Competitive Binding Assay
In a competitive binding assay, the binding of two unlabeled molecules is measured indirectly by the replacement of a labeled molecule (competition for the same binding site). It allows the comparison of relative affinities of two or more molecules for one target. The competitive binding assay finds application for interactions in which none of the binding partners can be labeled as well as in screening approaches. One example can be found in this p38-alpha - inhibitor protocol.
A requirement for this type of assay is that labeled and unlabeled ligand bind to the same binding site of the target. The assay yields an EC50 which is the concentration at which 50% of the labeled molecule is displaced from the binding site. The affinity of the non-labeled ligand to the target (Ki) can then be calculated from the Kd between labeled ligand and target and the known total concentrations of labeled ligand and target in the assay.
General procedure
A ligand L (cyan) binds to a target T (red):
A fluorescent competitor C (orange) binds to the target at the same binding site as L:
Step 1
The binding affinity between C and T is quantified in a regular binding experiment. The fluorescent competitor C (used at a final concentration around or below the expected Kd) is kept constant in all capillaries, while the unlabeled target T is titrated (up to a concentration of at least 20x of the expected Kd). The dose response plot can be fitted using the law of mass action to extract the dissociation constant of this interaction (Kd).
Step 2
The ligand of interest L is titrated against a complex of T and C in a competition experiment. T is used at a final concentration that is sufficiently high for complex formation (~1-2x Kd) and constant in all capillaries, while C is used at the same concentration as in the previous measurement. L is then titrated to the complex of C and T and the displacement of C by L is measured. The resulting data is fitted with the Hill Equation and an EC50 is derived for the displacement. Note that the resulting curve has an opposite direction to the previous one.
From the data obtained in both experiments, the Ki between T and L (not Kd since it is determined in a competition experiment) can be calculated:
with
where Kd is the measured affinity between target T and fluorescent competitor C in step 1, while [T]t and [C]t are the total concentrations of T and C used in step 2.
For a mathematical derivation of Equations (I) and (II), see the Appendix.
Appendix: Mathematical equations
Two equilibriums are established in competitive binding:
Total concentrations:
From (1) and (3):
From (5) and (6):
From (2), (6) and (7):
At ligand concentration [L]t = 0, it is also [TL] = 0 and [TC] = [TC]m (max), and it follows from (7):
Definition:
At ligand concentration , it is , and it follows from (8), (12) and (13):