Immunocytochemical localization of water-soluble glycoproteins, including Group 1 allergen, in pollen of ryegrass,Lolium perenne, using ferritin-labelled antibody

Abstract

The cellular sites of the glycoproteins Group 1 allergen (glycoprotein 1) and Antigen A (glycoprotein 2) in mature ryegrass pollen have been investigated by immunoelectron microscopy. Radioimmunoassays confirm previous findings of cross-reactivity between the purified glycoprotein antigens at the high immunoglobulin G (IgG) concentrations used for localization. Freeze-drying of anthers followed by anhydrous processing has been employed because of the water solubility and mobility of the glycoproteins. A double-embedding technique has been developed. This involves, first, embedding anthers in the water-soluble plastic resin JB-4, sectioning and incubating in ferritin-labelled antisera by the indirect method. The sections are then embedded in Spurr's resin for ultra-thin sectioning. Both glycoproteins are found in the following sites: (1) exine and intine wall layers; (2) pollen cytoplasm; (3) the orbicules and anther loculus; and (4) the anther cuticle. In the exine arcades and surface and in the anther loculus, the ferritin label is bound to pollenkitt. The finding that the glycoproteins are in similar sites is predictable in view of the cross-specificity of the antisera. The extent of antibody penetration of the plastic sections has been examined; labelling is confined to cut grains and absent from intact grains.

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

“…Here as in all other fields of cell biology, colloidal gold has replaced ferritin [7, 18] as an electron–dense marker.…”

“…Thus, alternative preparation techniques had to be developed. Grass pollen grains were fixed in absolute methanol [5] or a combination of freezing techniques and direct embedding into plastic was used [6, 7] thus avoiding aqueous fixatives and graded ethanol series. These studies localized pollen antigens and allergens in the wall and occasionally in the cytoplasm of the pollen grains.…”

Is the quantity of orbicules released by Dactylis glomerata and Cynosurus echinatus (Poaceae) big enough to play an allergenic role?

“…However, they have yet to be found in orbicules of many other clinically important species, including grasses. In Lolium perenne L., Vithanage et al (1982) found group 1 allergens in the orbicules, but no labelling was obtained by Taylor et al (1994) using monoclonal antibodies. Therefore, it is still unknown whether orbicules are always loaded with allergens and which pollen allergens, i.e., major and/or minor allergens they contain.…”

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

“…Classical methods for localizing allergens in pollen with the help of LM are based on immunofluorescence techniques (Takahashi et al, 1989;Vithanage et al, 1982;Howlett et al, 1981;Knox et al, 1970;Belin and Rowley, 1971). Because these techniques can mask the presence of antigens in the pollen cell wall owing to autofluorescence (Knox et al, 1970), we used immunogold-silver enhancement, which has the advantages of indirect immunolabeling originally developed for TEM and makes it possible to visualize gold particles by light microscopy due to the formation of a silver precipitate (Danscher and Norgaard, 1983).…”