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Short answer
Isothermal titration, short for isothermal titration calorimetry (ITC), measures the heat released or absorbed when two molecules bind. A ligand is injected in small portions into a solution of a biomolecule, and the heat of each injection is recorded. Fitting the resulting curve gives the binding affinity (KD), the stoichiometry (n) and the enthalpy (ΔH) from a single experiment, without labelling either partner.
Definition and scope
Instrument manufacturer Malvern Panalytical describes ITC as a method that works by "directly measuring the heat that is either released or absorbed during a biomolecular binding event". The measurement is made in solution, and the manufacturer states that it requires "no modification of binding partners, either with fluorescent tags or through immobilization."
That makes ITC a thermodynamic method rather than a structural or kinetic one. It reports how tightly two molecules bind and what the binding is made of in energetic terms. It is part of the wider toolkit used in biophysics to study molecular interactions, and it rests on the same heat-and-energy concepts covered in what is thermodynamics.
How an ITC experiment works in practice
Inside the instrument: cell, syringe and reference cell
According to Malvern Panalytical, the instrument has two cells and an injection device. The table below restates only what that page describes.
| Component | What it holds or does |
|---|---|
| Reference cell | Contains water and acts as the reference. |
| Sample cell | Contains the biomolecule of interest, such as a protein. |
| Syringe in the injection device | Holds the ligand. It is inserted into the sample cell and delivers small portions (aliquots) of ligand in a series of injections. |
| Heat sensors and heaters | The two cells are kept at the same temperature. Sensors detect any temperature difference during binding and heaters compensate for it. |
The same page reports that the heat changes detected are "a few millionths of a degree Celsius."
Reading the isotherm
Each injection produces a heat peak. The area of each peak is integrated and plotted against the molar ratio of ligand to protein, and that plot is the isotherm. As the ratio rises the protein becomes saturated, so the heat change from each injection decreases.
Three results are then read from the isotherm, as the manufacturer describes it:
- KD (binding affinity): obtained by fitting the isotherm to a binding model.
- n (stoichiometry): read from the molar ratio at the centre of the isotherm.
- ΔH (enthalpy): taken directly from the isotherm as the heat released per mole of ligand bound.
The manufacturer adds that the method also determines the entropy of binding (ΔS), which completes the thermodynamic profile.
What each ITC output tells a lab
A result sheet from an ITC run is easier to use when each number is tied to the question it answers. This checklist is built from the parameter list above.
| Output | Question it answers | How it is obtained |
|---|---|---|
| KD | How strong is the interaction? | Fitting the isotherm to a binding model |
| n | How many ligand molecules bind per biomolecule? | Molar ratio at the centre of the isotherm |
| ΔH | How much heat does binding release or absorb per mole of ligand? | Directly from the isotherm |
| ΔS | What does the entropy of binding contribute to the full profile? | Determined by the method, completing the thermodynamic profile |
The manufacturer lists the following applications: quantifying binding affinity, candidate selection and optimisation, measuring thermodynamics and active concentration, and measuring enzyme kinetics.
Types and related methods
Other techniques report on the same kind of question from a different angle. Another calorimetry method is covered in differential scanning calorimetry. Surface plasmon resonance is a separate route to the dissociation constant: see surface plasmon resonance binding kinetics and KD calculation. For reading binding curves more generally, see the Hill coefficient guide.
Where it matters for labs: standard procedures and calibration
Because ITC is a quantitative method run on many different instruments, comparability between labs depends on shared practice. Bastos, Velazquez-Campoy and colleagues published "Isothermal titration calorimetry (ITC): a standard operating procedure (SOP)" in European Biophysics Journal in 2021. According to its abstract in Europe PMC, the work came from a COST Action working group, notes that ITC is widely used for studying binding interactions, and argues that a shared standard procedure helps produce comparable results. It covers experimental design and instrument calibration, with particular attention to chemical calibration.
For a lab or core facility, the practical reading is that an ITC result is only as comparable as the calibration and experimental design behind it. When a method is documented in a lab’s own SOP, citing the community procedure it follows makes the work easier to review and repeat.
Common misunderstandings
"ITC needs a label or an immobilised partner."
It does not. The manufacturer describes ITC as label-free, run in solution with both binding partners unmodified.
"The instrument reads KD directly."
The instrument records heat. KD comes from fitting the isotherm to a binding model, whereas ΔH is taken directly from the isotherm.
"It is a kinetic method."
ITC is presented as a thermodynamic method. The manufacturer lists enzyme kinetics among its applications, but the core outputs are KD, n, ΔH and ΔS.
FAQ
What is isothermal titration used for?
It is used to quantify how a ligand binds to a biomolecule: affinity, stoichiometry and the thermodynamics of binding, in solution and without labels.
What does "isothermal" mean here?
The instrument keeps the sample and reference cells at the same temperature, using heaters to compensate for the heat of binding. The heat the heaters must supply or withhold is what is measured.
Which parameters does one experiment give?
KD, n, ΔH and ΔS. The manufacturer states that ITC can determine all of these simultaneously in a single experiment.
Where can I find a standard procedure?
The 2021 ITC SOP by Bastos, Velazquez-Campoy and colleagues in European Biophysics Journal is the community procedure described above.
Sources
- Malvern Panalytical, "Isothermal titration calorimetry (ITC)" technology page: https://www.malvernpanalytical.com/en/products/technology/microcalorimetry/isothermal-titration-calorimetry. Checked 2026-10-29. This is a manufacturer’s description, so statements about what the method can do are the manufacturer’s own.
- Bastos M, Velazquez-Campoy A, et al. "Isothermal titration calorimetry (ITC): a standard operating procedure (SOP)." European Biophysics Journal, 2021. Abstract on Europe PMC (PMID 33665758, DOI 10.1007/s00249-021-01509-5). Checked 2026-10-29.
