Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Concept Videos

The Equilibrium Binding Constant and Binding Strength02:18

The Equilibrium Binding Constant and Binding Strength

The equilibrium binding constant (Kb) quantifies the strength of a protein-ligand interaction. Kb can be calculated as follows when the reaction is at equilibrium:
Ligand Binding Sites02:40

Ligand Binding Sites

Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...
Ligand Binding Sites02:40

Ligand Binding Sites

Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...
Data Validation01:15

Data Validation

Method validation is a crucial process in analytical chemistry designed to confirm that a given method consistently produces reliable and high-quality results. This process is essential when a method is applied to different sample matrices or when procedural modifications are made, ensuring that the results meet acceptable standards across various applications.
Key parameters for method validation include:
Ligand Binding and Linkage00:49

Ligand Binding and Linkage

Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked.  In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence the...
Ligand Binding and Linkage00:49

Ligand Binding and Linkage

Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked.  In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence the...

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

ISR: background, evolution and implementation, with specific consideration for ligand-binding assays.

Bioanalysis·2014
Same author

Specificity of bioanalytical methods for pharmacokinetic support of biological macromolecular therapeutics is important.

Bioanalysis·2011
Same author

Specificity and accuracy data for ligand-binding assays for macromolecules should be interpreted with caution.

The AAPS journal·2008
Same author

Appropriate calibration curve fitting in ligand binding assays.

The AAPS journal·2007
Same author

Bioanalytical method validation for macromolecules in support of pharmacokinetic studies.

Pharmaceutical research·2005
Same author

Disposition of a specific cyclooxygenase-2 inhibitor, valdecoxib, in human.

Drug metabolism and disposition: the biological fate of chemicals·2002

Related Experiment Video

Updated: Jun 27, 2026

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets
10:16

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

Published on: March 12, 2019

Some important considerations for validation of ligand-binding assays.

John W A Findlay1

  • 1Gilead Sciences, Inc., Clinical Pharmacology, 4 University Place, 4611 University Drive, Durham, NC 27707-3458, USA. john.w.findlay@gilead.com

Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
|November 22, 2008
PubMed
Summary

Ligand-binding assays (LBAs) for macromolecules require careful validation due to complex standards and potential interferences. Accuracy of LBA results, especially pharmacokinetic data, should be cautiously interpreted.

More Related Videos

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects
13:57

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

Published on: February 18, 2014

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions
08:40

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

Published on: March 14, 2016

Related Experiment Videos

Last Updated: Jun 27, 2026

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets
10:16

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

Published on: March 12, 2019

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects
13:57

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

Published on: February 18, 2014

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions
08:40

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

Published on: March 14, 2016

Area of Science:

  • Pharmacokinetics
  • Analytical Chemistry
  • Biotechnology

Background:

  • Ligand-binding assays (LBAs) are crucial for quantifying analytes in biological samples.
  • Macromolecule LBAs present unique challenges compared to small molecule assays, including complex reference standards and potential interferences.
  • Non-linear calibration curves in LBAs necessitate careful selection of curve-fitting algorithms.

Purpose of the Study:

  • To highlight the complexities and limitations of calibrating and validating ligand-binding assays (LBAs) for macromolecules.
  • To emphasize the need for cautious interpretation of LBA-derived data, particularly pharmacokinetic parameters for proteins.
  • To advocate for rigorous validation approaches in LBA development and implementation.

Main Methods:

  • Discussion of challenges in LBA calibration curve fitting for non-linear data.
  • Comparison of validation strategies for small molecule versus macromolecule LBAs.
  • Exploration of potential interferences in macromolecule LBAs from degradation products.
  • Consideration of total error and confidence intervals for LBA validation.

Main Results:

  • Macromolecule reference standards are more complex than those for small molecules.
  • Cross-validation with chromatography is more straightforward for small molecule LBAs than for macromolecule LBAs.
  • Unknown interferences from biotransformation products can compromise the accuracy of macromolecule LBA results.
  • Pharmacokinetic data derived from macromolecule LBAs should be viewed with caution.

Conclusions:

  • The accuracy of macromolecule concentrations and pharmacokinetic data determined by LBAs requires careful consideration due to potential interferences.
  • Rigorous validation methods, including total error and confidence interval assessments, are essential for the reliable implementation of LBAs.
  • Further research into understanding and mitigating interferences in macromolecule LBAs is warranted.