Homogeneous dual-parameter assay for prostate-specific antigen based on fluorescence resonance energy transfer.

Three monoclonal antibodies (Mab) specific for prostate-specific antigen (PSA) were used to design a homogeneous dual-parameter immunoassay based on fluorescence resonance energy transfer (FRET). One antibody was labeled with terbium(III) chelate, which acted as a donor, and the other two antibodies were labeled with fluorescent acceptor dyes (either Alexa Fluor (AF) 488 or Alexa Fluor 680). Due to the selection of the antibodies, sensitized emission of the AF488 could be measured only if uncomplexed, free PSA (PSA-F) was present in the sample.

Terbium(III) chelate as an efficient donor for multiple-wavelength fluorescent acceptors.

The emission spectra of the lanthanide chelates enable them to act as a donor for several acceptors emitting at different wavelengths. Fluorescence resonance energy transfer between terbium(III) chelate labeled antibody Fab fragment (donor) and a 17beta-estradiol conjugated to Alexa Fluor 488, 555, 594 or 680 (acceptor) was employed to study the functionality of the terbium(III) chelate as an efficient donor for several acceptors emitting from green to far-red. During measurement, the sensitized emission of the acceptor was measured at acceptor specific wavelength.

Particulate and soluble Eu(III)-chelates as donor labels in homogeneous fluorescence resonance energy transfer based immunoassay.

Many well-established homogeneous separation free immunoassays rely on particulate label technologies. Particles generally contain a high concentration of the embedded label and they have a large surface area, which enables conjugation of a large amount of protein per particle. Eu(III)-chelate dyed nanoparticles have been successfully used as labels in heterogeneous and homogeneous immunoassays.

Homogeneous noncompetitive immunoassay for 17beta-estradiol based on fluorescence resonance energy transfer.

We previously presented a homogeneous noncompetitive assay principle based on quenching of the fluorescence of europium(III) chelate. This assay principle has now been applied to the measurement of 17beta-estradiol (E2) using europium(III) chelate labeled estradiol specific antibody Fab fragment (Eu(III)-Fab) as a donor and E2 conjugated with nonfluorescent QSY21 dye as an acceptor. Fluorescence could be measured only from those Eu(III)-Fab that were bound to E2 of the sample, while the fluorescence of the Eu(III)-Fab that were not occupied by E2 were quenched by E2-QSY21 conjugates.

Homogeneous non-competitive bioaffinity assay based on fluorescence resonance energy transfer.

A homogeneous non-competitive assay principle for measurement of small analytes based on quenching of fluorescence is described. Fluorescence resonance energy transfer (FRET) occurs between the donor, intrinsically fluorescent europium(III)-chelate conjugated to streptavidin, and the acceptor, quencher dye conjugated to biotin derivative when the biotin-quencher is bound to Eu-streptavidin. Fluorescence can be measured only from those streptavidins that are bound to biotin of the sample, while the fluorescence of the streptavidins that are not occupied by biotin are quenched by quencher-biotin conjugates.