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Related Experiment Video

Updated: Jul 11, 2026

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity
08:45

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Published on: September 7, 2011

Appropriate calibration curve fitting in ligand binding assays.

John W A Findlay1, Robert F Dillard

  • 1Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and Development, Groton, CT, USA. john.w.findlay@gilead.com

The AAPS Journal
|October 3, 2007
PubMed
Summary

Calibration curves in ligand binding assays typically show a sigmoidal relationship. The 4-parameter logistic (4-PL) model is standard, but a 5-parameter logistic (5-PL) model can improve fit for asymmetric curves.

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Assay Development

Background:

  • Ligand binding assays commonly exhibit nonlinear, sigmoidal calibration curves.
  • The 4-parameter logistic (4-PL) model is the established standard for analyzing these curves.
  • Optimizing assay performance requires careful consideration of model selection and assay design.

Purpose of the Study:

  • To evaluate the performance of the 4-parameter logistic (4-PL) and 5-parameter logistic (5-PL) models for calibration curves in ligand binding assays.
  • To investigate the impact of weighting and assay design parameters on model accuracy and precision.
  • To provide guidance on selecting appropriate models for different calibration curve characteristics.

Main Methods:

  • Analysis of sigmoidal calibration curves using 4-PL and 5-PL models.
  • Evaluation of model performance with and without weighting.
  • Assessment of assay design parameters, including calibrator placement and plate layout.
  • Comparison of model-predicted data with nominal calibrator values.

Main Results:

  • The 4-PL model is the reference standard, optimizing accuracy and precision across the usable range.
  • Incorporating weighting generally improves calibration curve performance.
  • The 5-PL model can enhance goodness of fit for asymmetric curves.
  • 4-PL and 5-PL models typically outperform alternatives, especially at concentration extremes.

Conclusions:

  • The 4-PL and 5-PL models are robust for ligand binding assay calibration curves.
  • Weighting and careful assay design are crucial for optimal model application.
  • Model selection should consider curve asymmetry and performance across the entire concentration range.