Why is such a good idea to perform global fitting even for 1:1 interactions?

Advantages of performing a global fitting of several Isothermal Titration Calorimetry isotherms

We have remarked the relevance of global fitting as an indispensable tool to perform a robust analysis of complex binding interactions. Actually, global fitting is a very advantageous tool for the analysis of simpler binding modes like standard 1:1 interactions and it can be used to identify the source of potential differences observed between experiment repeats.
Here we will see how to use global fitting together with the parameters rA and rM of AFFINImeter to identify the source of discrepancies between repeats of the same experiment, to ultimately end up with reliable results.
The isotherms of figure 1 are replicates of the same ITC experiment where a 1:1 interaction between the titrant (A) and the titrate (M) is monitored, but individual fitting of each isotherm to the simple model M + A ↔ MA (Figure 2) yield different KA (association constant) and ΔH values. In fact, from a visual inspection of the isotherms one can presume that  the saturation degree reached in replicate 1 is less than in replicate 2 and that the mid titration point is below the expected stoichiometric equivalence point (At/M= 1), most probably because the titrant (A) and/or titrate (M) concentration was not accurately determined in this experiment.


Figure 1. A)  Global fitting 2 where KΔH  and rA are global parameters and rM is individual between isotherms. B) Global fitting 1 where KAΔH and rM are global parameters and rA is individual between isotherms.


global fitting of the two isotherms was performed in which KAΔH  and rA are shared fitting parameters amongst the isotherms; moreover, rM* is set as individual fitting parameter to check for potential deviations between nominal and true concentration of M in replicate 1; similarly, a global fitting was performed in which KAΔH and rM are shared parameters, and rA* is an individual fitting parameter to check for potential deviations between nominal and true concentration of (figure 1).

Figure 2. Representation of the experiment using the AFFINImeter AM code.


Comparison of the two global fittings clearly shows that a good result is only achieved when deviations in the concentration of A is considered. The value of rA obtained indicates that the active concentration of A is 79% times the nominal concentration (table).

Table: Results obtained from global fitting 2.


This protocol using AFFINImeter has already been proven to be useful in drug discovery programs, working with ligand samples of inaccurate concentration.

We would like to thank Dr. Eric Ennifar (University of Strasbourg) for kindly providing the ITC experiments described herein.


*rM, rA, are scaling parameters employed to correct for potential differences between the nominal and true concentration of compounds M and A, respectively.

The course of Isothermal Titration Calorimetry data analysis

Isothermal Titration Calorimetry (ITC) is widely considered the gold standard technique for the direct determination of binding thermodynamics.
Nowadays, getting kinetic information directly from the thermogram is also possible thanks to the method KinITC implemented in AFFINImeter. Besides KinITC, AFFINImeter offers a number of advanced analytical tools to take the most out of ITC data.

Aiming to show researchers how to make use of these tools, we have prepared a course on ITC data analysis, consisting of a series of video tutorials imparted by Eva Muñoz (Senior Scientist at AFFINImeter) and that we will release in three parts.

The following picture shows the scheme of the course:

The first part, we present three video tutorials dedicated to the analysis of ITC data with KinITC and the multi-temperature tools. Here we are attaching the video tutorials.

  1. Introduction-KinITC automatic processing
  2. KinITC Manual process
  3. Multi-temperature analysis

In the following parts of this course, we will be focused on the design of two binding models for ITC curve fitting with AFFINImeter:

KinITC: Obtain Thermodynamic and Kinetic Data from your ITC Measurements in just five clicks

In AFFINImeter we have implemented KinITC, this is a new method to obtain kinetic information from Isothermal Titration Calorimetry Data. With one single titration experiment it calculates the kinetic constants (kon and koff) and the thermodynamic data (KD and ΔH) of 1:1 binding interactions.

Request an AFFINImeter Online Demo

AFFINImeter is a software designed to further exploit the potential of your Isothermal Titration Calorimetry instrument.

Contact us to request an Online Demo

We want to offer our help to guide you through AFFINImeter. If you are interested in the analysis of a particular ITC data and you don’t know which binding model you should apply or how to design it with AFFINImeter, do not hesitate to contact us!



Meanwhile you can consult our educational material (Videotutorial, Cases of Use, Notes and Webinars) in our Web Page or read the Tutorials and examples section.

Remember that as a launching promotion you can use AFFINImeter free during the first 6 months!

Global fitting analysis of a protein-ligand binding experiment

Global fitting analysis of a protein-ligand binding experiment

A few weeks ago AFFINImeter launched an Isothermal Titration data Analysis challenge of the analysis of a protein-ligand binding experiments. The participant had to globally analyze a set of Isothermal Titration Calorimetry experiments using AFFINImeter and get the thermodynamic and structural parameters of the interaction between both molecules (the receptor protein and the ligand).

The participants in this contest had the opportunity to demonstrate their ability to propose the right model for a given binding isotherm as well as to get the corresponding parameters upon fitting using AFFINImeter. On their side, less experienced participants had the opportunity to:

The results recently published by Henlz et al in [Methods 59 (2013) 336-348] were taken as a reference to generate the set of ITC isotherms selected for this contest.


Complete guide of a global analysis of Isothermal Titration Calorimetry data in AFFINImeter

A few days ago AFFINImeter launched an Isothermal Titration Calorimetry (ITC) data analysis challenge. Here the participant had to globally analyse a set of Isothermal Titration Calorimetry experiments using AFFINImeter and get the thermodynamic and structural parameters of the interaction between both molecules (the receptor protein and the ligand).

The participants in this contest had the opportunity to demonstrate their ability to propose the right model for a given binding isotherm as well as to get the corresponding parameters upon fitting using AFFINImeter. On their side, less experienced participants had the opportunity to:

During the last days we though it might be useful to help you throughout the fittings proposed for the contest. Therefore, we have prepared a video that explains, step by step, How to fit CURVE 1. IMPORTANTLY, this information will be of great help to solve step 2, the global fitting of CURVES 1-4.

The data proposed for the contest will remain available here, in case you want to learn more about it or to try it by yourself.


Stoichiometric and site constants: two approaches to analyze data with AFFINImeter.

The interaction between two species, i.e. a protein and its ligand, is defined by means of the equilibria existing between free and bound species and the binding constant(s) associated to each equilibrium. This scenario can be described in terms or reaction schemes following two approaches:

a) Based on equilibria between existing stoichiometric species, to obtain stoichiometric binding constants and

b) Based on equilibria between the ligand and specific interaction site(s) of the protein, to obtain site binding constants.


The understanding of both approaches/type of binding constants is key for a correct interpretation of the results after data analysis, in order to get key structural and mechanistic information of the binding event; i.e. the presence or absence of cooperative interactions when a ligand binds to a multivalent receptor.
The design of binding models for ITC curve fitting with AFFINImeter can be done following these two approaches, to perform analysis based on stoichiometric and/or site binding constants.

The scientific team of AFFINImeter has just released three NOTES regarding this subject to guide users into the right selection of binding model approach and a better understanding of stoichiometric vs site binding constants.

Comparative table of the two approaches for binding model design available in AFFINImeter
Characteristics of the two approaches for binding model design available in AFFINImeter



Or visit the RESOURCES section of AFFINImeter web page where you find tutorials, webinars, cases of use, among others.

How to perform a Global Fitting Analysis?

The Global fitting of multiple isotherms is one of the advanced tools that AFFINImeter offers to facilitate the analysis and interpretation of isothermal titration experiments and to expand the range of applications of this technique.

The following video tutorial describes the global fitting of three isotherms of a displacement assay describing, a receptor interacting with a tight ligand, with a weak ligand, or with both ligands simultaneously, in a competitive experiment where the ligands are mixed in the syringe of the ITC equipment.


If you want to know more about global fittings with AFFINImeter you can also download the case of use “Global Analysis in ITC Displacement Titrations with AFFINImeter” that describes a Displacement Titration Assay to determine the thermodynamics of HIV-protease with indinavir, a high-affinity binder, and with acetyl-pepstatin, a weaker ligand.

ITC displacement titrations offer an attractive alternative to standard assays when working with ultra-high or ultra-low- affinity interacting systems. The method requires the fitting of at least two isotherms that share various adjustable parameters. The case study exemplifies the potential advantages of using AFFINImeter in ITC displacement assays. The software offers unique advanced tools that enhance the robustness of the method and makes it more versatile, facilitating the acquisition of reliable thermodynamic data from ultra-high of ultra-low affinity systems. Thus, it opens a door for new applications of the displacement assay.




Competitive Binding Assay

Competition assays

Competitive binding assays where two (or more) ligands bind to the same receptor have become common experiments in many research areas, from basic investigations to innovation in the pharmaceutical industry. These assays can be done in different formats, i.e. through a displacement assay where ligand “L1” is displaced by ligand “L2” from a preformed complex “L1-receptor” or via titration of a receptor solution with a mixture of L1 +L2. Either way, the competitive binding assay provides rich thermodynamic and structural information of the various binding events taking place during the course of the experiment. Thus, Isothermal Titration Calorimetry (ITC) competition assays performed in a displacement format have been revealed as an efficient tool for the quantitative analysis of very high- / low- interactions, with application in the field of fragment based drug screening (ref).

Analysis of competition experiments with AFFINImeter

The versatility of the experimental setup in AFFINImeter-ITC permits the analysis of ITC competition experiments in its various formats. As an illustration, the following lines describe the analysis of an ITC isotherm resulting from a competitive experiment where a solution of a receptor in titrated with mixture of two competing ligands.

AFFINImeter contains a series of examples with which users can practice and learn the overall process of data fitting: from equipment and data uploading to fitting model design and data fitting.

In this post we will review an example of a competitive model fitting model used to analyze the experiment data of two ligands in the syringe competing for binding to the same receptor.

Practical case: competitive binding model

The AFFINImeter example “competitive binding model” illustrates an ITC experiment where two ligands, “A” and “B” compete with each other for binding to the receptor “M”.

Drawing of a competitive binding model
Competitive Binding model scheme

This situation corresponds to a binding model consisting of three free species (A, B and M) two binding equilibria representing the interaction of M with A and M with B

The model was designed with the “reaction builder” and stored in “models”.The equipment used is decribed and stored in “equipments”. The dataseries is uploaded and stored in “dataseries”. When the dataseries is uploaded, the user has to complete the information relative to the equipment used and the species concentration. In this particular case (where there is a competitor “B”).

How to fit a project with AFFINImeter


1- Go to PROJECT MANAGEMENT and create a new PROJECT an a new FIT SUBPROJECT.

  • Add the dataseries created previously to the subproject.
  • Select the model created previously.
  • Keep all the default values in FitSetting.

2- Press Run button.

This Steps are described in the following Video Tutorial:

You can follow this tutorial in AFFINImeter, the Experimental Data and Binding model are stored in your own AFFINImeter account. If you hasn’t registered yet go to the AFFINImeter Software WebPage to get your account.



Ref: W. B. Turnbull, Divided we fall? Studying low-affinity fragments of ligands by ITC. GE Healthcare Life Sciences protocol, 2011, pp 1-11.