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

Abstract

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 Major Allergen Bet V 1 inBetulaPollen Using Monoclonal Antibody Labelling

“…Belin & Rowley (1971) concluded that the allergens in Behrln are probably stored inside the pollen protoplasm and diffuse through the aperture during germination. Staff et al (1990), Taylor et al (1994) and Knox & Suphioglu (1996) localized allergens in the starch grains and cytosol of rye-grass pollen. Our aim with the present study is to determine the localization of the allergens in the cytoplasmic organelles and pollen wall of Berulupnidirln, using monoclonal antibody and immunocytochemistry.…”

Immunogold probes for light and electron microscopic localization of Ole e I in several Oleaceae pollens.

“…The MAbs 1D8 and l6G2 also recognized proteins in these areas in all species tested here (Figures 4-6). This nuclear labeling, likewise observed in rye grass pollen (Staff et al, 1990) and birch pollen (Grote 1991), may reflect the nonspecific attraction of the gold probe to nuclear material (Staff et al, 1990). Alternatively, nuclear labeling may be considered evidence of free dlffusion of the small allergen molecule into the nucleus via the nuclear pores, as suggested by Grote (1991).…”

“…Earlier studies with light microscopy (LM) and transmission electron microscopy (TEM) have reported the localization of allergens in pollen from ragweed, ryegrass, mugwort, birch, alder, Japanese cedar, and hazel, among other allergenic species (Avjioglu et al, 1994;Grote et al, 1994;Miki-Hirosige et al, 1994;Kos et al, 1993;Vrtala et al, 1993;Singh et al, 1991;Staff et al, 1990).…”

Doença alérgica polínica: polens alergógenos e seus principais alérgenos

“…Other clinically relevant allergens are those of groups 2, 3, 4 and 13, which are recognized by over 50% of grass pollen allergic individuals 22 . Lol p 1 and Lol p 5, the main cloned and sequenced groups 1 and 5 L. perenne allergens [23][24][25] , are located in different compartments: Lol p 1 is found in the cytosol 26,27 and Lol p 5 is associated with grass pollen starch grains 25 . Additionally, the occurrence of many antigen components with a similar molecular mass within a same species may be due to isoforms, such as those seen in Lol p 1 and Lol p 5 (isoforms 4 and 8, respectively) 28 .…”