Isothermal Titration Calorimetry (ITC) Assessment Service

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.

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What is Isothermal Titration Calorimetry?

Isothermal Titration Calorimetry (ITC) is a sophisticated analytical technique that measures the heat generated or absorbed when molecular interactions occur in solution, particularly between biomolecules such as proteins, enzymes, DNA, and small ligands. As the only technique capable of simultaneously determining all binding parameters in a single experiment, ITC stands out for its ability to provide comprehensive thermodynamic profiles without requiring fluorescent tags or immobilization of binding partners. This powerful method directly measures critical parameters including binding affinity (Ka), enthalpy changes (ΔH), and binding stoichiometry (n), making it invaluable for applications across biochemical, medical, genetic, and pharmacological fields, especially in drug development where understanding molecular binding mechanisms is crucial.

The fundamental principle of ITC relies on measuring the power required to maintain constant temperature between two cells - a reference cell and a sample cell containing the biomolecules of interest - as a ligand solution is titrated into the sample cell. This process generates binding curves that reveal detailed thermodynamic parameters including dissociation constant (Kd), molar free energy change (ΔG), and molar entropy change (ΔS). The technique's versatility and accuracy make it particularly valuable for studying protein mutations' effects on ligand binding, developing structure-affinity relationships, and conducting competition experiments to understand inhibitor mechanisms. Unlike other methods such as fluorescence assays, NMR, or SPR, ITC offers distinct advantages in terms of ease of use, cost-effectiveness, and the ability to study molecules in their native states without limitations related to solution clarity, molecular weight, temperature, or pH.

Principle of ITC Assay

ITC operates on a remarkably precise yet straightforward principle: the direct measurement of heat changes that occur during molecular binding events. The instrument consists of two identical cells - a sample cell and a reference cell - maintained at a constant temperature through an adiabatic shield. When molecules interact, they either release heat (exothermic) or absorb heat (endothermic), causing minute temperature fluctuations that the instrument detects with exceptional sensitivity.

The reference cell, typically filled with buffer or water, serves as a thermal reference point while the sample cell contains one of the binding partners (usually a macromolecule like a protein). The other binding partner (typically a ligand) is systematically injected into the sample cell through a highly precise syringe. As binding occurs, the instrument measures the time-dependent power input required to maintain thermal equilibrium between the two cells. This power compensation, precisely measured in micro-watts, directly correlates to the heat of binding for each injection. The resulting data appear as a series of peaks, with each peak representing an injection. As the macromolecule becomes saturated with ligand, the magnitude of these peaks progressively decreases until only the heat of dilution remains, producing a complete binding isotherm that contains rich information about the thermodynamics of the interaction.

Fig. 1 Basic principle of isothermal titration calorimetry.¹

Suitable Sample Matrices

• Proteins
• DNA
• Enzymes
• Drug molecules
• Biological ligands

Applications of ITC Analysis

Advantages of Our ITC Analysis

• Enables analysis without the need for labeling or sample modification.
• Allows determination of multiple thermodynamic binding parameters in one experiment.
• Requires minimal sample amounts, preserving sample integrity while offering high sensitivity and precision.
• Capable of detecting a wide range of binding affinities.
• Provides rapid detection with straightforward operation.

ITC vs. DSC - What Are the Differences?

Differential Scanning Calorimetry (DSC) and Isothermal Titration Calorimetry (ITC) are both thermal analysis techniques, yet they serve distinct purposes. DSC measures how a sample responds to a programmed temperature change, recording the energy required to alter its temperature. This reveals how the sample absorbs energy compared to a reference, making DSC ideal for studying molecular stability under varying temperature conditions, such as protein stability under stress.

In contrast, ITC operates at a constant temperature, focusing on the direct observation of chemical interactions within the sample. ITC is specifically suited to measure thermodynamic parameters during binding events, rather than examining how these interactions respond to temperature changes. Thus, DSC is preferred for stability studies under fluctuating conditions, while ITC excels in analyzing the fundamental thermodynamics of molecular binding.

Why Choose Creative Biolabs?

Creative Biolabs offers high-precision Isothermal Titration Calorimetry (ITC) assessment services, providing label-free, accurate measurement of binding interactions. With tailored protocols, minimal sample usage, and comprehensive thermodynamic data in a single experiment, we support diverse research needs in biopharmaceutical and diagnostic applications. Our expert team and end-to-end project support make us a trusted partner for reliable, reproducible results.

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FAQ

1. 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.

2. 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.

3. 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.
4. 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.
5. 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.

6. 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.

7. 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.
8. 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.

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