Leena Kokko

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. The amount of total PSA (PSA-T) was obtained by measuring the sensitized emission of AF680. Thus, the assay could simultaneously measure the amount of both PSA-F and PSA-T from a single sample. The lowest limits of detection with buffer calibrators were 0.74 and 0.60 ng/mL for PSA-F and PSA-T, respectively. Both of the assays were linear up to 100 ng/mL. The performance of the assay was also tested against heterogeneous single-parameter assays using spiked female plasma samples. The Pearsons correlations were 0.994 for PSA-F and 0.997 for PSA-T assays. However, due to the calibrator matrix being different from the sample matrix, the obtained concentrations with homogeneous assay, especially for PSA-F, were slightly less than with heterogeneous assays. In conclusion, it was shown that a homogeneous dual-parameter assay based on the measurement of the sensitized emission of two different acceptors in combination with a single donor can be performed. The assay was done in a single step using only one excitation wavelength and was functional within the clinically important analyte concentrations.

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 17β-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. All the tested dyes proved to be efficient acceptors, and they were successfully used in the competitive homogeneous E2 assay. The highest signal to background ratio and the best assay performance was obtained with Alexa Fluor 680, due to the very low donor emission background at the far-red area. In addition, the sensitized emission of both Alexa Fluor 488 and 680 could be measured simultaneously without significant cross talk.

Enzyme Inhibitor Screening Using a Homogeneous Proximity-Based Immunoassay for Estradiol

The authors have previously reported a homogeneous time-resolved fluorescence proximity immunoassay for estradiol. The assay was based on luminescence resonance energy transfer between a long lifetime fluorescent europium(III) chelate-dyed nanoparticle donor and a short lifetime, near-infrared fluorescent acceptor. The energy transfer prolonged the lifetime of the sensitized acceptor emission, and the fluorescence of the acceptor was measured using a time-resolved detection. The developed immunoassay was employed to screen inhibitors for enzyme 17β-hydroxysteroid dehydrogenase type 1. The enzyme overexpressed in MCF-7 cells catalyzed a reversible conversion of estroneto17β-estradiol. The inhibition efficiency of the tested molecule was obtained by comparing the final concentration of converted estradiol after 60 min of conversion reaction in a sample and in a conversion control not containing an inhibitor. The Zβ factor calculated using the E2 concentrations of the homogeneous assay was 0.64, demonstrating a relatively good performance of the assay. The results from the homogeneous assay were comparable with the results obtained using radioactively labeled estrone as a substrate and high-performance liquid chromatography (HPLC) separation of estrone and converted estradiol after the enzyme reaction. Thus, this homogeneous assay can simplify the primary screening of potential new drug molecules by replacing a tedious radiometric HPLC method.