Isothermal Titration Calorimetry (ITC) Assessment Service

Introduction Published Data FAQ Resources

Creative Biolabs offers isothermal titration calorimetry (ITC) services, which provide incomparable sensitivity with high quality binding data for biomolecular interactions of interest. Our ITC services have been used extensively in studying macromolecule interactions with studies looking at antibody-antigen, protein-protein, protein-ligand, DNA-ligand and RNA-macromolecule studies.

Introduction

In biology, particularly in studies relating the structure of macromolecules to their functions, two of the most important questions are (i) how tightly does a small molecule bind to a specific interaction site and (ii) how fast does the reaction take place if the molecule is a substrate and is converted to a product? ITC is a quantitative technique that can determine the binding affinity (Ka), enthalpy changes (ΔH), and binding stoichiometry (n) of the interaction between two or more molecules in solution. ITC is now routinely used to directly characterize the thermodynamics of macromolecule binding interactions and the kinetics of enzyme-catalyzed reactions.

ITC has advantages over other techniques such as fluorescence assays, NMR and SPR for studying complex formation in terms of ease of use and cost. It does not require any fluorescent probes or radioactive tags for data analysis. Immobilization and chemical modification of protein is not required. Also, it does not have limitations associated with clarity of the solution, molecular weight, temperature or pH. It is one of the best methods for determining the thermodynamic parameters of ligand binding.

Other optional antibody analysis services:

Published Data

The Binding Determination of Tau and IgG2a-BCR by ITC

Fig. 2 ITC binding profile of BCR with taurine. (Wanli Song, 2022)

Taurine (Tau) is abundant in lymphocytes and is widely used as a dietary supplement because of its sulfur-containing properties. Here, the researchers found that Tau can regulate B cell receptor (BCR)-mediated signal transduction and induce B cell activation. Injection of Tau into ovalbumin immunized mice showed that it could also increase the production of IgG. In addition, the results of isothermal titration calorimetry and surface plasmon resonance analysis showed that Tau could specifically bind to IgG2a-BCR. The results of the fluorescence spectrum analysis showed that Tau could bind to the F(ab')2 region of IgG, while the molecular docking analysis showed that Tau was bound to the framework regions (FRs) of the variable regions of the heavy chains (VH) and in the light chains (VL) of IgG2a-BCR.

Reference

Song, Wanli, et al. "Taurine promotes B-cell activation by interaction with the VH/VL framework regions of B‐cell receptor." Immunology 169.2 (2023): 141-156.

FAQ

What is isothermal titration calorimetry (ITC) and how is it used in antibody analysis?
ITC is a biophysical technique used to measure the thermodynamics of molecular interactions, including binding affinity, stoichiometry, enthalpy, and entropy changes. In antibody analysis, ITC can be employed to quantitatively assess the binding strength and thermodynamic properties between an antibody and its antigen. This information is crucial for understanding the efficiency and mechanism of antibody binding, which can aid in the design and optimization of therapeutic antibodies.
Why is ITC preferred for studying antibody-antigen interactions?
ITC is preferred for its ability to provide a complete thermodynamic profile of the binding interaction without the need for any label or modification of the components. This method is highly sensitive and can detect even weak interactions, making it ideal for detailed analysis of antibody-antigen interactions. Additionally, ITC can be used with complex biological fluids, allowing interactions to be studied under near-physiological conditions.
What are the key parameters measured by ITC in antibody analysis?
In antibody analysis, ITC measures several key parameters:
- Ka (association constant): Reflect the affinity between the antibody and antigen.
- ΔH (change in enthalpy): Indicate the heat absorbed or released during binding.
- n (stoichiometry): Show the number of antigens bound per antibody.
- ΔS (change in entropy): Help in understanding the non-covalent forces driving the binding, such as hydrogen bonding and hydrophobic effects.
What challenges might researchers face when using ITC for antibody analysis?
- High sample concentration requirements: ITC typically requires relatively high concentrations of both antibody and antigen, which may be difficult to achieve with high-affinity antibodies or limited sample availability.
- Lengthy experimental setup and analysis: Each ITC experiment can be time-consuming to set up and analyze, particularly when optimizing experimental conditions.
- Interpretation of complex data: The data obtained from ITC can be complex, especially in cases of multiple binding sites or heterogeneous antibody preparations, requiring careful analysis and expertise in thermodynamics.
How do temperature variations affect ITC measurements in antibody analysis?
Temperature plays a critical role in ITC measurements as it influences both the thermodynamics and kinetics of antibody-antigen interactions. Variations in temperature can affect the binding affinity and thermodynamic parameters such as enthalpy and entropy. Researchers must carefully select the temperature that closely mimics physiological conditions or the specific conditions under which the antibody is intended to function. Additionally, temperature control during the experiment is crucial to ensure accurate and reproducible data.
Can ITC differentiate between high-affinity and low-affinity antibodies?
ITC is capable of differentiating between high-affinity and low-affinity antibodies. This is because the technique directly measures the binding constant (Ka), which is indicative of the affinity. High-affinity antibodies typically result in stronger and more exothermic or endothermic peaks at lower concentrations of antigen, whereas low-affinity interactions may require higher concentrations of antigen to achieve measurable binding curves. ITC provides precise quantification of affinity across a wide range, making it ideal for comparing antibodies based on their binding strengths.
What sample preparation steps are recommended for effective ITC analysis of antibodies?
- Purity: Ensure that both the antibody and antigen are highly purified to avoid non-specific binding and aggregation that could skew results.
- Concentration: Accurately determine the concentrations of both interacting partners. The antibody is typically used in the cell (the part of the instrument where the sample is placed), and the antigen is titrated into it.
- Buffer matching: Both the antibody and antigen should be in the same buffer to prevent heat effects due to dilution or buffer mismatch, which can interfere with the measurement of binding-specific heat changes.
- Degassing: Before the experiment, solutions should be degassed to remove air bubbles that could interfere with the calorimetric measurements.
What are the limitations of using ITC for antibody analysis?
- Sample consumption: ITC can be sample-intensive, requiring significant amounts of both antibody and antigen, which may be a limitation when dealing with expensive or scarce materials.
- Low throughput: Compared to other techniques like ELISA or surface plasmon resonance (SPR), ITC has lower throughput due to the time required for each measurement and the manual setup involved.
- Sensitivity to changes: ITC is sensitive to small changes in experimental conditions such as buffer composition and temperature, which require rigorous control to ensure consistent results.
- Complex data analysis: The data obtained, especially in cases of complex binding models or weak interactions, can be challenging to interpret, requiring substantial expertise in thermodynamics and data analysis.