Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction

Summary

A detailed protocol to perform a titration ELISA is described. Moreover, a novel algorithm is presented to evaluate titration ELISAs and to obtain a dissociation constant of binding of a soluble ligand to a microtiter plate-immobilized receptor.

Abstract

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial parameter to compare different ligands, e.g., competitive inhibitors, protein isoforms and mutants, for their binding strength to a binding partner. Dissociation constants are determined by plotting concentrations of bound versus free ligand as binding curves. In contrast, titration curves, in which a signal that is proportional to the concentration of bound ligand is plotted against the total concentration of added ligand, are much easier to record. The signal can be detected spectroscopically and by enzyme-linked immunosorbent assay (ELISA). This is exemplified in a protocol for a titration ELISA that measures the binding of the snake venom-derived rhodocetin to its immobilized target domain of α2β1 integrin. Titration ELISAs are versatile and widely used. Any pair of interacting proteins can be used as immobilized receptor and soluble ligand, provided that both proteins are pure, and their concentrations are known. The difficulty so far has been to determine the dissociation constant from a titration curve. In this study, a mathematical function underlying titration curves is introduced. Without any error-prone graphical estimation of a saturation yield, this algorithm allows processing of the raw data (signal intensities at different concentrations of added ligand) directly by mathematical evaluation via non-linear regression. Thus, several titration curves can be recorded simultaneously and transformed into a set of characteristic parameters, among them the dissociation constant and the concentration of binding-active receptor, and they can be evaluated statistically. When combined with this algorithm, titration ELISAs gain the advantage of directly presenting the dissociation constant. Therefore, they may be used more efficiently in the future.

Introduction

The dissociation constant K is a key parameter to describe the affinity of a receptor (R) for its ligand (L). Based on the law of mass action, K is defined for the equilibrium, in which the receptor-ligand complex RL dissociates into the receptor R and the ligand L:

            Equation 1

with the indices f indicating the free/unbound state of receptor and....

Protocol

1. Stock Solutions

1. To prepare 100 mL of 10x TBS pH 7.4 solution, dissolve 6.06 g of TRIS and 8.77 g of NaCl in 90 mL of deionized water, adjust the pH to 7.4 with 37% HCl solution, fill the volume to 100 mL with deionized water, and filter the solution.
2. To prepare 100 mL of 1 M HEPES/NaOH, pH 7.4 solution, dissolve 23.83 g of HEPES in 90 mL of deionized water, adjust the pH to 7.4 with 1.5 M NaOH, fill the volume to 100 mL with deionized water, and filter the solution.
3. To prepare 100 mL of 5 M NaCl solution, dissolve 29.2 g of NaCl in 90 mL of deionized water. Fill the volume to 100 mL with deionized water and filter the solution.<....

Results

After the ELISA has been developed, the yellow color of the converted alkaline phosphatase substrate, para-nitrophenolate, indicates that the amount of bound rhodocetin ligand decreases with decreasing concentrations of added rhodocetin from columns 1 to 11 (Figure 1). The colorless wells in the rhodocetin-free wells in column 12 show a low background signal.

Photometric quantification at 4.......

Discussion

The titration ELISA is a versatile test system to determine the dissociation of a receptor-ligand interaction. As the titration ELISA circumvents the necessity to separate free and bound ligands effectively and to analyze their concentrations quantitatively, substantially more studies and publications have employed titration ELISAs instead of recording binding curves. Moreover, titration ELISAs are easy to perform and require reasonably low amounts of receptor and ligand. For accurate analysis of dissociation constants, .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
TRIS	neoFrox	1125KG001
HEPES	Sigma-Aldrich	H4034
NaCl	Applichem	1,316,591,214
MgCl2	Merck	172571
integrin a2A, wild-type and mutant, recombinant		isolated in author's lab
NiNTA superflow column	Qiagen, Germany	30821
Coomassie-Brilliant Blue R250	Serva	35050
bicinchoninic acid assay (BCA), protein concentration determination kit	Fisher Scientific	23225
bovine serum albumine (BSA), fraction V	Applichem	A1391
25 % solution of glutaraldehyde	Merck	354400
anti-rabbit immunglobulin-antibodies from goat, conjugated with alkaline phosphatase	Sigma-Aldrich	A9919
Glycine	Applichem	A1377
Zn(II)-acetate	Applichem	A4324
NaOH	Applichem	A1551
Alkaline phosphatase substrate tablet (5 mg)	Sigma-Aldrich	S0942
Costar half-area microtiter plate	Thermo Scientific	Corning 3690
micro reaction tubes	Eppendorf	30120086
Microplate ELISA reader	BioTek	Synergy HT