TY - JOUR
T1 - Reactivity of monoclonal antibodies EG1 and EG2 with eosinophils and their granule proteins
AU - Nakajima, Hirokazu
AU - Loegering, David A.
AU - Kita, Hirohito
AU - Kephart, Gail M.
AU - Gleich, Gerald J.
PY - 1999
Y1 - 1999
N2 - 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 μg/mL stained 95-100% of nonstimulated eosinophils, regardless of fixative; EG1 and EG2 at 0.1 μg/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.
AB - 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 μg/mL stained 95-100% of nonstimulated eosinophils, regardless of fixative; EG1 and EG2 at 0.1 μg/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.
KW - Cellular activation immunochemistry radioimmunoassay
KW - Human
UR - http://www.scopus.com/inward/record.url?scp=0032870091&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0032870091&partnerID=8YFLogxK
U2 - 10.1002/jlb.66.3.447
DO - 10.1002/jlb.66.3.447
M3 - Article
C2 - 10496315
AN - SCOPUS:0032870091
SN - 0741-5400
VL - 66
SP - 447
EP - 454
JO - Journal of Leukocyte Biology
JF - Journal of Leukocyte Biology
IS - 3
ER -