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 Experiment Videos

Competition BIAcore for measuring true affinities: large differences from values determined from binding kinetics

L Nieba1, A Krebber, A Plückthun

  • 1Biochemisches Institut der Universität Zürich, Switzerland.

Analytical Biochemistry
|February 15, 1996
PubMed
Summary

BIAcore measurements of antibody-binding constants are unreliable due to rebinding and avidity effects. Preincubating antibodies with antigen offers a valid alternative for accurate binding constant determination in solution.

Related Concept Videos

You might also read

Related Articles

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

Sort by
Same author

Unravelling Receptor and RGD Motif Dependence of Retargeted Adenoviral Vectors using Advanced Tumor Model Systems.

Scientific reports·2019
Same author

Folding in vitro and transport in vivo of pre-β-lactamase are SecB independent.

Molecular microbiology·2017
Same author

AFM structural study of the molecular chaperone GroEL and its two-dimensional crystals: an ideal "living" calibration sample.

Ultramicroscopy·2002
Same author

Forces and energetics of hapten-antibody dissociation: a biased molecular dynamics simulation study.

Journal of molecular biology·2002
Same author

Protein-fold evolution in the test tube.

Trends in biochemical sciences·2001
Same author

Zooming in on the hydrophobic ridge of H-2D(b): implications for the conformational variability of bound peptides.

Journal of molecular biology·2001

Area of Science:

  • Biochemistry
  • Immunology
  • Analytical Chemistry

Background:

  • Surface plasmon resonance (SPR) instruments like BIAcore are widely used to study molecular interactions.
  • Accurate determination of binding constants (K_a) is crucial for understanding biological systems.
  • Traditional BIAcore methods rely on measuring association (on-rates) and dissociation (off-rates) of molecules.

Purpose of the Study:

  • To evaluate the accuracy of BIAcore-derived binding constants compared to solution-based measurements.
  • To identify and quantify artifacts affecting BIAcore kinetic measurements.
  • To develop a reliable method for determining binding constants using BIAcore.

Main Methods:

  • Utilized BIAcore instrument to measure on- and off-rates for haptens/peptides binding to antibodies.

Related Experiment Videos

  • Compared BIAcore-derived binding constants with solution-based equilibrium measurements.
  • Investigated the impact of varying coupling densities on kinetic measurements.
  • Developed and validated a method involving antibody preincubation with antigen.

    • Main Results:

    • BIAcore measurements of binding constants did not correlate with solution-based values.
    • Rebinding and avidity effects significantly skewed BIAcore results, by up to 500-fold.
    • Antibody preincubation with antigen allowed for reproducible binding constant determination.
    • Established conditions under which the preincubation method is valid.

    Conclusions:

    • Direct measurement of on- and off-rates using BIAcore can be misleading for absolute or relative binding constant determination.
    • Rebinding and avidity effects must be carefully controlled or accounted for in SPR studies.
    • Antibody preincubation with antigen provides a robust alternative for accurate binding constant determination in solution using BIAcore.