Competitive assays play a vital role in antibody characterization due to their ability to provide valuable insights into the binding properties, specificity, and stability of antibodies. By introducing competition between multiple antibodies for the same antigen, these assays allow for a comprehensive analysis of their binding affinities, epitope mapping, and functional characterization. This information is crucial for selecting lead candidates for therapeutic development, understanding the mechanisms of action, and optimizing antibody design. Furthermore, competitive assays enable researchers to evaluate the potential cross-reactivity of antibodies, assess their suitability for specific applications, and compare the performance of different candidates. By employing competitive assays, scientists can make informed decisions and ensure the successful development of safe and effective antibody-based therapeutics.
Measuring binding affinity by competition (Jarmoskaite et al., 2020).
BioLayer interferometry (BLI) binding studies with wildtype gp42 (Sathiyamoorthy et al., 2014)
MST is a versatile biophysical method that measures the movement of molecules across a temperature gradient. It quantifies the binding affinity between an antibody and its target antigen by tracking the migration of fluorescently labeled molecules.MST allows researchers to determine the dissociation constant (KD) with high sensitivity, even for low-affinity interactions. In addition, it provides information on stoichiometric and thermodynamic parameters. The ability to study complex interactions in solution makes MST a powerful tool for antibody characterization.
BLI is an optical technique that measures the interference pattern of light waves reflected from the sensor surface. It provides real-time monitoring of biomolecular interactions, including antibody-antigen binding. BLI provides rapid kinetic analysis, affinity determination, and epitope classification. Its label-free nature and ability to analyze crude samples make it the first choice for antibody characterization.
nanoDSF is a thermal transfer-based method for assessing protein stability and monitoring conformational changes. nanoDSF enables researchers to assess antibody stability, identify regions prone to aggregation, and evaluate the effect of formulation conditions on antibody integrity. It provides valuable insights for antibody quality control and formulation development.
|Microscale Thermophoresis (MST)||Bio-Layer Interferometry (BLI)||nano Differential Scanning Fluorimetry (nanoDSF)|
|Sample Requirements||Can handle crude samples; suitable for challenging matrices||Can handle crude samples; suitable for challenging matrices||Can handle crude samples; may require higher concentrations|
|Key Applications||Binding affinity determination, stoichiometry, thermodynamics||Real-time kinetics analysis, affinity determination, epitope mapping||Protein stability assessment, conformational changes, formulation development|
|Artifact Potential||Requires labeling of target molecule||Label-free analysis||Label-free analysis|