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Letters to the Editor |
Department of Biotechnology, University of Turku, Turku, Finland
aAddress correspondence to this author at: Department of Biotechnology, University of Turku, Tykistökatu 6A, FIN-20520 Turku, Finland. Fax 358-2-333-8050; e-mail katri.kuningas{at}utu.fi
To the Editor:
Many medical conditions are related to alterations in protein or hormone concentrations in blood. These changes are commonly detected with heterogeneous immunoassays that require a separation step before signal measurement. A simple homogeneous assay performable directly in whole blood would be useful in clinical diagnostics, but the performance of homogeneous fluorescence-based immunoassays has been severely limited by autofluorescence and strong absorption of ultraviolet and visible light in whole blood. These problems might be avoided by the use of near-infrared excited upconversion fluorescence. We recently described a novel homogeneous assay method based on upconversion fluorescence resonance energy transfer (FRET). In this method an upconverting phosphor (UCP) is used as a donor and a fluorescent protein or a small-molecular fluorescent dye is used as an acceptor (1)(2). We report here the use of a competitive homogeneous immunoassay for 17ß-estradiol (E2) in whole blood as a model to demonstrate the application of upconversion FRET using a near-infrared fluorescent acceptor dye (Fig. 1A
; and see Fig. 1 in the Data Supplement that accompanies the online version of this Letter at http://www.clinchem.org/content/vol53/issue1).
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A UCP (Luminophor SPF) with a structural composition of La2O2S:Yb3+,Er3+ was coated as described earlier (1)(2)(3) with an E2-specific recombinant antibody Fab fragment with a known cross-reactivity profile(4)(5). A Fab fragment was used to provide more favorable donor–acceptor distances for resonance energy transfer(5). The average size of the UCP donor particle was 
390 nm, determined with a Coulter N4 Plus submicron particle size analyzer (Beckman Coulter). A succinimidyl ester of a small-molecular acceptor dye, Alexa Fluor 680 (AF680) (Molecular Probes, Invitrogen Corp.), was coupled with a 2.5-fold molar excess of the amino-derivative of E2 (6-oxoestradiol 6-[O-(6-aminohexyl)oxime]) as described(2). Whole blood was collected in lithium heparin anticoagulated tubes (Venoject; Terumo Europe) from male volunteers at the Department of Biotechnology, University of Turku, and used in the assay with their informed consent. The E2 immunoassay was carried out in buffer, plasma, and blood according to an assay principle previously described(2). Blood and plasma samples were diluted in buffer to comprise 20% of the total 45-µL reaction volume. The reactions were incubated at +30 °C by rotating the microtiter plate 6 times per minute around its horizontal axis to prevent erythrocyte sedimentation. Donor emission from the UCP and sensitized acceptor emission from the fluorescent dye were measured at 665 nm and at 740 nm, respectively, under continuous laser excitation at 980 nm using Plate Chameleon (Hidex Oy)(3).
Examination of the ratio of maximum (without E2) and minimum (with 10 000 nmol/L E2) energy-transfer signals in buffer and whole blood with a 15 mg/L phosphor donor was optimal when 4 nmol/L of acceptor dye was used (see Fig. 2 in the online Data Supplement). Binding of the acceptor to the donor was even observed with a 31.25 pmol/L acceptor with a ratio of 8 in buffer and 6 in blood. Standard curves (Fig. 1B
) and interassay precision profiles (see Fig. 3 in the online Data Supplement) for the E2-immunoassay, obtained with optimal donor and acceptor concentrations, were very similar in buffer, heparin plasma, and whole blood. Detection limits of the assay (defined here as concentrations that inhibited 20% of the maximum signal) in the 3 matrices were estimated to be 0.21 nmol/L (60 ng/L), 0.46 nmol/L (120 ng/L) and 0.49 nmol/L (130 ng/L), respectively. Analytical recoveries for E2 in plasma and blood are presented in Table 1 of the online Data Supplement. Lower recoveries and higher detection limits in plasma and blood compared with buffer were assumed to result from the presence of endogenous steroid-binding proteins in biological matrices, increasing the total binder concentration in assay reactions (2). Minimum signals (background signals of the assay), originating from nonspecific binding interactions and nonproximity-based reabsorptive energy transfer(1)(2), were slightly increased in whole blood compared with other sample matrices, most likely because of stronger nonspecific binding interactions in blood.
Under infrared excitation, no autofluorescence from blood was detected at visible wavelengths, owing to the unique nature of upconversion. Because of the relative transparency of blood to light at wavelengths above 650 nm, the excitation of the UCP was not attenuated (see Fig. 4 in the online Data Supplement). The measurement of the sensitized acceptor emission above 700 nm completely eliminates the background arising from blood and from possible donor emission, even without temporal resolution in fluorescence detection. Moreover, in the upconversion FRET-based assay no emission from the acceptor alone is produced upon infrared excitation. The assay is relatively simple and can be performed in a standard microtiter plate assay format using uncomplicated detection instrumentation. The ability to perform homogeneous fluorescence-based immunoassays directly in whole blood provides new possibilities for biomedical research and clinical diagnostics.
Acknowledgments
This study was supported by the Finnish Funding Agency for Technology and Innovation (Tekes), the Academy of Finland (Grant No. 209417) and the Graduate School of In Vitro Diagnostics in Finland. A grant from the Instrumentarium Science Foundation denoted to K.K. is also gratefully acknowledged. The authors are thankful for technological support from Hidex Oy in anti-Stokes photoluminescence measurement and from Pirjo Laaksonen in drawing blood samples.
References
The following articles in journals at HighWire Press have cited this article:
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  S. Jiang, M. K. Gnanasammandhan, and Y. Zhang Optical imaging-guided cancer therapy with fluorescent nanoparticles J R Soc Interface, January 6, 2010; 7(42): 3 - 18. [Abstract] [Full Text] [PDF]
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, dashed line) and plasma (
, narrow solid line) were obtained after 45 min of incubation using 15 mg/L UCP donor and 4 nmol/L acceptor conjugate. IC50 values (concentration that inhibited 50% of the maximum signal) of the assay were 0.72 nmol/L, 1.25 nmol/L, and 1.35 nmol/L, respectively. Standard curves were fitted to the data based on means of 4 replicates using the program Origin 6.0 (OriginLab Corporation) and the logistic function y = (A1 – A2)/[1 + (x/x0)p)] + A2, where A1 and A2 correspond to maximum and minimum values of the response, respectively, p is the slope and x0 is the IC50 value. The error bars indicate the SD of the value. cts, counts.