Anal. Chem. 2008, 80, 9763–9768
Homogeneous Dual-Parameter Assay for Prostate-Specific Antigen Based on Fluorescence Resonance Energy Transfer Tiina Kokko,* Tuomo Liljenba ¨ ck, Mari T. Peltola, Leena Kokko, and Tero Soukka Department of Biotechnology, University of Turku, Turku, Finland Three monoclonal antibodies (Mab) specific for prostatespecific 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 singleparameter 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. Fluorescence resonance energy transfer (FRET) is defined as a nonradiative energy transfer from an excited donor molecule to an acceptor fluorophore.1 For FRET to occur, short distance between donor and acceptor as well as spectral overlapping between the emission of the donor and the excitation of the acceptor are required (Fo¨rster theory).2 Intrinsically fluorescent lanthanide chelates are a group of interesting donors for FRET * Corresponding author. E-mail:
[email protected]. Phone: +358 2 333 8084. Fax: +358 2 333 8050. (1) Selvin, P. R.; Rana, T. M.; Hearst, J. E. J. Am. Chem. Soc. 1994, 116, 6029– 6030. (2) Fo ¨rster, T. Ann. Phys. 1948, 6, 55–74. 10.1021/ac801875a CCC: $40.75 2008 American Chemical Society Published on Web 11/14/2008
due to their many special properties.3 The slow decay of the lanthanide luminescence extends the observed lifetime of the fluorescent acceptors, and the sensitized emission of the acceptor can be measured using temporal delay with little or no interfering background.4 They also have an unusually large Stokes’ shift and usually more than one emission peak. Furthermore, these peaks are narrow and well separated from each other.5 These properties have been used to create a homogeneous assay utilizing the FRET between two labeled antibodies (sandwich-type assay). Blomberg et al. have created a human chorionic gonadotropin (βhCG) assay using FRET between terbium(III) chelate and Alexa Fluor 546.6 Furthermore, a similar technique is used commercially in Kryptor systems by Brahms (Henningsdorf, Germany).7 Lanthanide chelates have been used to create multiparameter assays. These assays are often heterogeneous and utilize the sharp emission peaks of the different lanthanides measuring the emissions at lanthanide-specific wavelengths (for additional information, see, for example, Hemmila¨ and Mukkala8). Karvinen et al. created a multiparameter assay for caspases using FRET to quench the emission of europium(III), terbium(III), and samarium(III) chelates. Thus, the assay could simultaneously measure the activity of three different caspases.9 However, creating a homogeneous multiplexed assay that simultaneously utilizes different lanthanides as donors together with appropriate fluorescent dyes as acceptors is challenging because the emission maxima and the emission valleys of the different lanthanides coincide. Therefore, there could be donor emission within the whole measurement range making the selection of the appropriate measurement wavelengths for sensitized emission of the acceptors very difficult. However, it has recently been shown that a lanthanide chelate (terbium(III) chelate) can act as a single donor for multiple acceptors. Futhermore, if the acceptors are chosen appropriately, it is possible to simultaneously measure the sensitized emission of different acceptors at acceptor-specific (3) Hemmila¨, I.; Laitala, V. J. Fluoresc. 2005, 15, 529–542. (4) Selvin, P. R.; Hearst, J. E. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 10024– 10028. (5) Gudgin Dickson, E. F.; Pollak, A.; Diamandis, E. P. J. Photochem. Photobiol., B 1995, 27, 3–19. (6) Blomberg, K.; Hurskainen, P.; Hemmila¨, I. Clin. Chem. 1999, 45, 855– 861. (7) Mathis, G. Clin. Chem. 1993, 39, 1953–1959. (8) Hemmila¨, I.; Mukkala, V.-M. Crit. Rev. Clin. Lab. Sci. 2001, 38, 441–519. (9) Karvinen, J.; Elomaa, A.; Ma¨kinen, M. L.; Hakala, H.; Mukkala, V. M.; Peuralahti, J.; Hurskainen, P.; Hovinen, J.; Hemmila¨, I. Anal. Biochem. 2004, 325, 317–325.
Analytical Chemistry, Vol. 80, No. 24, December 15, 2008
9763
wavelengths without crosstalk from the other acceptors.10 Recently, Kupcho et al. have published a research where terbium(III) chelate was used as a single donor for two separate acceptors to study ligand-specific interactions of peptides with nuclear receptor ligand binding domains.11 Prostate-specific antigen (PSA) is a member of the human kallikrein protease family.12 PSA is produced by prostatic epithelial cells13 and occurs mainly complexed to R1-antichymotrypsin (65-95% of the total PSA in blood). Some of the PSA (5-35%) is present in a free, unbound, form of PSA.14,15 It has been shown that the ratio between total PSA (PSA-T) and the free PSA (PSAF) is less in cancer cases than in benign prostatic hyperplasia (BPH).14-16 Thus, there is a diagnostic demand to measure both free and total PSA from a single sample. Two separate dualparameter heterogeneous immunoassays for PSA-F and PSA-T have been published. Leinonen et al. used samarium(III) and europium(III) chelate labeled antibodies achieving functional ranges of around 2-490 and 1-53 ng/mL for PSA-T and PSA-F, respectively,17 whereas Mitrunen et al. constructed a similar assay using terbium(III) and europium(III) chelates obtaining ∼0.5-50 ng/mL functional range for both free and total PSA.18 Typically, healthy young men (
