FLUORESCENCE RESONANCE ENERGY-TRANSFER ON SINGLE LIVING CELLS - APPLICATION TO BINDING OF MONOVALENT HAPTENS TO CELL-BOUND IMMUNOGLOBULIN-E

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 K(int) of 9 . 10(8), 8 . 10(8), and 8 . 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.