TY - JOUR

T1 - Functional and immunochemical characterisation of different antibodies against the erythropoietin receptor

AU - Kirkeby, Agnete

AU - van Beek, Johan

AU - Nielsen, Jacob

AU - Leist, Marcel

AU - Helboe, Lone

PY - 2007/8/15

Y1 - 2007/8/15

N2 - Since it was discovered that the hematopoietic hormone erythropoetin (EPO) exerts neuroprotective effects in the CNS, many studies on the EPO receptor (EPOR) function and localisation in the CNS have been performed. For this purpose, commercially available anti-EPOR antibodies have often been applied. As the literature data on these antibodies show inconsistencies, we here systematically compared six frequently used, commercially available EPOR antibodies for different applications. Five of the antibodies appeared to specifically recognize recombinant rat and human EPOR in HEK293 cells by Western blotting, but the same antibodies yielded different and inconsistent results when human UT-7 cells or rat brain tissue were applied. Immunocytochemical staining of EPOR-transfected HEK cells only produced consistent results with three of the six antibodies. All antibodies stained neurons in rat brain sections, but with large differences in the staining pattern and only the C-20 EPOR antibody was found to label astrocytes. Since EPOR antibodies have been applied in several studies as EPOR antagonists, we further tested the antibodies for their capacity to functionally block the EPO-EPOR interaction in a cellular signalling system with STAT-5 phosphorylation as readout. Here, only the MAB307 antibody showed a partial effect at concentrations of 5-50 μg/ml.

AB - Since it was discovered that the hematopoietic hormone erythropoetin (EPO) exerts neuroprotective effects in the CNS, many studies on the EPO receptor (EPOR) function and localisation in the CNS have been performed. For this purpose, commercially available anti-EPOR antibodies have often been applied. As the literature data on these antibodies show inconsistencies, we here systematically compared six frequently used, commercially available EPOR antibodies for different applications. Five of the antibodies appeared to specifically recognize recombinant rat and human EPOR in HEK293 cells by Western blotting, but the same antibodies yielded different and inconsistent results when human UT-7 cells or rat brain tissue were applied. Immunocytochemical staining of EPOR-transfected HEK cells only produced consistent results with three of the six antibodies. All antibodies stained neurons in rat brain sections, but with large differences in the staining pattern and only the C-20 EPOR antibody was found to label astrocytes. Since EPOR antibodies have been applied in several studies as EPOR antagonists, we further tested the antibodies for their capacity to functionally block the EPO-EPOR interaction in a cellular signalling system with STAT-5 phosphorylation as readout. Here, only the MAB307 antibody showed a partial effect at concentrations of 5-50 μg/ml.

KW - Antibodies

KW - Brain

KW - EPOR antagonist

KW - Erythropoietin receptor

KW - Immunoblotting

KW - Immunohistochemistry

UR - http://www.scopus.com/inward/record.url?scp=34250622423&partnerID=8YFLogxK

U2 - 10.1016/j.jneumeth.2007.03.026

DO - 10.1016/j.jneumeth.2007.03.026

M3 - Journal article

C2 - 17524492

AN - SCOPUS:34250622423

VL - 164

SP - 50

EP - 58

JO - Journal of Neuroscience Methods

JF - Journal of Neuroscience Methods

SN - 0165-0270

IS - 1

ER -