A FILTRATION-BASED ASSAY TO QUANTITATE GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR-BINDING

Filtration-based binding assays have numerous advantages over centrifugation-based assays, yet they have not been established for many protein ligands due to the high nonspecific binding of the protein to the membrane filter. This paper describes a vacuum filtration method that permits quantitative evaluation of [I-125]GM-CSF binding to its receptor on intact cells. The method includes the use of glass fiber filters presoaked in a solution of polyvinylpyrrolidone and Tween 20 to greatly reduce nonspecific binding of the protein ligand. The ratio of specific:nonspecific binding observed with this filtration assay was comparable to values reported for centrifugation assays. [I-125]GMCSF binding to HL-60 cells was shown to be time-dependent, saturable, and specific. The estimated K-d (70 pM) and B-max (160 r/cell) were similar 60 values reported using centrifugation assays. This filtration method is much less labor-intensive, has greater sample throughput, and allows for a more rapid determination of GM-CSF binding compared to the centrifugation-based assay, Although developed to quantitate the binding of GM-CSF to its receptor on intact cells, this assay is also applicable to other cytokines and can be used with both intact cells and isolated plasmamembrane preparations. (C) 1995 Academic Press, Inc.