Most purported antibodies to the human granulocyte colony-stimulating factor receptor are not specific.
OBJECTIVE
Antibodies to human granulocyte colony-stimulating factor receptor (HuG-CSFR) are widely available and have been used in numerous studies to evaluate the expression of this protein on normal and malignant cells of hematopoietic and nonhematopoietic origin. Spurred by recent studies that demonstrated that two commonly used antibodies against the erythropoietin and thrombopoietin receptors can in fact bind to completely unrelated and more broadly expressed proteins, we screened 27 commercially available monoclonal and polyclonal antibodies with claimed specificity to HuG-CSFR to determine if they are specific to this receptor.


METHODS
Antibodies were evaluated by Western blotting, flow cytometry, and immunohistochemistry using 293T cells engineered to overexpress HuG-CSFR protein, immortalized human hematopoietic cell lines expressing endogenous G-CSFR, and purified human neutrophils.


RESULTS
Only two monoclonal antibodies and one polyclonal antibody could be employed using defined Western blotting or flow cytometry protocols to detect G-CSFR protein in cell lysates or on the surface of cells that express G-CSFR messenger RNA with no binding to cells that did not express the gene. None of the antibodies were suitable for immunohistochemistry. Competitive inhibition with soluble G-CSFR extracellular domain and small inhibitory RNA-mediated knock-down of G-CSFR messenger RNA further demonstrated the limited specificity of these antibodies for HuG-CSFR expressed on the cell surface.


CONCLUSIONS
Most commercially available anti-HuG-CSFR antibodies do not bind specifically to this protein. These studies highlight the need for investigators to validate antibodies in their own systems to avoid the inadvertent use of nonspecifically binding antibodies that could lead, as exemplified in this case with a hematopoietic growth factor receptor, to erroneous conclusions about protein expression.
deBruin
Cortney
C
Hematology/Oncology Research Therapeutic Area, and Department of Protein Sciences, Amgen Inc., Thousand Oaks, CA 91320, USA.
Lincoln
Patrice
P
Hartley
Cynthia
C
Shehabeldin
Amro
A
Van
Gwyneth
G
Szilvassy
Stephen J
SJ
D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D000918:Antibody Specificity / Q000276:immunology*; D001667:Binding, Competitive / Q000276:immunology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D005434:Flow Cytometry; D015973:Gene Expression Regulation, Leukemic; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry; D007938:Leukemia / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D009504:Neutrophils / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding / Q000276:immunology; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D020298:U937 Cells
D000911:Antibodies, Monoclonal; D012333:RNA, Messenger; D016188:Receptors, Granulocyte Colony-Stimulating Factor