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 [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.
Scott
C W
CW
Pharmacology Department, Zeneca Pharmaceuticals, Wilmington, Delaware 19897, USA.
Gomes
B C
BC
Hubbs
S J
SJ
Koenigbauer
H C
HC
D002460:Cell Line; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism*; D006801:Humans; D011869:Radioligand Assay / Q000379:methods; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism*; D012680:Sensitivity and Specificity
D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor