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Intact Transition Epitope Mapping - Thermodynamic Weak-force Order (ITEM - TWO).

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|November 5, 2019
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Summary

We developed a new electrospray mass spectrometry method to determine antibody affinity in gas-phase experiments. This technique rapidly maps epitopes and measures immune complex dissociation energies with minimal solution handling.

Keywords:
Antibody affinity determinationAntibody specificity determinationMass spectrometric epitope mappingNano-electrospray mass spectrometryNative MSQ-ToF analyzer

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Area of Science:

  • Analytical Chemistry
  • Biochemistry
  • Immunology

Background:

  • Determining antibody affinity and epitope specificity is crucial in immunology and drug development.
  • Existing methods often require extensive in-solution handling and can be time-consuming.

Purpose of the Study:

  • To develop and validate a novel electrospray mass spectrometry (MS) method for gas-phase determination of antibody affinity.
  • To enable rapid epitope mapping and characterization of immune complexes.

Main Methods:

  • Electrospray ionization mass spectrometry (ESI-MS) was employed to analyze antibody-peptide immune complexes in the gas phase.
  • Collision-induced dissociation (CID) experiments were performed by varying voltage in a collision cell to dissociate immune complexes.
  • Analysis of ion intensity ratios allowed for the calculation of gas-phase dissociation constants and Gibbs energies of activation.

Main Results:

  • The method successfully determined antibody affinities in the gas phase, with results correlating well with in-solution measurements.
  • Gas-phase dissociation constants were independent of peptide source and instrument used.
  • The technique enabled identification of epitope-containing peptides within a mixture.

Conclusions:

  • This electrospray MS method provides a rapid and efficient means for epitope mapping and antibody affinity determination.
  • The gas-phase approach offers a valuable alternative to traditional in-solution methods, minimizing sample handling.
  • The method accurately characterizes antibody specificity and affinity, key properties for therapeutic and diagnostic applications.