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 α (GMRα) and a biotinylated GM-CSF (bGM-CSF). Using this system, competitive inhibition of bGM-CSF binding to soluble GM-CSF receptor α (sGMRα) 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.