Antibody-mediated fluorescence enhancement based on shifting the intramolecular dimer<-->monomer equilibrium of fluorescent dyes.
A novel concept is described for directly coupling fluorescence emission to protein-ligand binding. It is based on shifting the intramolecular monomer<-->dimer equilibrium of two fluorescent dyes linked by a short spacer. A 13-residue peptide, recognized by a monoclonal antibody against human chorionic gonadotrophin (hCG), was labeled with fluorescein (F) and tetramethylrhodamine (T) at its N- and C-terminus, respectively. Spectral evidence suggests that when the conjugate is free in solution, F and T exist as an intramolecular dimer. Fluorescence quenching of fluorescein and rhodamine is approximately 98% and approximately 90%, respectively, due to dimerization. When the double-labeled peptide is bound to anti-hCG, however, the rhodamine fluorescence increases by up to 7.8-fold, depending upon the excitation wavelength. This is attributed to the dissociation of intramolecular dimers brought about by conformational changes of the conjugate upon binding. Fluorescein fluorescence, on the other hand, was still quenched because of excited-state energy transfer and residual ground-state interactions. Antibody binding also resulted in a approximately 3.4-fold increase in fluorescence anisotropy of the peptide. These changes in intensity and anisotropy allow direct measurement of antigen-antibody binding with a fluorescence plate reader or a polarization analyzer, without the need for separation steps and labeling antibodies. Because recent advances in peptide technology have allowed rapid and economical identification of antigen-mimicking peptides, the double-labeled peptide approach offers many opportunities for developing new diagnostic assays and screening new therapeutic drugs. It also has many potential applications to techniques involving recombinant antibodies, biosensors, cell sorting, and DNA probes.
Wei
A P
AP
Department of Pharmaceutics, University of Utah, Salt Lake City 84108.
Blumenthal
D K
DK
Herron
J N
JN
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D006063:Chorionic Gonadotropin / Q000276:immunology; D000939:Epitopes; D005453:Fluorescence; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D006063:Chorionic Gonadotropin; D000939:Epitopes; D005456:Fluorescent Dyes