Recent Developments in Isothermal Titration Calorimetry Label Free Screening

Peters, William B.; Frasca, Verna; Brown, Richard K.

doi:10.2174/138620709789104889

Cited by 30 publications

(21 citation statements)

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“…It should be emphasized that these results are specific to the VP-ITC instrument we studied, and different results could be obtained for different calorimeters. Indeed, a prior study of this type for the iTC200 instrument does not show a clear pattern of proportional error 42 , although the scatter in the data points makes this difficult to pin down. Fortunately, the succinic acid experiment is straightforward and the materials are inexpensive, so it may easily be used to characterize the heat error for whatever instrument one is using.…”

Section: Discussionmentioning

confidence: 97%

“…Following a previously published procedure 42 , we determined the uncertainty of the heat release per injection by repeatedly injecting fixed amounts of succinic acid solution into an excess of NaOH and determining the variance of the integrated heats across the series of injections. In principle, all injections should yield essentially the same heat, due to the excess of NaOH.…”

Section: Methodsmentioning

confidence: 99%

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Evaluation and Minimization of Uncertainty in ITC Binding Measurements: Heat Error, Concentration Error, Saturation, and Stoichiometry

Kantonen

Henriksen

Gilson

2017

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background Isothermal titration calorimetry (ITC) is uniquely useful for characterizing binding thermodynamics, because it straightforwardly provides both the binding enthalpy and free energy. However, the precision of the results depends on the experimental setup and how thermodynamic results are obtained from the raw data. Methods Experiments and Monte Carlo analysis are used to study how uncertainties in injection heat and concentration propagate to binding enthalpies in various scenarios. We identify regimes in which it is preferable to fix the stoichiometry parameter, N, and evaluate the reliability of uncertainties provided by the least squares method. Results The noise in the injection heat is mainly proportional in character, with ~1% and ~3% uncertainty at 27C and 65C, respectively; concentration errors are ~1%. Simulations of experiments based on these uncertainties delineate how experimental design and curve fitting methods influence the uncertainty in the final results. Conclusions In most cases, experimental uncertainty is minimized by using more injections and by fixing N at its known value. With appropriate technique, the uncertainty in measured binding enthalpies can be kept below ~2% under many conditions, including low C values. General Significance We quantify uncertainties in ITC data due to heat and concentration error, and identify practices to minimize these uncertainties. The resulting guidelines are important when ITC data are used quantitatively, such as to test computer simulations of binding. Reproducibility and further study are supported by free distribution of the new software developed here.

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Section: Discussionmentioning

confidence: 97%

Section: Methodsmentioning

confidence: 99%

Evaluation and Minimization of Uncertainty in ITC Binding Measurements: Heat Error, Concentration Error, Saturation, and Stoichiometry

Kantonen

Henriksen

Gilson

2017

Biochimica et Biophysica Acta (BBA) - General Subjects

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“…DSC of two glycoforms exhibited two transition temperatures, representing the unfolding of the C H 2 and C H 3 domains. [20][21][22][23][24][25][26][27][28][29][30]. SPR showed that the increase in the binding affinity was due to an enhanced association rate.…”

Section: Effect Of Antibody Glycosylation To Binding Activitymentioning

confidence: 98%

Biophysical characterization of antibodies with isothermal titration calorimetry

Frasca¹

2016

J Appl Bioanal

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REVIEWAntibodies play a key role in the immune response. Since antibodies bind antigens with high specificity and tight affinity, antibodies are an important reagent in experimental biology, assay development, biomedical research and diagnostics. Monoclonal antibodies are therapeutic drugs and used for vaccine development. Antibody engineering, biophysical characterization, and structural data have provided a deeper understanding of how antibodies function, and how to make better drugs. Isothermal titration calorimetry (ITC) is a label-free binding assay, which measures affinity, stoichiometry, and binding thermodynamics for biomolecular interactions. When thermodynamic data are used together with structural and kinetic data from other assays, a complete structure-activity-thermodynamics profile can be constructed. This review article describes ITC, and discusses several applications on how data from ITC provides insights into how antibodies function, guide antibody engineering, and aid design of new therapeutic drugs.

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“…A van’t Hoff analysis requires multiple measurements across a wide range of temperatures, which is both time consuming and costly, and technical challenges are faced in robust data analysis [44]. In contrast, the widespread availability of sensitive modern microcalorimeters has made isothermal titration calorimetry (ITC) an attractive approach for the study of binding thermodynamics [45,46]. A single ITC experiment can provide estimates of both K a and Δ H (and hence also Δ G and T Δ S ) from a single experiment [47].…”

Section: Experimental Evidence For Compensationmentioning

confidence: 99%

Entropy-Enthalpy Compensation: Role and Ramifications in Biomolecular Ligand Recognition and Design

Chodera

Mobley

2013

Annu. Rev. Biophys.

453

440

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Recent calorimetric studies of small molecule interactions with biomolecular targets have generated renewed interest in the phenomenon of entropy-enthalpy compensation. In these studies, entropic and enthalpic contributions to binding are observed to vary substantially and in an opposing manner as the ligand or protein is modified while the binding free energy varies little. In severe examples, engineered enthalpic gains can lead to completely compensating entropic penalties, frustrating ligand design. Here, we examine the evidence for compensation, as well as its potential origins, prevalence, severity, and ramifications for ligand engineering. We find the evidence for severe compensation to be weak in light of the large magnitude of and correlation between errors in experimental measurements of entropic and enthalpic contributions to binding, though a limited form of compensation may be common. Given the difficulty of predicting or measuring entropic and enthalpic changes to useful precision, or using this information in design, we recommend ligand engineering efforts instead focus on computational and experimental methodologies to directly assess changes in binding free energy.

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Recent Developments in Isothermal Titration Calorimetry Label Free Screening

Cited by 30 publications

References 0 publications

Evaluation and Minimization of Uncertainty in ITC Binding Measurements: Heat Error, Concentration Error, Saturation, and Stoichiometry

Evaluation and Minimization of Uncertainty in ITC Binding Measurements: Heat Error, Concentration Error, Saturation, and Stoichiometry

Biophysical characterization of antibodies with isothermal titration calorimetry

Entropy-Enthalpy Compensation: Role and Ramifications in Biomolecular Ligand Recognition and Design

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