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

Filtration-based binding assays have numerous advantages over centrifu gation-based assays, yet they have not been established for many prote in ligands due to the high nonspecific binding of the protein to the m embrane filter. This paper describes a vacuum filtration method that p ermits quantitative evaluation of [I-125]GM-CSF binding to its recepto r on intact cells. The method includes the use of glass fiber filters presoaked in a solution of polyvinylpyrrolidone and Tween 20 to greatl y reduce nonspecific binding of the protein ligand. The ratio of speci fic:nonspecific binding observed with this filtration assay was compar able to values reported for centrifugation assays. [I-125]GMCSF bindin g to HL-60 cells was shown to be time-dependent, saturable, and specif ic. The estimated K-d (70 pM) and B-max (160 r/cell) were similar 60 v alues reported using centrifugation assays. This filtration method is much less labor-intensive, has greater sample throughput, and allows f or a more rapid determination of GM-CSF binding compared to the centri fugation-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.