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Functional and immunochemical characterisation of different antibodies against the erythropoietin receptor

Kirkeby, Agnete LU ; van Beek, Johan; Nielsen, Jacob; Leist, Marcel and Helboe, Lone (2007) In Journal of Neuroscience Methods 164(1). p.50-58
Abstract

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.... (More)

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. © 2007 Elsevier B.V. All rights reserved.

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author
publishing date
type
Contribution to journal
publication status
published
keywords
Antibodies, Brain, EPOR antagonist, Erythropoietin receptor, Immunoblotting, Immunohistochemistry
in
Journal of Neuroscience Methods
volume
164
issue
1
pages
9 pages
publisher
Elsevier
external identifiers
  • Scopus:34250622423
ISSN
0165-0270
DOI
10.1016/j.jneumeth.2007.03.026
language
English
LU publication?
no
id
b421f4bf-64e1-441c-a12d-19926e8928a7
date added to LUP
2016-06-02 09:30:41
date last changed
2016-11-07 15:01:13
@misc{b421f4bf-64e1-441c-a12d-19926e8928a7,
  abstract     = {<p>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. © 2007 Elsevier B.V. All rights reserved.</p>},
  author       = {Kirkeby, Agnete and van Beek, Johan and Nielsen, Jacob and Leist, Marcel and Helboe, Lone},
  issn         = {0165-0270},
  keyword      = {Antibodies,Brain,EPOR antagonist,Erythropoietin receptor,Immunoblotting,Immunohistochemistry},
  language     = {eng},
  month        = {08},
  number       = {1},
  pages        = {50--58},
  publisher    = {ARRAY(0x8a4bb70)},
  series       = {Journal of Neuroscience Methods},
  title        = {Functional and immunochemical characterisation of different antibodies against the erythropoietin receptor},
  url          = {http://dx.doi.org/10.1016/j.jneumeth.2007.03.026},
  volume       = {164},
  year         = {2007},
}