Reactivity of monoclonal antibodies EG1 and EG2 with eosinophils and their granule proteins

Use of the murine monoclonal antibodies EG1 and EG2 has been based on the assumption that EG2 recognizes activated eosinophils. We examined the reactivity of EG1 and EG2 with eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), and stimulated and nonstimulated eosinophils from normal donors, By radioimmunoassay, EG1 recognized only ECP, whereas EG2 recognized both ECP and EDN, By Western blot, EG1 reacted with ECP, EG2 reacted with both ECP and EDN, but EG2 could not distinguish between lysates of stimulated and nonstimulated eosinophils. By immunofluorescence, EG1 and EG2 at 20 mu g/mL stained 95-100% of nonstimulated eosinophils, regardless of fixative; EG1 and EG2 at 0.1 mu g/mL stained 61-90% of acetone- and paraformaldehyde-fixed and only 5-21% of methanol-fixed nonstimulated eosinophils, Thus, the reactivity of EG1 and EG2 with eosinophils depends on the method of fixation and antibody concentration; and EG2, in contrast to previous reports, cannot reliably discriminate between resting and activated eosinophils.