Immunoautoradiographic and protein-A/gold labelling experiments for localization of pollen allergens using antisera from atopic human individuals

Fromme, Hans Georg; Grote, Monika; Sinclair, N. J.; Kalveram, K. J.

doi:10.1007/bf00494069

Cited by 8 publications

(7 citation statements)

References 27 publications

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“…However, the fixation procedure was aqueous and movement of water soluble allergens was likely before precipitation, although these workers claim to have prevented diffusion of proteins highly soluble in water, and glycoproteins from pollen grains. Fromme et al (1985) and Grote and Fromme (1986) used human IgE antibodies from strongly birch pollen-allergic patients on similar specimens, to confirm the localization of the allergens in the exine with gold-labelled antibodies. V61ker et al (1986) attempted to localize the hazelnut pollen allergen by predpitation with cuprolinic blue in phosphatebuffered saline, followed by immunogold labelling.…”

Section: Introductionmentioning

confidence: 96%

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

et al. 1990

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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.

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Section: Introductionmentioning

confidence: 96%

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

et al. 1990

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“…Instead of animal antibodies, human IgE antibodies have been used for the detection of allergens in pollen grains [11, 19, 20, 21]. This involves the use of a bridge antibody (anti–human IgE antibody) which is coupled with a third antibody directed against the species in which the bridge antibody was raised.…”

Section: Anhydrous Fixation: a Major Requirement To Trace Pollen Allementioning

confidence: 99%

In situ Localization of Pollen Allergens by Immunogold Electron Microscopy: Allergens at Unexpected Sites

Grote¹

1999

Int Arch Allergy Immunol

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The windborne pollen grains of many trees and grasses contain a number of highly water–soluble (glyco)proteins which upon moistening, e.g. on the human respiratory mucosa, rapidly diffuse out of the pollen grain. In susceptible individuals, they may cause allergic reactions. Because of their rapid release from the pollen, pollen allergens were expected to be located in the outermost layers of the pollen grain, i.e. on the surface and in the wall (exine, intine). First attempts to localize allergens by microscopic methods in the pollen grain supported this view. However, since conventional preparation methods were used, artificial mobilization of allergens could not be excluded. Based on new, completely anhydrous preparation protocols and on the immunogold–labeling technique, pollen allergens were electron–microscopically shown to be located in the interior of the pollen grain, i.e. in the cytoplasm, often within ribosome–rich areas. This unexpected result was obtained in all strictly anhydrously prepared pollen species. The biological function of these allergens is largely unknown, however, clues as to possible cellular functions have been obtained for the birch pollen major allergen Bet v 1.

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“…On the basis of quantitative immunoassays and immunocytochemistry, Southworth et al (1988) established that purified exines are antigenic and that the polyclonal and monoclonal antibodies bind to exines of some species of angiosperms and gymnosperms. Due to the rapidity with which the allergens were released on contact with water it was earlier assumed that allergens appear mainly in the exine (Fromme et al 1985, Grote andFromme 1986). This theory has not been altogether disregarded.…”

Section: Discussionmentioning

confidence: 98%

Localization and release of allergens from tapetum and pollen grains ofBetula pendula

et al. 1999

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Summary.Although intact pollen grains are assumed to be the primary carrier of pollen allergens, specific immunoreactive components have been found in other aerosol fractions, e.g., starch grains and remains of tapetal cells Cryo-scanning-electron-microscopy results demonstrate the presence of a clear network of strands connecting the tapetum with the microspores. The distribution of protein in tapetal orbicules, pollen wall, and pollen cytoplasm was tested by histochemical stains for light microscopy and transmission electron microscopy. The protein is mainly localized at the apertures and starch grains in the cytoplasm of pollen and in the core and on the surface of tapetal orbicules. Monoclonal antibodies Bv-10, BIP3, and BIP4 have been used to locate the cellular sites of pollen and tapetal allergens in Betula pendula (syn. B. verrucosa). The application of rapid-freeze fixation prevented relocation of allergens from their native sites. The allergens are predominantly found in the starch grains and to lesser extent in the exine. We also tested interactions between mature birch pollen and human fluids: saliva, nostrils fluid, and eyes solution. The aim was to mimic more closely the in vivo situation during allergenic response. In all cases we observed several pollen grains that were burst and had released their cytoplasmic contents. In the nose the allergens are released from the pollen within minutes. In rhinitis, nasal pH is increased from the normal pH 6.0 to 8.0. When we used nasal fluid at pH 8.0, the number of ruptured pollen grains increased. The mechanism that might induce formation of small allergen-bearing particles from living plant cells is discussed.

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Immunoautoradiographic and protein-A/gold labelling experiments for localization of pollen allergens using antisera from atopic human individuals

Cited by 8 publications

References 27 publications

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

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

In situ Localization of Pollen Allergens by Immunogold Electron Microscopy: Allergens at Unexpected Sites

Localization and release of allergens from tapetum and pollen grains ofBetula pendula

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