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Affinity Measurement Services for Bivalent Antigens

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With strong expertise and extensive experience in affinity and kinetics measurement for bivalent antigens, scientists of Creative Biolabs are capable of satisfying your any specific demand. Based on Biacore, ProteOn and Octet systems, Creative Biolabs provides Surface Plasmon Resonance (SPR) and Bio-Layer Interferometry (BLI) services for high-efficiency label-free affinity measurements. You can count on us to assist you all through your project.

The real-time determination of interaction kinetics is the most important application of Biacore system. However, the results of kinetics analysis are only relative to the context of the interaction model chosen for evaluation. It is not really possible to know a binding model from the kinetics detected, although the shape of the sensorgram can sometimes provide hints for the choice of appropriate models.

Figure 1. Fitting experimental data to a mathematical model does not mean that the model is appropriate, because the same group of data may also fit several other models. In this example, the closeness of fit cannot distinguish confidently between a bivalent analyte model (left) and a heterogeneous ligand model (right). (Biacore Assay Hand Book)Figure 1. Fitting experimental data to a mathematical model does not mean that the model is appropriate, because the same group of data may also fit several other models. In this example, the closeness of fit cannot distinguish confidently between a bivalent analyte model (left) and a heterogeneous ligand model (right). (Biacore Assay Hand Book)

For the reason mentioned above, Creative Biolabs performs affinity measurement services for bivalent antigens using different kinetics models. Currently, there are two situations for bivalent antigens and antibodies interactions assay: monovalent antibodies binding to bivalent antigens; bivalent antibodies binding to bivalent antigens.

1. Monovalent antigen-monovalent antibody binding model
This model describes one antigen A molecule binding to one antibody B molecule:

Ka is the association rate constant, and Kd is the dissociation rate constant.Ka is the association rate constant, and Kd is the dissociation rate constant.

2. Bivalent antigen-monovalent antibody binding model
This model describes the interaction between a monovalent antibody and an antigen that carried two separate binding sites.

Ka1 and Kd1 are the association and dissociation rate constants for the first site, while Ka2 and Kd2 are the association and dissociation rate constants for the second site respectively.Ka1 and Kd1 are the association and dissociation rate constants for the first site, while Ka2 and Kd2 are the association and dissociation rate constants for the second site respectively.

Once binding occurs at the first analyte site, the binding that happens at the second site is facilitated by the proximity of antigen and antibody. Similarly, antigens captured at both sites are not released from the surface until the dissociation occurs at both sites, so the observed dissociation rate is much slower than that of single antigen binding site. Remarkably, the association rate constant of the second site is reported in units of RU-1s-1 (Response Unit). This is because both of the interacting components are present on the surface and are measured in RU, not in true concentrations.

3. Bivalent antigen-Bivalent antibody binding model
This model describes the interaction of an antigen that carries two separate binding sites with an bivalent antibody. Crosslinking between bivalent antigens and bivalent antibodies leads to the formation of the microclusters or macroclusters (Monte Carlo study).

Figure 2. An example of bivalent antigen-antibody interactions with Monte Carlo study. Representation of various forms of B-cell clustering by antigen crosslinking. (Cell. & Mol. Immunol., 2011)Figure 2. An example of bivalent antigen-antibody interactions with Monte Carlo study. Representation of various forms of B-cell clustering by antigen crosslinking. (Cell. & Mol. Immunol., 2011)

Reference:
A. S. Reddy et al. (2011). Monte Carlo study of B-cell receptor clustering mediated by antigen crosslinking and directed transport. Cell. & Mol. Immunol. 8: 255-264. 
Biacore assay Handbook 29-0194-00 Edition AA, GE Healthcare, P9-P14.




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