Competitive binding assays for high-affinity binders in the presence of endogenous ligands: application to biotin-binding proteins.
Endogenous ligands complicate radioligand-binding assays of high-affinity binding proteins by obscuring binding sites or by diluting the labeled ligand. We have developed a mathematical model for such systems where radioligand and endogenous ligand are structurally identical. Data which relate radioligand binding at equilibrium as a function of sample volume can be plotted such that the concentrations of endogenous ligand and binder are graphically determined; however, a more precise determination may be done by nonlinear regression with the aid of a microcomputer. The method is demonstrated for the assay of biotin-binding proteins in the presence of a range of endogenous biotin concentrations below and above that required to saturate the binding sites.
Schreiber
R W
RW
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Letavic
M A
MA
McGahan
T J
TJ
White
H B
HB
D000818:Animals; D001360:Avidin / Q000032:analysis; D001665:Binding Sites; D001710:Biotin / Q000032:analysis*; D002645:Chickens; D002852:Chromatography, Ion Exchange; D008956:Models, Chemical; D011869:Radioligand Assay / Q000379:methods*
D001360:Avidin; D001710:Biotin