Receptor specific adhesion assay for the quantification of integrin-ligand interactions in intact cells using a microplate based, label-free optical biosensor

摘要

Highlights•A label-free approach for determination of the affinity of ligands to cell adhesion receptors is presented.•The integrin-ligand binding affinity is measured in intact living cells without the need for isolating the receptors.•The IC50value of echistatin binding to integrins was determined in a rapid and cost effective manner.AbstractIn this proof of principle study, a sensitive, label-free, whole cell approach for determination of the affinity of ligands to cell adhesion receptors is presented. The employed optical biosensor is capable of recording, in real time, the kinetics of cell adhesion with high resolution in a microplate based format. The assay was made receptor specific by measuring cell adhesion on integrin ligand displaying surfaces while tuning the concentration of the integrin ligand under study in the employed cell suspensions. As a case study, Arg-Gly-Asp (RGD) displaying polymer surfaces were used and cell adhesion was quantified by measuring the antagonistic action of echistatin, a disintegrin also containing the RGD motif, on the adhesion of cancer cells. Using this novel methodology the half maximal inhibitory concentration (IC50) for echistatin in living cancer cells was determined to be in the range of 20–40nM. The introduced methodology is fast, sensitive, and does not require isolated receptors because biological effects are measured in intact, living cells. The utility of this method for study of other type of adhesion receptors and ligands is discussed.