. A newly developed anhydrous fixation technique in a mixture of glutaraldehyde, paraformaldehyde and 2,2-dimethoxypropane followed by embedding in LR Gold resin resulted in both improved infiltration of pollen grains compared with existing techniques and the localization of these water-soluble antigens in their original sites compared with diffusion artefacts following aqueous methods. After anhydrous fixation, Lol p I was predominantly located in the electron-opaque regions of the cytosol of the vegetative cell of the tricellular pollen grains (24 counts microns-2), whereas Lol p IX was detected mainly within starch granules (16 counts microns-2). For both Lol p I and Lol p IX, similar labelling was detected in the cells of the endothecium and middle layer (18 counts microns-2), but none was found in the tapetal cells or orbicules.

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.

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.