title: Localization of the two major allergens in rye-grass pollen using specific monoclonal antibodies and quantitative analysis of immunogold labelling.
abstract: 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.
authors.affiliation: School of Botany, University of Melbourne, Parkville, Victoria, Australia.
authors.forename: P E
authors.initials: PE
authors.lastname: Taylor
authors.forename: I A
authors.initials: IA
authors.lastname: Staff
authors.forename: M B
authors.initials: MB
authors.lastname: Singh
authors.forename: R B
authors.initials: RB
authors.lastname: Knox
mesh_terms: D000485:Allergens / Q000032:analysis* / Q000276:immunology; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; D001665:Binding Sites; D003600:Cytosol / Q000737:chemistry; D007150:Immunohistochemistry; D008129:Lolium / Q000737:chemistry; D010940:Plant Proteins / Q000032:analysis; D011058:Pollen / Q000737:chemistry / Q000648:ultrastructure; D016707:Tissue Fixation / Q000379:methods*
chemical_list: D000485:Allergens; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; C042814:Lol p I protein, Lolium perenne; D010940:Plant Proteins