Application of Fluorescence Correlation Spectroscopy to Hapten-Antibody Binding

  • Theodore L. Hazlett
  • Qiaoqiao Ruan
  • Sergey Y. Tetin
Part of the Methods in Molecular Biology™ book series (MIMB, volume 305)


Two-photon fluorescence correlation spectroscopy 2P—FCS has received a large amount of attention over the past ten years as a technique that can monitor the concentration, the dynamics, and the interactions of molecules with single molecule sensitivity. In this chapter, we explain how 2P—FCS is carried out for a specific ligand-binding problem. We briefly outline considerations for proper instrument design and instrument calibration. General theory of autocorrelation analysis is explained and straightforward equations are given to analyze simple binding data. Specific concerns in the analytical methods related to IgG, such as the presence of two equivalent sites and fractional quenching of the bound hapten—fluorophore conjugate, are explored and equations are described to account for these issues. We apply these equations to data on two antibody—hapten pairs: antidigoxin IgG with fluorescein—digoxin and antidigitoxin IgG with Alexa488—digitoxin. Digoxin and digitoxin are important cardio glycoside drugs, toxic at higher levels, and their blood concentrations must be monitored carefully. Clearly, concentration assays based on IgG rely on accurate knowledge of the hapten—IgG binding strengths. The protocols for measuring and determining the dissociation constants for both IgG—hapten pairs are outlined and discussed.

Key Words

Fluorescence immunoglobulin G (IgG) autocorrelation digoxin digitoxin fluorescein FCS equilibrium constant dissociation constant diffusion constant hapten fluorescence correlation spectroscopy 


  1. 1.
    Matayoshi E. D. and Swift K. M. (2001) Application of FCS to protein-ligand interactions: comparison with fluorescence polarization, in: Fluorescence Correlation Spectroscopy. Theory and Applications. (Rigler R. and Elson E. S., ed.) Springer-Verlag, Berlin, Germany, pp. 84–98.Google Scholar
  2. 2.
    Tetin S. Y., Swift K. M., and Matayoshi E. D. (2002) Measuring antibody affinity and performing immunoassay at the single molecule level. Anal. Biochem. 321, 183–187.CrossRefGoogle Scholar
  3. 3.
    Elson E. L. and Magde D. (1974) Fluorescence correlation spectroscopy. I. Conceptual basis and theory. Biopolymers 13, 1–27.CrossRefGoogle Scholar
  4. 4.
    Magde D., Elson E. L., and Webb W. W. (1972) Thermodynamic fluctuations in a reacting system—measurement by fluorescence correlation spectroscopy. Phys. Rev. Let. 29, 705–708.CrossRefGoogle Scholar
  5. 5.
    Magde D., Elson E. L., and Webb W. W. (1974) Fluorescence correlation spectroscopy. II. An eExperimental realization. Bioploymers 13, 20–61.CrossRefGoogle Scholar
  6. 6.
    Thompson N.L. (1991) Fluorescence correlation spectroscopy, in: Topics in Fluorescence Spectroscopy. Volume 1(Lakowicz J.R., ed.) Plenum Press, New York, pp.337–378.Google Scholar
  7. 7.
    Thompson N. L., Lieto A. M., and Allen N. W. (2002) Recent advances in fluorescence correlation spectroscopy. Curr. Opin. Struct. Biol. 12, 634–641.PubMedCrossRefGoogle Scholar
  8. 8.
    Brock R., Hink M. A., and Jovin T. M. (1998) Fluorescence correlation microscopy of cells in the presence of autofluorescence. Biophys. J. 75, 2547–2557.PubMedCrossRefGoogle Scholar
  9. 9.
    Berland K. M., So P. T. C, and Gratton E. (1995) Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment. Biophys. J. 68, 694–701.PubMedCrossRefGoogle Scholar
  10. 10.
    Pramanik A., Olsson M. Langel U., Bartfai T., and Rigler R. (2001) Fluorescence correlation spectroscopy detects galanin receptor diversity on insulinoma cells. Biochemistry 40, 10,839–10,845.PubMedCrossRefGoogle Scholar
  11. 11.
    Schwille P., Haupts U., Maiti S., and Webb W. W. (1999) Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one-and two-photon excitation. Biophys. J. 11, 2251–2265.CrossRefGoogle Scholar
  12. 12.
    Weiss M., Hashimoto H., and Nilsson T. (2003) Anomalous protein diffusion in living cells as seen by fluorescence correlation spectroscopy. Biophys. J. 84, 4043-1052.Google Scholar
  13. 13.
    Bismuto E., Gratton E., and Lamb D. C. (2001) Dynamics of ANS binding to tuna apomyoglobin measured with fluorescence correlation spectroscopy. Biophys. J. 81, 3510–3521.PubMedCrossRefGoogle Scholar
  14. 14.
    Rigler R., Edman L., Foldes-Papp Z., and Wennmalm S. (2001) Fluorescence correlation spectroscopy in single-molecule analysis: enzymatic catalysis at the single molecule level. Single Mol. Spec: Nobel Conf. Lects. 67, 177–194.Google Scholar
  15. 15.
    Magde D., Webb W. W., and Elson E. L. (1978) Fluorescence correlation spectroscopy. III. Uniform translation and laminar flow. Biopolymers 17, 361–376.CrossRefGoogle Scholar
  16. 16.
    Lumma D., Best A., Gansen A., Feuillebois F., Radler J. O., and Vinogradova O.I. (2003) Flow profile near a wall measured by double-focus fluorescence crosscorrelation. Phys. Rev. E. 6705, 6313–6318.Google Scholar
  17. 17.
    Muller J. D., Chen Y., and Gratton E. (2000) Resolving heterogeneity on the single molecular level with the photon counting histogram. Biophysical J. 76, 474–486.CrossRefGoogle Scholar
  18. 18.
    Muller J. D., Chen Y., and Gratton E. (2001) Photon counting histogram statistics, in: Fluorescence Correlation Spectroscopy. Theory and Applications (Rigler R. and Elson E. L., eds.) Springer-Verlag, Berlin, Germany, pp. 410–437.Google Scholar
  19. 19.
    Kask P., Palo K., Ullmann D., and Gall K. (1999) Fluorescence-intensity distribution analysis and its application in biomolecular detection technology. Proc. Natl. Acad. Sci. USA 96, 1379-1376.Google Scholar
  20. 20.
    Sanchez S. A., Chen Y., Muller J. D., Gratton E., and Hazlett T. L. (2001) Solution and interface aggregation states of Crotalus atrox venom phospholipase A2 by two-photon excitation fluorescence correlation spectroscopy. Biochemistry 40, 6903–6911.PubMedCrossRefGoogle Scholar
  21. 21.
    Patel R. C, Kumar U., Lamb D. C, Eid J. S., Rocheville M., Grant M., et al. (2002) Ligand binding to somatostatin receptors induces receptor-specific oligomer formation in live cells. Proc. Natl. Acad. Sci. USA 99, 3294–3299.PubMedCrossRefGoogle Scholar
  22. 22.
    Kim S. A., Heinze K. G., Waham M. N., and Schwille P. (2004) Intracellular calmodulin availability accessed with two-photon cross-correlation. Proc. Natl. Acad. Sci. USA 101, 105–110.PubMedCrossRefGoogle Scholar
  23. 23.
    Hess S. T. and Webb W. W. (2002) Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy. Biophys. J. 83, 2300–2317.PubMedCrossRefGoogle Scholar
  24. 24.
    Chen Y., Muller J. D., Berland K. M., and Gratton E. (1999) Fluorescence correlation spectroscopy. Methods 19, 234–252.PubMedCrossRefGoogle Scholar
  25. 25.
    Pramanik A. and Rigler R. (2001) FCS Analysis of ligand-receptor interactions in living cells, in: Fluorescence Correlation Spectroscopy. Theory and Applications, (Rigler R. and Elson E. S., eds.) Springer-Verlag, Berlin, Germany, pp. 101–131.Google Scholar
  26. 26.
    Johnson M. L. (1992) Analysis of ligand-binding data with experimental uncertainties in independent variables, in: Numerical Computer Methods (Brand L. and Johnson M., eds.). Academic Press, New York, NY, pp. 68–87.Google Scholar
  27. 27.
    Klotz I. M. and Hunston D. L. (1984) Mathematical models for ligand-receptor binding. J. Biol. Chem. 259, 10,060–10,062.PubMedGoogle Scholar
  28. 28.
    Tetin S. Y. and Hazlett T. L. (2000) Optical spectroscopy in studies of antibodyhapten interactions. Methods 20, 341–361.PubMedCrossRefGoogle Scholar
  29. 29.
    Winzor D. J. and Sawyer W. H. (1995) Quantitative Characterization of Ligand Binding. Wiley-Liss, Inc., New York, NY.Google Scholar
  30. 30.
    Weber G. and Anderson S. R. (1965) Multiplicity of binding. Range of validity and practical rest of Adair’s equation. Biochemistry 4, 1942–1947.CrossRefGoogle Scholar
  31. 31.
    Palo K., Mets U., Jager S., Kask P., and Gall K. (2000) Fluorescence intensity multiple distributions analysis: concurrent determination of diffusion times and molecular brightness. Biophys. J. 79, 2858–2866.PubMedCrossRefGoogle Scholar
  32. 32.
    Chen Y., Muller J. D., Tetin S. Y., Tyner J. D., and Gratton E. (2000) Probing ligand protein binding equilibria with fluorescence correlation spectroscopy. Biophys. J. 79, 1074–1084.PubMedCrossRefGoogle Scholar
  33. 33.
    Adamczyk M. and Grote J. (1999) Efficient synthesis of 3-aminodigoxigenin and 3-aminodigitoxigenin probes. Bioorg. Med. Chem. Lett. 9, 771–774.PubMedCrossRefGoogle Scholar
  34. 34.
    Hazlett T. and Gratton E. (2004) Photon counting and analog data acquisition in fluorescence correlation spectroscopy: issues of sensitivity and dynamic range. Biophys. J. 86, 157A.Google Scholar
  35. 35.
    Praissman M. and Rupley J. A. (1968) Comparison of proteinstructure in the crystal and in solution. 3. Tritium-hydrogen exchange of lysozyme and a lysozyme-saccharide complex. Biochemistry 7, 2446–2450.PubMedCrossRefGoogle Scholar
  36. 36.
    Klonis N. and Sawyer W. H. (2000) Effect of solvent-water mixtures on the prototropic equilibria of fluorescein and on the spectral properties of the monoanion. Photochem. Photobiol. 72, 179–185.PubMedCrossRefGoogle Scholar
  37. 37.
    Zhou M., Jin L., Chen B., Ding Y., Ma H., and Chen D. (2003) Afterpulsing and its correction in fluorescence correlation spectroscopy experiments. Appl. Op. 42, 4031–4036.CrossRefGoogle Scholar
  38. 38.
    Widengren J., Mets U., and Rigler R. (1995) Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study. J. Phys. Chem. 99, 13,368–13,379.CrossRefGoogle Scholar
  39. 39.
    Berland K. and Shen G. (2003) Excitation saturation in two-photon fluorescence correlation spectroscopy. Appl. Op. 42, 5566–5576.CrossRefGoogle Scholar
  40. 40.
    Cantor C. R. and Schimmel P. R. (1980) Biophysical Chemistry. Part II: Techniques for the Study of Biological Structure and Function. W. H. Freeman and Company, San Francisco, CA, p. 584.Google Scholar
  41. 41.
    Wohland T., Rigler R., and Vogel H. (2001) The standard deviation in fluorescence correlation spectroscopy. Biophys. J. 80, 2987–2999.PubMedCrossRefGoogle Scholar
  42. 42.
    Meseth U., Wohland T., Rigler R., and Vogel H. (1999) Resolution of fluorescence correlation measurements. Biophys. J. 76, 1619–1631.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2005

Authors and Affiliations

  • Theodore L. Hazlett
    • 1
  • Qiaoqiao Ruan
    • 2
  • Sergey Y. Tetin
    • 2
  1. 1.Laboratory for Fluorescence Dynamics, Department of PhysicsUniversity of Illinois at Urbana-ChampaignUrbana
  2. 2.Core R&D Biotechnology, Abbott Diagnostic DivisionAbbott LaboratoriesAbbott Park

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