Journal article
Fluorescence resonance energy transfer on single living cells. Application to binding of monovalent haptens to cell-bound immunoglobulin E
Biophysical journal, Vol.60(2)
Aug 1991
PMCID: PMC1260066
PMID: 1832974
Featured in Collection : UN Sustainable Development Goals @ Drexel
Abstract
We have determined the specific binding of 2,4-dinitrophenyl (DNP)-haptens to two different monoclonal immunoglobulin (IgE) molecules bound to Fc epsilon-receptors on the cell surface of single, living rat basophilic leukemia cells subclone 2H3 cells. The measurements were performed at 4 degrees, 15 degrees, and 25 degrees C using a recently developed technique that permits the quantitative determination of fluorescence resonance energy transfer between two fluorophores on single cells in a microscope from the photobleaching kinetics of the donor fluorophore. We introduce here a method for performing binding studies on individual attached cells. At 25 degrees C, the titration studies yielded equilibrium binding constants Kint of 9 x 10(8), 8 x 10(8), and 8 x 10(7) M-1 for the monovalent haptens N-2,4-DNP-epsilon-amino-n-caproic acid, N epsilon-2,4-DNP-L-lysine, and N-2,4-DNP-gamma-amino-n-butyric acid, respectively. Our data indicate that the affinity constants for the first two haptens binding to IgE on adherent cells are 4 to 11 times larger than that of the corresponding values obtained by fluorescence quenching experiments with the same haptens and IgE molecules either in solution or bound to cells in suspension.
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Details
- Title
- Fluorescence resonance energy transfer on single living cells. Application to binding of monovalent haptens to cell-bound immunoglobulin E
- Creators
- U Kubitscheck - Institute for Experimental Physics, University of Bremen, GermanyM Kircheis - Institute for Experimental Physics, University of Bremen, GermanyR Schweitzer-Stenner - Institute for Experimental Physics, University of Bremen, GermanyW Dreybrodt - Institute for Experimental Physics, University of Bremen, GermanyT M Jovin - Institute for Experimental Physics, University of Bremen, GermanyI Pecht - Institute for Experimental Physics, University of Bremen, Germany
- Publication Details
- Biophysical journal, Vol.60(2)
- Publisher
- Elsevier
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- Chemistry
- Identifiers
- 991014877838004721
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