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 (K A) and thermodynamic parameters for both mAb were ascertained by monitoring the steady-state intrinsic and fluorescein fluorescence. Both techniques were in good agreement and gaveK A values in the 109 M −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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
E. Day (1992)Advanced Immunochemistry, Wiley Liss, New York.
D. R. Davies, E. A. Padlan, and S. Sheriff (1990)Annu. Rev. Biochem. 59, 439–473.
E. A. Kabat, T. T. Wu, and H. Bilofsky (1977)J. Biol. Chem. 252, 6609–6616.
E. A. Padlan (1994)Mol. Immunol. 31, 169–217.
S. Tonegowa (1983)Nature 30, 575–581.
E. A. Padlan (1990)Proteins 7, 112–124.
I. Mian, A. Bradwell, and A. Olson (1991)J. Mol. Biol. 217, 133–151.
C. Chothia and A. M. Lesk (1987)J. Mol. Biol. 196, 901–917.
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.
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.
J. Anglister, R. Levy, and T. Scherf (1989)Biochemistry 28, 3360–3365.
W. D. Bedzyk and E. W. Voss, Jr. (1991)Mol. Immunol. 28, 27–34.
J. Novotny, R. E. Bruccoleri, and F. A. Saul (1989)Biochem Instr. 28, 4735–4749.
C. A. Hunter and J. K. Sanders (1990)J. Am. Chem. Soc. 112, 5525–5534.
G. Cilento and K. Zinner (1968) in B. Pullman (Ed.),Molecular Associations in Biology, Academic Press, New York, pp. 309–321.
J. R. Lakowicz (1991)Principles of Fluorescence Spectroscopy, Plenum Press, New York, Vol. 2, pp. 341–339.
S. F. Velick, C. W. Parker, and H. N. Eisen (1960)Proc. Natl. Acad. Sci. USA 46, 1470–1482.
R. Watt and E. Voss, Jr. (1977)Immunochemistry 14, 533–541.
E. F. G. Templeton and W. R. Ware (1985)Mol. Immunol. 22, 45–55.
A. K. Rudolph, P. D. Burrows, and M. R. Wahl (1981)Eur. J. Immunol. 11, 527–529.
W. D. Bedzyk, J. N. Herron, A. B. Edmundson, and E. W. Voss, Jr. (1990)J. Biol. Chem. 265, 133–138.
J. N. Herron, X. He, M. L. Mason, E. W. Voss, Jr., and A. B. Edmundson (1989)Proteins 5, 271–280.
C. L. Kirkemo and M. T. Shipchandler (1986)U. S. Patent #14,614,823.
C. L. Kirkemo and M. T. Shipchandler (1985)U. S. Patent #14,510,251.
M. T. Shipchandler, J. R. Fino, L. D. Klein, and C. L. Kirkemo (1987)Anal. Biochem. 162, 89–101.
P. H. Kussie, G. Albright, and D. S. Linthicum (1989)Meth. Enzymol. 178, 49–63.
R. A. Stinson and J. J. Holbrook (1973)Biochem. J. 131, 719–728.
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.
S. K. Burley and G. A. Petsko (1986)FEBS Lett. 203, 139–143.
H. Edelhoch (1967)Biochemistry 6, 1948–1954.
P. R. Droupadi, J. M. Anchin, E. A. Meyers, and D. S. Linthicum (1993)J. Mol. Recog. 5, 173–179.
J. Herron N., D. M. Kranz, D. M. Jameson, and E. W. J. Voss, Jr. (1986)Biochemistry 25, 4602–4609.
S. Shifrin (1968) in B. Pullman (Ed.),Molecular Associations in Biology, Academic Press, New York, pp. 323–341.
M. M. Martin and L. Lindqvist (1975)J. Luminesc. 10, 381–390.
V. G. Omelyanenko, W. Jiskoot, and J. N. Herron (1993)Biochemistry 32, 10423–10429.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Droupadi, P.R., Nanavaty, T., Smith, C. et al. Fluorescence spectroscopy of monoclonal antibodies produced against the fluorescyl hapten conjugated through the xanthene ring. J Fluoresc 5, 273–277 (1995). https://doi.org/10.1007/BF00723898
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00723898