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Related Experiment Video

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Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms
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Using Antigen-antibody Binding Kinetic Parameters to Understand Single-Molecule Array Immunoassay Performance.

Trinh L Dinh1, Kevin C Ngan1, Charles B Shoemaker2

  • 1Department of Chemistry, Tufts University , Medford, Massachusetts 02155, United States.

Analytical Chemistry
|October 26, 2016
PubMed
Summary
This summary is machine-generated.

Optimizing single-molecule array (Simoa) immunoassays involves selecting antibodies with specific binding kinetics. Lower dissociation (koff) rates for detection antibodies significantly enhance Simoa assay performance and signal intensity.

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Area of Science:

  • Biotechnology
  • Immunotechnology
  • Assay Development

Background:

  • Single-molecule array (Simoa) immunoassays are powerful tools, but their performance varies widely.
  • Antibody selection is crucial for Simoa performance, yet the impact of specific antibody-antigen binding kinetics remains unclear.

Purpose of the Study:

  • To investigate how antibody-antigen binding kinetic parameters influence the performance of Simoa immunoassays.
  • To identify key kinetic factors for optimizing Simoa assay development.

Main Methods:

  • Examined the effects of association (kon) and dissociation (koff) rate constants of capture and detector antibodies on Simoa performance.
  • Compared six antibodies with a wide range of equilibrium dissociation constants (KD).

Main Results:

  • Simoa assay performance showed an inverse relationship with the detection antibody's koff.
  • Highest fluorescent signals were observed when detection antibody koff was < 10-5 s-1.
  • Capture antibody koff < 10-3 s-1 was preferred, while kon values were not significant factors.

Conclusions:

  • Assay optimization can be accelerated by prioritizing detection antibodies with the slowest dissociation (koff) rates.
  • Understanding antibody kinetics is key to improving Simoa immunoassay performance.