Macrophage specificity of three anti-CD68 monoclonal antibodies (KP1, EBM11, and PGM1) widely used for immunohistochemistry and flow cytometry

Objectives: To investigate the specificity of three anti-CD68 monoclonal antibodies (mAbs) for macrophages (Mφ) in immunohistochemistry (IHC) and flow cytometry (FACS). Methods: IHC was performed on cryostat sections of rheumatoid arthritis (RA) and osteoarthritis (OA) synovial membranes using the a...

Full description

Saved in:
Bibliographic Details
Published in:Annals of the rheumatic diseases 2004-07, Vol.63 (7), p.774-784
Main Authors: Kunisch, E, Fuhrmann, R, Roth, A, Winter, R, Lungershausen, W, Kinne, R W
Format: Article
Language:English
Subjects:
Aged
Aged, 80 and over
Antibodies, Monoclonal - immunology
Antigens, CD - immunology
Antigens, Differentiation, Myelomonocytic - immunology
Arthritis - immunology
Arthritis, Rheumatoid - immunology
Biological and medical sciences
CD68
Cell Line
Diseases of the osteoarticular system
DMEM
Dulbecco’s modified Eagle’s medium
Enzymes
Extended Report
FACS
FCS
Female
fetal calf serum
fibroblast(s)
Fibroblasts
Flow cytometry
Flow Cytometry - methods
fluorescence activated cell sorter
Humans
IHC
Immunoglobulins
immunohistochemistry
Immunohistochemistry - methods
joint trauma
mAb
macrophages
Macrophages - immunology
Male
Mathematical problems
Medical sciences
Middle Aged
monoclonal antibody
osteoarthritis
Osteoarthritis - immunology
paraformaldehyde
Patients
PBS
PFA
phosphate buffered saline
Power plants
rheumatoid arthritis
Rheumatology
room temperature
Sensitivity and Specificity
sodium citrate/sodium chloride
SSC
synovial fibroblasts
synovial macrophages
synovial membrane
Synovial Membrane - immunology
Citations: Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Objectives: To investigate the specificity of three anti-CD68 monoclonal antibodies (mAbs) for macrophages (Mφ) in immunohistochemistry (IHC) and flow cytometry (FACS). Methods: IHC was performed on cryostat sections of rheumatoid arthritis (RA) and osteoarthritis (OA) synovial membranes using the anti-CD68 mAbs KP1, EBM11, and PGM1, and the fibroblast (FB) markers CD90 and prolyl 4-hydroxylase. Expression of CD68 was also analysed by FACS on the monocytic cell lines THP-1 and U937, as well as on synovial fibroblasts (SFB), skin FB, and gingival FB (both surface and intracellular staining). Results: In IHC, there was an overlap between CD68 (mAbs KP1 and EBM11) and the FB markers CD90/prolyl 4-hydroxylase in the lining layer, diffuse infiltrates, and stroma of RA and OA synovial membranes. In FACS analysis of THP-1 and U937 cells, the percentage of cells positive for the anti-CD68 mAbs KP1 and EBM11 progressively increased from surface staining of unfixed cells, to surface staining of pre-fixed cells, to intracellular staining of the cells. Upon intracellular FACS of different FB, nearly all cells were positive for KP1 and EBM11, but only a small percentage for PGM1. In surface staining FACS, a small percentage of FB were positive for all three anti-CD68 mAbs. Conclusion: An overlap between CD68 (mAbs KP1 or EBM11) and the FB markers CD90 or prolyl 4-hydroxylase may prevent unequivocal identification of Mφ in synovial tissue by IHC or in monocytic cells and FB upon intracellular FACS. This may be due to sharing of common markers by completely different cell lineages.
ISSN:0003-4967
1468-2060
DOI:10.1136/ard.2003.013029