Journal of Fluorescence

, Volume 5, Issue 3, pp 273–277 | Cite as

Fluorescence spectroscopy of monoclonal antibodies produced against the fluorescyl hapten conjugated through the xanthene ring

  • P. R. Droupadi
  • T. Nanavaty
  • C. Smith
  • D. D. Johnson
  • M. Adamczyk
  • D. S. Linthicum
Article

Abstract

Two mouse anti-fluorescyl monoclonal antibodies (mAb), clones FL43.1 and FL55.3, were produced to the fluorescein hapten, which was conjugated to the carrier protein through the 4′ position of the xanthene ring. Association constants (KA) and thermodynamic parameters for both mAb were ascertained by monitoring the steady-state intrinsic and fluorescein fluorescence. Both techniques were in good agreement and gaveKA values in the 109M−1 range. Ligand-induced intrinsic fluorescence quenching showed a hypsochromic shift for mAb FL43.1, but not for FL55.3, suggesting that the ligand interacts with different tryptophan residues in each mAb. Because these mAb are directed toward the phenylcarboxylate portion of fluorescein, the different ionic and structural forms should be useful as indicators of antibody binding site pH and buffering capacity near the binding site.

Key words

Monoclonal antibody fluorescein complex fluorescence quenching intrinsic fluorescence 

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References

  1. 1.
    E. Day (1992)Advanced Immunochemistry, Wiley Liss, New York.Google Scholar
  2. 2.
    D. R. Davies, E. A. Padlan, and S. Sheriff (1990)Annu. Rev. Biochem. 59, 439–473.Google Scholar
  3. 3.
    E. A. Kabat, T. T. Wu, and H. Bilofsky (1977)J. Biol. Chem. 252, 6609–6616.Google Scholar
  4. 4.
    E. A. Padlan (1994)Mol. Immunol. 31, 169–217.Google Scholar
  5. 5.
    S. Tonegowa (1983)Nature 30, 575–581.Google Scholar
  6. 6.
    E. A. Padlan (1990)Proteins 7, 112–124.Google Scholar
  7. 7.
    I. Mian, A. Bradwell, and A. Olson (1991)J. Mol. Biol. 217, 133–151.Google Scholar
  8. 8.
    C. Chothia and A. M. Lesk (1987)J. Mol. Biol. 196, 901–917.Google Scholar
  9. 9.
    C. Chothia, A. M. Lesk, A. Tramontano, M. Levitt, S. J. Smith-Gill, G. Air, S. Sheriff, E. A. Padlan, D. Davies, W. R. Tulip, P. M. Colman, S. Spinelli, P. M. Alzari, and R. J. Poljak (1989)Nature 342, 877–883.Google Scholar
  10. 10.
    E. A. Kabat, T. T. Wu, H. M. Perry, K. S. Gottesman, and C. Foeller (1991) U. S. Department of Health and Human Services NIH Publication 91-3242.Google Scholar
  11. 11.
    J. Anglister, R. Levy, and T. Scherf (1989)Biochemistry 28, 3360–3365.Google Scholar
  12. 12.
    W. D. Bedzyk and E. W. Voss, Jr. (1991)Mol. Immunol. 28, 27–34.Google Scholar
  13. 13.
    J. Novotny, R. E. Bruccoleri, and F. A. Saul (1989)Biochem Instr. 28, 4735–4749.Google Scholar
  14. 14.
    C. A. Hunter and J. K. Sanders (1990)J. Am. Chem. Soc. 112, 5525–5534.Google Scholar
  15. 15.
    G. Cilento and K. Zinner (1968) in B. Pullman (Ed.),Molecular Associations in Biology, Academic Press, New York, pp. 309–321.Google Scholar
  16. 16.
    J. R. Lakowicz (1991)Principles of Fluorescence Spectroscopy, Plenum Press, New York, Vol. 2, pp. 341–339.Google Scholar
  17. 17.
    S. F. Velick, C. W. Parker, and H. N. Eisen (1960)Proc. Natl. Acad. Sci. USA 46, 1470–1482.Google Scholar
  18. 18.
    R. Watt and E. Voss, Jr. (1977)Immunochemistry 14, 533–541.Google Scholar
  19. 19.
    E. F. G. Templeton and W. R. Ware (1985)Mol. Immunol. 22, 45–55.Google Scholar
  20. 20.
    A. K. Rudolph, P. D. Burrows, and M. R. Wahl (1981)Eur. J. Immunol. 11, 527–529.Google Scholar
  21. 21.
    W. D. Bedzyk, J. N. Herron, A. B. Edmundson, and E. W. Voss, Jr. (1990)J. Biol. Chem. 265, 133–138.Google Scholar
  22. 22.
    J. N. Herron, X. He, M. L. Mason, E. W. Voss, Jr., and A. B. Edmundson (1989)Proteins 5, 271–280.Google Scholar
  23. 23.
    C. L. Kirkemo and M. T. Shipchandler (1986)U. S. Patent #14,614,823.Google Scholar
  24. 24.
    C. L. Kirkemo and M. T. Shipchandler (1985)U. S. Patent #14,510,251.Google Scholar
  25. 25.
    M. T. Shipchandler, J. R. Fino, L. D. Klein, and C. L. Kirkemo (1987)Anal. Biochem. 162, 89–101.Google Scholar
  26. 26.
    P. H. Kussie, G. Albright, and D. S. Linthicum (1989)Meth. Enzymol. 178, 49–63.Google Scholar
  27. 27.
    R. A. Stinson and J. J. Holbrook (1973)Biochem. J. 131, 719–728.Google Scholar
  28. 28.
    J. Anglister, M. W. Bond, T. Frey, D. Leahy, M. Levitt, H. M. McConnell, G. S. Rule, J. Tomasello, and M. Whittaker (1987)Biochemistry 26, 6058–6064.Google Scholar
  29. 29.
    S. K. Burley and G. A. Petsko (1986)FEBS Lett. 203, 139–143.Google Scholar
  30. 30.
    H. Edelhoch (1967)Biochemistry 6, 1948–1954.Google Scholar
  31. 31.
    P. R. Droupadi, J. M. Anchin, E. A. Meyers, and D. S. Linthicum (1993)J. Mol. Recog. 5, 173–179.Google Scholar
  32. 32.
    J. Herron N., D. M. Kranz, D. M. Jameson, and E. W. J. Voss, Jr. (1986)Biochemistry 25, 4602–4609.Google Scholar
  33. 33.
    S. Shifrin (1968) in B. Pullman (Ed.),Molecular Associations in Biology, Academic Press, New York, pp. 323–341.Google Scholar
  34. 34.
    M. M. Martin and L. Lindqvist (1975)J. Luminesc. 10, 381–390.Google Scholar
  35. 35.
    V. G. Omelyanenko, W. Jiskoot, and J. N. Herron (1993)Biochemistry 32, 10423–10429.Google Scholar

Copyright information

© Plenum Publishing Corporation 1995

Authors and Affiliations

  • P. R. Droupadi
    • 1
  • T. Nanavaty
    • 1
  • C. Smith
    • 1
  • D. D. Johnson
    • 2
  • M. Adamczyk
    • 2
  • D. S. Linthicum
    • 1
  1. 1.Department of Veterinary Pathobiology, College of Veterinary MedicineTexas A&M UniversityCollege Station
  2. 2.Abbott Diagnostics DivisionAbbott LaboratoriesAbbott Park

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