RECEPTOR QUANTIFICATION
Quantify the number of accessible target sites in a binding kinetic assay
To understand the biological and pathological functions driven by a cell surface molecule, it is important to know the cellular expression level. By inducing depletion conditions in an assay (Many targets, few ligands, and low to sub-nanomolar affinity) and using the OneToOneDepletionCorrected model for data evaluation, the number of targets can be estimated.
Inducing depletion conditions is difficult for low affinity binders and, in this case, the interaction kinetic extrapolation (KEX) method can be an alternative.
Experimental parameters were adjusted to drive the system toward ligand depletion. As a result, the One-To-One fitting model proved suboptimal for analysing the binding kinetics of the CD44V6-targeting antibody to HCT-116 cancer cells, whereas the One-To-One Depletion corrected model provided an accurate fit.
Gained information
- Affinity of the interaction (KD)
- Kinetic rate constants (ka and kd)
- Ligand residence time (t1/2)
- Total number of binding sites (nB)
- Number of targets per cell or per tissue
Key publications
Modelling ligand depletion for simultaneous affinity and binding site quantification on cells and tissue.
Weber J, Djurberg K, Lundsten Salomonsson S, Kamprath M, Hoehne A, Westin H, Vergara F, Bondza S.
Sci Rep. 2023 Jun 20;13(1):10031.
Summary
PubMed
Circumventing the requirement of binding saturation for receptor quantification using interaction kinetic extrapolation.
Barta P, Björkelund H, Andersson K.
Nucl Med Commun. 2011. 32(9):863-7.
Summary
PubMed
Examples of insights
