Homogeneous dual-parameter assay for prostate-specific antigen based on fluorescence resonance energy transfer.
Publication/Presentation Date
12-15-2008
Abstract
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 Pearson's 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.
Volume
80
Issue
24
First Page
9763
Last Page
9768
ISSN
1520-6882
Published In/Presented At
Kokko, T., Liljenbäck, T., Peltola, M. T., Kokko, L., & Soukka, T. (2008). Homogeneous dual-parameter assay for prostate-specific antigen based on fluorescence resonance energy transfer. Analytical chemistry, 80(24), 9763–9768. https://doi.org/10.1021/ac801875a
Disciplines
Medicine and Health Sciences
PubMedID
19012418
Department(s)
Department of Pathology and Laboratory Medicine
Document Type
Article