Bifluorophoric molecules as fluorescent beacons for antibody–antigen binding

Abstract

The quenching of fluorescence (up to 98%) by anti-fluorescein antibodies is well documented in the literature. Here we report a system where, instead of quenching, bifluorophoric molecules are designed to increase in fluorescence upon binding by an anti-fluorescein antibody. Bifluorophoric molecules are made of fluorescein (F) linked to tetramethylrhodamine (T) via varying numbers of methylene units, denoted as F-(CH(2))(n)-T. These F-(CH(2))(n)-T conjugates are almost nonfluorescent when free in solution due to intramolecular dimerization and stacking. Upon binding to an anti-fluorescein antibody, however, up to 110-fold increase in fluorescence was observed from the rhodamine moiety. This increase is believed to result from intramolecular dimer dissociation that dequenches the rhodamine fluorescence. Fluorescein fluorescence, on the other hand, remains quenched due to binding and intramolecular resonance energy transfer. Moreover, the excitation wavelength was at the absorption maxima of fluorescein, giving a Stoke's shift of about 90 nm. This system couples directly molecular recognition with a concurrent increase in fluorescence emission, obviating wash and incubation steps required by most assays. It is an important molecular reporter system for developing homogeneous assays.

Chemical Synthesis of Triple‐Labelled Three‐Helix Bundle Binding Proteins for Specific Fluorescent Detection of Unlabelled Protein

“…11–13 Fluorescent biosensors can be composed of a binding molecule, such as an antibody or enzyme, derivatized with a single fluorescent probe, which is sensitive to changes in the local environment 1416 In other biosensors the binding molecule is labelled with two fluorophores 17–19. In these molecules, the basis for signalling is that the interaction between the two fluorescent probes is altered upon ligand binding, either through changes in the conformation or in the local environment.…”

A Mouse Monoclonal Antibody Against Alexa Fluor 647

“…While many anti-fluorophore antibodies quench fluorescence, (15,16) an increase in fluorescence intensity by anti-fluorophore antibody binding is not unheard of. For example, the anti-dansyl antibody A-6398 (Life Technologies) enhances the fluorescence of the fluorophore to which it binds.…”