ABSTRACT
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 [125I]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. [125I] GM-CSF binding to HL-60 cells was shown to be time-dependent, saturable, and specific. The estimated Kd (70 pM) and Bmax (160 r/cell) were similar to 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 plasma membrane preparations.
