How can we define Kd?
KD is the equilibrium dissociation constant, a calculated ratio of koff/kon, between the antibody and its antigen. The association constant (kon) is used to characterize how quickly the antibody binds to its target. The dissociation constant (koff) is used to measure how quickly an antibody dissociates from its target.
KD and affinity are inversely related, since affinity is defined as the strength of binding of the antibody to its ligand. A high affinity interaction is characterized by a low KD, a fast recognizing (high kon) and a strong stability of formed complexes (low koff).
But be careful reaching early conclusions when comparing two references! Two antibodies can have the same affinity, but one may have both a high on- and off-rate constant, while the other may have both a low on- and off-rate constant.
How can we correlate Kd and sensitivity?
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. That’s how we can correlate KD to sensitivity, speaking in:
Ø micromolar (µM, KD between 10-4 to 10-6),
Ø nanomolar (nM, KD between 10-7 to 10-9),
Ø picomolar (pM, KD between 10-10 to 10-12),
Ø and femtomolar (fM, KD between 10-13 to 10-15).
We typically consider a low affinity antibody to be in a micromolar range, when specific species can allow the generation oh high affinity antibodies (pico to femtomolar, obtained with rabbit and guinea pig monoclonal antibodies).
How does Surface Plasmon Resonance work?
At Diaclone, we measure KD using the SPR (Surface Plasmon Resonance) technology and the Octet instrument. This technique can help you to rank your antibodies but also to better understand the performance of your antibodies.
Surface Plasmon Resonance (SPR) is a spectroscopic method, which consist in immobilizing a ligand on a thin metal film and measuring the change in refractive index (RI) upon binding of the analyte. A sensorgram is simply a graphical trace of refractive index changes to the sensor chip surface. Since the RI change is directly proportional to mass changes caused by analyte binding to immobilized ligand, it enables us to measure binding and unbinding events. Most significantly, these measurements are made in “real-time,” and thus, it is possible to extract accurate values for the rates of both association and dissociation of Ab–Ag complexes.
Fig1. A classical sensorgram and related binding and unbinding events
Concrete case study of Kd measurement
For example, this ELISA development showed that the pair worked only when mAb1 is coated (Fig.2). The determination of KD and association/ dissociation profiles of antibodies by Octet permitted us to understand why the pair works only in one way.
Fig. 2: mAb1-mAb2 pairing evaluation
The mAb#2 has a quick dissociation time (Fig. 3), so, when it’s coated and after all the washing steps, the antigen doesn’t stay on mAb2. Using this antibody as revelation avoids this dissociation which is linked to accumulated washing steps.
Fig. 3: Profiles of association dissociation of mAb#1 (left) and mAb#2 (right)
Octet analysis can also help to validate the accessibility of tags (HIS, GST…), to study the interaction receptor-ligand and the potential inhibition of interaction with an antibody, and to perform epitope binning (also known as epitope mapping or pairing).
The advantage of Octet compared to the highly popular Biacore technique is the lower price, the rapid execution of experiments and the small amount of proteins required.
Whatever the interaction to study, Octet is a valuable tool.
The Octet technology is fully integrated into our custom monoclonal antibody development so that it can be implemented to further study monoclonal antibody candidates.
A large range of custom services, dedicated to your needs:
- Biological activity : agonist or antagonist, secretion enhancer, blocking signal transduction, cellular growth activation or inhibition,….or any new model to design
- Effector activity : ADCC, ADCP or CDC
- Antibody applications : ELISA, Western Blotting, Flow cytometry…
- Antibody labelling
Contact us to discover how Diaclone can support your activity