title: Monoclonal antibody EG2 does not provide reliable immunohistochemical discrimination between resting and activated eosinophils.
abstract: The monoclonal antibody (mAb) EG2 has been considered to identify activated eosinophils and several immunohistochemical reports of EG2+ eosinophils in various allergic and other inflammatory disorders have suggested an important pathogenic role for such cells. This study showed that cellular EG2 reactivity, both in peripheral blood and mucosal tissue preparations, depends mainly on the method of sample preparation. Nearly 100% of blood eosinophils from normal individuals were strongly EG2+ when prepared by formalin fixation, whereas only a fraction reacted in the unfixed (64%) or acetone-fixed (60%) state. A significantly increased (p < 0.03) number of EG2+ cells were likewise detected in cryo-sections of inflamed nasal mucosa after formalin fixation compared with acetone fixation. Moreover, virtually all eosinophils were EG2+ in cryo-sections of normal jejunal mucosa fixed in periodate-lysine-(0.5%) paraformaldehyde prior to freezing. Conversely, EG2 reacted only weakly, or failed to react, with many eosinophils in cryo-material not subjected to such pre-fixation, in contrast to adjacent non-eosinophilic cells which were brightly stained. Two-colour immunofluorescence consistently revealed overlapping labelling with EG2 and mAb EG1 or a polyclonal antibody to eosinophil cationic protein in sections of formalin-fixed, paraffin-embedded normal gastrointestinal mucosa. Our findings thus showed that EG2 does not provide reliable immunohistochemical discrimination between resting and activated eosinophils. When optimal pre-fixation of tissue specimens was omitted, EG2 reactivity appeared to be caused, at least in part, by leached antigen adsorbed to adjacent non-eosinophilic cells.
authors.affiliation: Laboratory for Immunohistochemistry and Immunopathology (LIIPAT), University of Oslo, Norway.
authors.forename: F L
authors.initials: FL
authors.lastname: Jahnsen
authors.forename: P
authors.initials: P
authors.lastname: Brandtzaeg
authors.forename: T S
authors.initials: TS
authors.lastname: Halstensen
mesh_terms: D000328:Adult; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001798:Blood Proteins / Q000276:immunology; D047091:Eosinophil Granule Proteins; D004804:Eosinophils / Q000276:immunology* / Q000378:metabolism*; D005260:Female; D005404:Fixatives / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D005629:Frozen Sections; D006652:Histological Techniques; D006801:Humans; D007124:Immunoenzyme Techniques; D008297:Male; D008875:Middle Aged; D012260:Ribonucleases
chemical_list: D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D047091:Eosinophil Granule Proteins; D005404:Fixatives; D012260:Ribonucleases