The measurement of the reaction rate constants can be used to define an equilibrium or affinity constant (1/K D). The binding of an antibody to its antigen is a reversible process, and the rate of the binding reaction is proportional to the concentrations of the reactants.Īt equilibrium, the rate of complex formation is equal to the rate of dissociation into its components +. It is typically measured and reported by the equilibrium dissociation constant (K D), which is used to evaluate and rank order strengths of bimolecular interactions. What is the relationship between K D and antibody affinity?Īffinity is the strength of binding of a single molecule to its ligand. The K D values listed on the website were determined by measuring the k on and k off rates of a specific antibody/antigen interaction and then using a ratio of these values to calculate the K D value. K D is the ratio of the antibody dissociation rate (k off), how quickly it dissociates from its antigen, to the antibody association rate (k on) of the antibody, how quickly it binds to its antigen. What does the K D value represent? How is it calculated? How does this method compare with other methods for measuring K D such as Biacore?.What is the typical K D value for an antibody? What would one expect to be a good K D value?.What is the relationship between K D and antibody affinity?.What does the K D value represent? How is it calculated?.The ratio of the experimentally measured off- and on- rates (K off / K on) is used to calculate the K D value.Also, each peptide spot was printed in duplicate on the microarray resulting in separate curves. Every antibody was tested at several different concentration, each corresponding to a different color curve on the graph.Steeper downside = faster off-rate/weaker antibody bindingĪt the dotted line, the solution was switched back to empty buffer solution and run across the peptide microarray. Dissociation Reaction (K off) - This part of the reaction is used to calculate the "off-rate" (K off), a constant used to characterize how quickly an antibody dissociates from its target.įlatter slope = slower off-rate/stronger antibody binding.Antibody solution at a specific concentration was injected at time zero and run across the peptide microarray for 15 min. Association Reaction (K on) - This part of the reaction is used to calculate the "on-rate" (K on), a constant used to characterize how quickly the antibody binds to its target.
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