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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.
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 value||Molar concentration (sensitivity)|
|10-4 to 10-6||Micromolar (µM)|
|10-7 to 10-9||Nanomolar (nM)|
|10-10 to 10-12||Picomolar (pM)|
|10-13 to 10-15||Femtomolar (fM)|
Table 1. KD and Molar Values
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.
The specific KD values 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 KD value.
Fig 2. Description of KD value graph:
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 KD values 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.
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.
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.
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).
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.
|Antibody||OI-RD KD (M)||Biacore KD (M)|