Competition Assays of Antibody Characterization

Why Competitive Assays in Antibody Characterization?

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.

What We Can Do:

  • Determination of Binding Affinities: Competitive assays enable the quantification of binding affinities between antibodies and target antigens. By measuring the strength of antibody-antigen interactions, researchers can evaluate the efficacy of the antibody and its potential for therapeutic applications.
  • Measuring binding affinity by competition (JarmoskaiteMeasuring binding affinity by competition (Jarmoskaite et al., 2020).

  • Epitope Mapping: Competitive assays aid in the identification and mapping of epitopes recognized by antibodies. By competing with other antibodies for binding to the antigen, researchers can gain insights into the specific regions or amino acids on the antigen that are targeted by the antibody.
  • Affinity Ranking and Selection: Competitive testing allows for the ranking and selection of antibodies based on their relative binding affinity. This information helps to identify high-affinity antibody candidates that exhibit optimal target engagement and potential therapeutic efficacy.
  • Kinetic analysis: such as bio-layer interferometry (BLI), allows real-time monitoring of antibody-antigen binding kinetics. This analysis provides information on the rate of binding and dissociation, as well as the overall binding strength between antibody and antigen.
  • BioLayer interferometry (BLI) binding studies with wildtype gp42BioLayer interferometry (BLI) binding studies with wildtype gp42 (Sathiyamoorthy et al., 2014)

  • Stability assessment: Competitive assays, including nano-differential scanning fluorescence (nanoDSF), allow assessment of the thermal stability of antibodies. This analysis helps researchers understand the impact of formulation conditions, storage and environmental factors on antibody integrity and stability.

Technology Platform for Antibody Stability Assessment at Creative Proteomics

Microscale Thermophoresis (MST):

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.

Bio-Layer Interferometry (BLI):

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.

Nano Differential Scanning Fluorometry (nanoDSF):

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)
Sensitivity High High High
Throughput Low High High
Label-free No Yes Yes
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
Time-consuming Yes No No
Artifact Potential Requires labeling of target molecule Label-free analysis Label-free analysis


  1. Jarmoskaite, Inga, et al. "How to measure and evaluate binding affinities." Elife 9 (2020): e57264.
  2. Sathiyamoorthy, Karthik, et al. "Assembly and architecture of the EBV B cell entry triggering complex." PLoS pathogens 10.8 (2014): e1004309.
* This service is for RESEARCH USE ONLY, not intended for any clinical use.