Bivalent Antigen Affinity Measurement Service

Background Monovalent-Monovalent Binding Model Bivalent-Monovalent Binding Model Bivalent-Bivalent Binding Model FAQ Resources

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.

Background

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)

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.

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.

Bivalent antigen-monovalent antibody binding model

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

K_a1 and K_d1 are the association and dissociation rate constants for the first site, while K_a2 and K_d2 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.

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)

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.

Other optional Antibody Affinity Measurement Services:

FAQ

How does bivalent antigen affinity measurement differ from monovalent antigen affinity measurement?
Bivalent antigen affinity measurement typically involves evaluating the binding strength between antigens with two binding sites and an antibody, which can lead to stronger and more stable interactions compared to monovalent antigens. This measurement is crucial in understanding the overall avidity and functional strength of an antibody-antigen complex, especially in multivalent binding scenarios often seen in immunological responses. Techniques such as surface plasmon resonance and ELISA are commonly used to assess these interactions, providing insights into the cooperative binding effects that are not observed with monovalent interactions.
What are some common challenges in studying bivalent antigens-antibody interactions?
Studying bivalent antigens-antibody interactions presents several challenges, including the complexity of interaction kinetics and the potential for cross-linking or aggregation. These interactions are not only dependent on the affinity of individual binding sites but also on the spatial arrangement and flexibility of the antigen's binding sites, which can influence overall binding strength and specificity. Additionally, accurately modeling these interactions requires sophisticated analytical techniques and interpretation, making it challenging to predict the behavior of these complexes in biological systems.
What role does valency play in the affinity measurement of antigens?
Valency significantly impacts the affinity measurement of antigens, especially in the context of bivalent or multivalent antigens. Bivalent antigens, having two binding sites, can engage more than one antibody molecule or two epitopes on the same antibody, potentially forming stronger and more stable complexes. This dual binding enhances the effective affinity through a phenomenon known as avidity. Avidity is the cumulative strength of multiple binding interactions, which can be much higher than the affinity of individual binding sites, leading to more effective and sustained immune responses.
Can bivalent antigen-antibody interactions be quantitatively analyzed in live cells?
Bivalent antigen-antibody interactions can be quantitatively analyzed in live cells using techniques like fluorescence microscopy, flow cytometry, and bioluminescence resonance energy transfer (BRET). These methods allow for the real-time monitoring of antigen-antibody binding dynamics within the cellular environment, providing insights into how these interactions influence cellular processes. Such analyses are crucial for understanding the biological significance of antibody binding in physiological and pathological conditions, aiding in the development of therapeutic antibodies and vaccines that effectively target specific antigens.
What techniques are commonly used to measure the affinity of bivalent antigens to antibodies?
Common techniques for measuring the affinity of bivalent antigens to antibodies include Surface Plasmon Resonance (SPR) and Biolayer Interferometry (BLI). Both techniques provide real-time, label-free analysis of the binding kinetics between antigens and antibodies. SPR measures changes in light reflection to assess binding events on a sensor chip, while BLI tracks changes in light interference caused by the formation of antigen-antibody complexes on an optical fiber.
How does the flexibility of a bivalent antigen affect its interaction with an antibody?
Flexibility affects the ability of the antigen's binding sites to properly align with the antibody's epitopes, which can enhance or inhibit the overall binding strength. More flexible linkers between the antigenic sites may allow for optimal orientation and increased binding avidity, improving the functional response of the antibody. Conversely, rigid structures might restrict this alignment, potentially reducing the effectiveness of the immune response. Understanding these dynamics is crucial for designing effective bivalent antigens for vaccines and therapeutic antibodies.
What are the implications of bivalent antigen-antibody interactions for therapeutic antibody design?
By understanding these interactions, researchers can design antibodies with optimized valency, affinity, and linker flexibility to target specific antigens more effectively. This can lead to enhanced efficacy in neutralizing pathogens or targeting diseased cells in conditions like cancer. Additionally, the design of bivalent antibodies can be tailored to improve half-life, reduce immunogenicity, and enhance tissue penetration, all of which are critical factors in the development of effective and safe therapeutic antibodies.