A cell-free assay to estimate the neutralizing capacity of granulocyte-macrophage colony-stimulating factor autoantibodies.
The aim of the project is to develop a novel method estimating granulocyte-macrophage colony-stimulating factor (GM-CSF) neutralizing capacity with high-throughput and good reproducibility. For that purpose, we designed a cell-free receptor binding assay consisting of a solid-phase recombinant soluble GM-CSF receptor alpha (GMRalpha) and a biotinylated GM-CSF (bGM-CSF). Using this system, competitive inhibition of bGM-CSF binding to soluble GM-CSF receptor alpha (sGMRalpha) by GM-CSF autoantibody or IgG fractions from the sera of patients with pulmonary alveolar proteinosis was examined, resulting in excellent reproducibility. Binding inhibition was correlated with growth inhibition of TF-1 cells, a GM-CSF dependent cell line. These results suggest that our cell-free system can be applied to estimate the neutralizing capacity of GM-CSF autoantibodies ex vivo.
Urano
Shinya
S
Niigata University Medical and Dental Hospital, Niigata, Japan.
Kaneko
Chinatsu
C
Nei
Takahito
T
Motoi
Natsuki
N
Tazawa
Ryushi
R
Watanabe
Masato
M
Tomita
Masahiro
M
Adachi
Takahiro
T
Kanazawa
Hiroko
H
Nakata
Koh
K
D001323:Autoantibodies / Q000302:isolation & purification / Q000378:metabolism*; D001667:Binding, Competitive / Q000187:drug effects* / Q000276:immunology; D001710:Biotin / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002474:Cell-Free System; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000276:immunology / Q000494:pharmacology*; D057166:High-Throughput Screening Assays; D006801:Humans; D011649:Pulmonary Alveolar Proteinosis / Q000097:blood / Q000175:diagnosis / Q000276:immunology*; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D015203:Reproducibility of Results
D001323:Autoantibodies; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D001710:Biotin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor