ANTIBODY-MEDIATED FLUORESCENCE ENHANCEMENT BASED ON SHIFTING THE INTRAMOLECULAR DIMER-REVERSIBLE-ARROW-MONOMER EQUILIBRIUM OF FLUORESCENT DYES

A novel concept is described for directly coupling fluorescence emissi on to protein-ligand binding. It is based on shifting the intramolecul ar monomer reversible arrow dimer equilibrium of two fluorescent dyes linked by a short spacer. A 13-residue peptide, recognized by a monocl onal antibody against human chorionic gonadotrophin (hCG), was labeled with fluorescein (F) and tetramethylrhodamine (T) at its N- and C-ter minus, respectively. Spectral evidence suggests that when the conjugat e is free in solution, F and T exist as an intramolecular dimer. Fluor escence quenching of fluorescein and rhodamine is similar to 98% and s imilar to 90%, respectively, due to dimerization. When the double-labe led 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 broug ht about by conformational changes of the conjugate upon binding. Fluo rescein fluorescence, an the other hand, was still quenched because of excited-state energy transfer and residual ground-state interactions. Antibody binding also resulted in a similar to 3.4-fold increase in f luorescence 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 nee d for separation steps and labeling antibodies. Because recent advance s in peptide technology have allowed rapid and economical identificati on of antigen-mimicking peptides, the double-labeled peptide approach offers many opportunities for developing new diagnostic assays and scr eening new therapeutic drugs. It also has many potential applications to techniques involving recombinant antibodies, biosensors, cell sorti ng, and DNA probes.