KD value: a quantitative measurement of antibody affinity

A guide to Kvalue and antibody affinity.

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 RabMAb antibodies, Abcam's line of Rabbit Monoclonal Antibodies, to assess not only their individual KD values but also to see the average affinity of a RabMAb antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, RabMAb antibodies appear to be on average 1-2 order of magnitude higher affinity (Figure 1). 

Fig 1. KD distribution RabMAb antibodies vs. mouse MAbs: In this comparison the KD values for 88 mouse MAbs were derived from published literature. The individual measurements for each mouse MAb KD value may have been developed using a number of various methods and approaches. The KD measurement values for the 863 RabMAb antibodies were all from the Ol-RD measurement project.

What is KD and how does it correlate to antibody affinity and sensitivity?

KD​ is the equilibrium dissociation constant, a ratio of koff/kon, between the antibody and its antigen. KD and affinity are inversely related. The KD value relates to the concentration of antibody (the amount of antibody needed for a particular experiment) and so the lower the KD value (lower concentration) and thus the higher the affinity of the antibody.

KD valueMolar concentration (sensitivity)
10-4 to 10-6Micromolar (µM)
10-7 to 10-9Nanomolar (nM)
10-10 to 10-12Picomolar (pM)
10-13 to 10-15Femtomolar (fM)

Table 1. KD and Molar Values

How were KD values measured?

The measurements were undertaken as a collaboration with J. Landry and X. Zhu, Dept. of Physics, UC Davis. Data processing, data analysis and report generation for OI-RD (Oblique-incidence reflectivity difference) binding affinity data were measured at UC Davis, Dept. of Physics and reviewed for measurement confidence by scientists at Abcam before posting.

Read the details of this novel approach for measuring antibody KD.

How to read KD value

The specific Kvalues and individual plots for each antibody have been added to each individual antibody product page for you to be able to see each antibodies measured K​value.

​​Fig 2. Description of Kvalue graph:

  1. Baseline - No antibody
  2. ​​Association Reaction (Kon) - This part of the reaction is used to calculate the "on-rate" (Kon), a constant used to characterize how quickly the antibody binds to its target. Antibody solution at a specific concentration was injected at time zero and run across the peptide microarray for 15 min.
  3. Dissociation Reaction (Koff- This part of the reaction is used to calculate the "off-rate" (Koff), a constant used to characterize how quickly an antibody dissociates from its target.

    Flatter slope = slower off-rate/stronger antibody binding
    Steeper downside = faster off-rate/weaker antibody binding

    At the dotted line, the solution was switched back to empty buffer solution and run across the peptide microarray.
  4. Every antibody was tested at several different concentration, each corresponding to a different color curve on the graph. Also, each peptide spot was printed in duplicate on the microarray resulting in separate curves.
  5. The ratio of the experimentally measured off- and on- rates (Koff / Kon) is used to calculate the KD value.

KD value - FAQs

  1. What does the Kvalue represent? How is it calculated?
  2. What is the relationship between Kand antibody affinity?
  3. What is the typical Kvalue for an antibody? What would one expect to be a good Kvalue?
  4. How were the KD values measured?
  5. How does this method compare with other methods for measuring Ksuch as Biacore?

What does the Kvalue represent? How is it calculated?

K​D is the ratio of the antibody dissociation rate (koff), how quickly it dissociates from its antigen, to the antibody association rate (kon) of the antibody, how quickly it binds to its antigen. The Kvalues listed on the website were determined by measuring the kon and koff rates of a specific antibody/antigen interaction and then using a ratio of these values to calculate the KD value.

What is the relationship between KD and antibody affinity?

Affinity is the strength of binding of a single molecule to its ligand. It is typically measured and reported by the equilibrium dissociation constant (KD), which is used to evaluate and rank order strengths of bimolecular interactions. 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.

At equilibrium, the rate of [antibody] [antigen] complex formation is equal to the rate of dissociation into its components [antibody] + [antigen]. The measurement of the reaction rate constants can be used to define an equilibrium or affinity constant (1/KD). In short, the smaller the KD value the greater the affinity of the antibody for its target.

What is the typical KD value for an antibody? What would one expect to be a good KD value?

Most antibodies have KD values in the low micromolar (10-6) to nanomolar (10-7 to 10-9) range. High affinity antibodies generally considered to be in the low nanomolar range (10-9) with very high affinity antibodies being in the picomolar (10-12) range. The median KD value for RabMAb antibodies based on over 850 measurements using the OI-RD measurement is approximately 7 x 10-11 M, demonstrating that on average RabMAb antibodies have very high affinity.

How were the KD values measured?

The KD values were measured using a novel label-free detection system developed by Dr. James Landry and Dr. Xiangdong Zhu at the University of California Davis, Department of Physics. This is a micro-array based system combined with a label-free optical scanner based on polarization-modulated oblique-incidence reflectivity difference (OI-RD) (reference).

Learn about the OI-RD method.

How does this method compare with other methods for measuring KD such as Biacore?

Kinetic measurements for six independent antibody-antigen pairs were performed using either OI-RD or a Biacore instrument. In this experiment, the antigen was captured to the solid support in duplicate. The binding data was generated by injecting the RabMAb antibodies at four concentrations. The model used to fit the experimental data was 1-to-1 Langmuir using global curve fitting analysis. The comparative analysis is summarized in table 2 and figure 2. The results indicate excellent correlation, within an order of magnitude, between the analysis methods.

AntibodyOI-RD KD (M)Biacore KD (M)
Cytokeratin 152.5E-105.6E​-10

Table 2. Comparative KD measurements obtained using OI-RD and Biacore.