A novel assay for comparing affinity constants of ligands with small differences in affinity: application to...

Analysis of the relevant mass action equations predicts that a dual-label approach can be used to compare quantitatively the equilibrium association constants of different ligands that bind to the same receptor. Indicator-dilution analysis predicts that the only source of error in such an approach will be that attributable to measurement of the label (e.g., gamma counting). Such an approach was designed and validated with respect to low-density lipoproteins (LDL). It was found that LDL with differences in affinity for the LDL receptor of as little as 10% could be readily distinguished, even in a large screening setting. Assessment of the affinities of LDL isolated from 100 subjects with a variety of lipoprotein disorders indicated that marked defects in the ability of LDL to bind to their receptor are not common. Furthermore, in these subjects a direct relationship was observed between the plasma LDL cholesterol concentration and the affinity of the circulating LDL for the LDL receptor, suggesting that in general LDL receptor number, rather than LDL affinity, plays the more important role in determining plasma LDL concentrations.