Determining antibody stoichiometry using time-integrated fluorescence cumulant analysis

Joseph P. Skinner, Bin Wu, Joachim D. Mueller, Sergey Y. Tetin

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

We applied fluorescence fluctuation spectroscopy to resolve the binding heterogeneity of fluorescently labeled ligand derived from brain natriuretic peptide (BNP), a widely used diagnostic marker of heart failure, to a corresponding monoclonal antibody. This system includes three species: (1) free ligand molecules, (2) antibody with a single site occupied, and (3) antibody with both sites occupied. The method we used, time-integrated fluorescence cumulant analysis (TIFCA), utilizes cumulants of fluorescence fluctuations to resolve subpopulations of multiple fluorescent species freely diffusing in a solution. The values of the cumulants depend on the concentration, molecular brightness and diffusion time of the fluorescent molecules. The number of molecules in each species reflects the antibody affinity. We apply TIFCA to successfully establish the stoichiometry of the system, estimate affinity, and identify the presence of an inactive fraction of antigen in a single titration experiment.

Original languageEnglish (US)
Pages (from-to)1131-1138
Number of pages8
JournalJournal of Physical Chemistry B
Volume115
Issue number5
DOIs
StatePublished - Feb 10 2011

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antibodies
Antibodies
Stoichiometry
stoichiometry
Fluorescence
fluorescence
Molecules
affinity
Ligands
molecules
ligands
Monoclonal antibodies
Brain Natriuretic Peptide
antigens
Antigens
Titration
titration
markers
peptides
brain

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Materials Chemistry
  • Surfaces, Coatings and Films

Cite this

Determining antibody stoichiometry using time-integrated fluorescence cumulant analysis. / Skinner, Joseph P.; Wu, Bin; Mueller, Joachim D.; Tetin, Sergey Y.

In: Journal of Physical Chemistry B, Vol. 115, No. 5, 10.02.2011, p. 1131-1138.

Research output: Contribution to journalArticle

Skinner, Joseph P. ; Wu, Bin ; Mueller, Joachim D. ; Tetin, Sergey Y. / Determining antibody stoichiometry using time-integrated fluorescence cumulant analysis. In: Journal of Physical Chemistry B. 2011 ; Vol. 115, No. 5. pp. 1131-1138.
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