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Abstracts of Oak Ridge Posters |
1 Department of Biotechnology, University of Turku, Tykistökatu 6A, Turku FIN-20520, Finland
aauthor for correspondence: fax 358-2-333-8050, e-mail Petri.huhtinen{at}utu.fi
Long-lifetime fluorescent europium(III) chelate nanoparticles have been shown to be applicable as labels in various heterogeneous and homogeneous immunoassays. Assay performance can be improved when these intrinsically labeled nanoparticles are used in combination with time-resolved fluorescence detection. In several previous publications, we have shown the potential of nanoparticle labels in different assay applications. In heterogeneous assays, the dynamic range of assays at low concentrations can be extended (1)(2)(3). Furthermore, the kinetic characteristics of assays can be improved by use of detection antibody-coated high-specific-activity nanoparticle labels instead of conventionally labeled detection antibodies (4). In homogeneous assays, europium(III) nanoparticles have been shown to be efficient donors in fluorescence resonance energy transfer, enabling simple and rapid high-throughput screening (5). Heterogeneous and homogeneous nanoparticle-label-based assays can be run with various sample matrixes, e.g., serum (3), heparin plasma (3), and mucus (S. Huopalahti, A. Valanne, T. Soukka, R. Vainionpää, T. Lövgren, and H. Härmä, University of Turku, unpublished data).
In the present work we describe a kinetic, heterogeneous, one-step immunoassay application in which europium(III) nanoparticle labels are used in an all-in-one dry-reagent format (Aio!TM) (6) to produce a simple, rapid, and sensitive sandwich-type assay for thyroid-stimulating hormone (TSH). Conventionally, the Aio! technology is based on the europium(III) chelate label, but in the present work the chelate label was replaced by a nanoparticle label. TSH, a widely used thyroid disease marker and the most important single determinant of thyroid status (7), was selected as a model analyte because of the requirement for a low limit of quantification for thyroid diagnostics. Although thyroid diseases are not immediately life-threatening, TSH is a valuable analyte for a rapid testing. It is measured frequently; therefore, high cost-effectiveness can be achieved by enabling diagnosis during a patient’s first visit to the doctor’s office and by reducing the number of return visits.
The TSH sandwich assay was performed with anti-TSH monoclonal antibodies (Mabs) 5405 and 5409 (Medix Biochemica) for capture and detection, respectively; europium(III) nanoparticle labels (Seradyn Inc.; 107 nm in diameter); and Aio! dry reagent microtitration wells (Innotrac Diagnostics). The procedure for coating nanoparticles with the detection Mab is described in detail elsewhere (4). Biotinylation and immobilization of the capture Mab on the microtitration wells were performed as described previously (8). Before label addition, the solid-phase Mab was insulated with 40 µL of Aio! insulation layer solution (Innotrac Diagnostics) in an overnight incubation (35 °C at 5% humidity). The nanoparticle labels were added to the wells in a 1-µL volume (1.0 x 108 nanoparticles/well), and the wells were dried under an air stream. The ready-to-use dry-reagent wells were stored in sealed aluminum bags at room temperature.
The kinetic one-step TSH assay was performed with the Aio! immunoassay system, in which 5 µL of serum sample was applied in a 30-µL assay volume into the dry-reagent wells and incubated for 10 min. The wells were then washed, and the long-lifetime fluorescence of particulate labels was detected to give a quantitative TSH result.
The design of the all-in-one, dry-reagent assay for TSH with use of the long-lifetime fluorescent nanoparticle label is shown in Fig. 1
. Because the kinetic assay was performed with an automated immunoassay system, assay times were relatively short (total assay time <15 min) with little hands-on time.
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The detection limits (mean + 2 SD of the zero calibrator) of the nanoparticle assays were 0.0012 mIU/L (7 fmol/L) and 0.02 mIU/L (111 fmol/L) when calibrators were diluted in assay buffer or serum-based matrix, respectively. The linear dynamic range of the assay was more than three orders of magnitude (<0.1 to 50 mIU/L). At the high end of the examined concentration range, we observed a deviation from linearity, but we detected no hook effect even at the highest concentration studied (100 mIU/L).
Within- and between-run imprecisions (CVs) were 4–14% and 6–17%, respectively. Recovery of 0, 0.5, 2.5, and 5 mIU/L TSH added to four serum samples was 90–110%, except one value that differed by +24%. Dilution of four samples with the zero calibrator in ratios of 1:2, 1:4, and 1:8 gave results within 10% of the expected values. For a method comparison of the nanoparticle-label-based application (y) and AutoDelfia® (x), an automated commercial assay (PerkinElmer Life and Analytical Sciences), the regression equation had a slope (SD) of 0.96 (0.021) and y-intercept (SD) of 0.12 (0.089) mIU/L. The mean values obtained with the nanoparticle and commercial assays were 2.82 and 2.83 mIU/L, respectively (Sy|x = 0.49 mIU/L; R = 0.988; n = 54).
As can be seen from the difference plot in Fig. 1B
, the mean percentage difference between the two methods was 
6%, and 48 of the 54 samples differed by <20%.
We have described a quantitative nanoparticle-label-based one-step TSH immunoassay. The detection limits correspond to those for the fourth- and third-generation TSH assays. The large dynamic range well below and above the reference interval for TSH (0.5 to 5.0 mIU/L) (9)(10) allows detection of hypo- as well as hyperthyroidism. The sample and assay solution volumes are small, only 5 µL and 25 µL, respectively, and the required hands-on time is short. Furthermore, full automation of the assay is possible.
We conclude that the developed kinetic, all-in-one dry-reagent immunoassay using time-resolved fluorometry has great potential as a rapid, cost-effective, and sensitive assay when antibody-coated, high-specific-activity europium(III) nanoparticles are used as labels. We believe that the analytical approach is applicable to various analytes and sample matrixes. Furthermore, this simple and rapid assay format could be beneficial, e.g., when time is critical concerning the life of a patient, such as for cardiac diagnostics.
References
The following articles in journals at HighWire Press have cited this article:
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  Y. Xu and Q. Li Multiple Fluorescent Labeling of Silica Nanoparticles with Lanthanide Chelates for Highly Sensitive Time-Resolved Immunofluorometric Assays Clin. Chem., August 1, 2007; 53(8): 1503 - 1510. [Abstract] [Full Text] [PDF]
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