Thus, while a response that reaches saturation will be at equilibrium, an equilibrium response may not be at saturation (2). The response at steady state (equilibrium) is denoted Req and is dependent on the number of ligand binding sites, the analyte concentration and the equilibrium dissociation constant. The best approach is to start with a low concentration, for instance 10 nM and work your way up until you obtain nice curves. Real kinetics can thus saturate the ligand at high analyte concentrations. To analyse a slow dissociation, the dissociation period should be long enough to have at least 5% signal decrease compared to the initial response (3). Theta (Θ) denotes the fraction of equilibrium. Landry JP, Sun YS, Guo XW and Zhu XD (2008). The example sensorgram consists of an association, steady state and dissociation phase. This requires knowledge of the kinetics. However, as the table shows, this can be a very long injection period and is mainly dependent on the dissociation rate constant. Slow dissociation and slow association kinetics are difficult to solve with short injection times. When you have established the concentration range, design an experiment with a dilution series. This is the constant which describes the drug / receptor interactions at equilibrium. When the analyte concentration equals the. Luckily, it is not important to know the Rmax to get meaningful kinetic results. KD 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 analyte concentration can be raised even further until all the ligand binding sites are occupied and the maximal response is reached. A number of times in this article, fast dissociation is indicated to suggest that equilibrium will be reached quickly. To get a better estimate for the ka it is better to prolong the association time (e.g. There is a distinct difference between the response at equilibrium (steady state) and at ligand saturation. Based on the comparison with published literature values for mouse monoclonal antibodies. Many thanks for the comprehensive resource, and apologies for the naive question - I'm learning this from scratch. 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). How do I calculate KD? What is the dissociation constant KD? . With three or more curves covering a wide analyte concentration range the fit will be more reliable. Because you know which ligand and analyte is used, you can calculate the theoretical Rmax with: Although a valid formula, in general it is not practical because the fraction active ligand is unknown. Nevertheless, at full ligand saturation the interaction is at equilibrium because complexes are formed and breaking up. Even at low concentrations, the curve will reach steady state. For instance, to reach 95% equilibrium, Θ = 0.95. To reach steady state quicker it is possible to raise the analyte concentration, but this will not always work. Thus, these sensorgrams clearly show the required (optimal) concentration of analyte to be used. For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome. Shouldn't it be when there is a fast association rate equilibrium will be reached quickly? So keep in mind that sensorgrams with a low response level (< 100 RU) are better than curves with a high response. KD is the equilibrium dissociation constant, a ratio of koff/kon, between the antibody and its antigen. Add repeats of the dilutions to show the system is stable. The K D value relates to the concentration of antibody (the amount of antibody needed for a particular experiment) and so the lower the K D value (lower concentration) and thus the higher the affinity of the antibody. At this point, the number of newly formed complexes equals the number of complexes breaking up. In the figure are five dissociation rates (10-1 – 10-5 s-1) with the same association rate (105 M-1s-1) and an analyte concentration of 1 times KD. Make your analyte dilution with a step 3 to go below the KD but this will give very little information since the curve will be almost linear. The response upon analyte binding is dependent on the number of ligand molecules immobilized and the size of the ligand and analyte (ligand - analyte molecular size ratio). The concentration range of the injected analyte is important. Landry JP, Zhu XD and Gregg JP (2004). Therefore, it is important to keep the experimental conditions constant and to mention these in your publication. 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. When it is real kinetics, the shape of the curves is almost totally determined by the dissociation rate. If Kd is the same for everything you are comparing, then ka and kd change proportionally, so if kd goes up, then ka goes up, which seems why equilibrium would be reached faster. The figure below shows sensorgrams with a) too high, b) optimal concentration and c) too low concentration of analyte. If the response keeps getting higher, other effects such as non-specific binding, bulk refractive distortion causes these high responses. Get resources and offers direct to your inbox. 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 shape of the curves is highly dependent on the dissociation rate constant. Oblique-incidence reflectively difference microscope for label-free high-throughput detection of biochemical reactions in a microarray format.