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 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.
Kubitscheck
U
U
Institute for Experimental Physics, University of Bremen, Germany.
Kircheis
M
M
Schweitzer-Stenner
R
R
Dreybrodt
W
W
Jovin
T M
TM
Pecht
I
I
D000818:Animals; D000944:Antigens, Differentiation, B-Lymphocyte / Q000378:metabolism; D055592:Biophysical Phenomena; D001703:Biophysics; D006241:Haptens / Q000378:metabolism*; D007073:Immunoglobulin E / Q000378:metabolism*; D015471:Leukemia, Basophilic, Acute / Q000276:immunology / Q000378:metabolism; D011961:Receptors, Fc / Q000378:metabolism; D017455:Receptors, IgE; D013050:Spectrometry, Fluorescence; D014407:Tumor Cells, Cultured / Q000276:immunology / Q000378:metabolism
D000944:Antigens, Differentiation, B-Lymphocyte; D006241:Haptens; D011961:Receptors, Fc; D017455:Receptors, IgE; D007073:Immunoglobulin E