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. 2000 May 9;97(10):5468-73.
doi: 10.1073/pnas.97.10.5468.

Ultrasensitive detection of pathological prion protein aggregates by dual-color scanning for intensely fluorescent targets

Affiliations

Ultrasensitive detection of pathological prion protein aggregates by dual-color scanning for intensely fluorescent targets

J Bieschke et al. Proc Natl Acad Sci U S A. .

Abstract

A definite diagnosis of prion diseases such as Creutzfeldt-Jakob disease (CJD) relies on the detection of pathological prion protein (PrP(Sc)). However, no test for PrP(Sc) in cerebrospinal fluid (CSF) has been available thus far. Based on a setup for confocal dual-color fluorescence correlation spectroscopy, a technique suitable for single molecule detection, we developed a highly sensitive detection method for PrP(Sc). Pathological prion protein aggregates were labeled by specific antibody probes tagged with fluorescent dyes, resulting in intensely fluorescent targets, which were measured by dual-color fluorescence intensity distribution analysis in a confocal scanning setup. In a diagnostic model system, PrP(Sc) aggregates were detected down to a concentration of 2 pM PrP(Sc), corresponding to an aggregate concentration of approximately 2 fM, which was more than one order of magnitude more sensitive than Western blot analysis. A PrP(Sc)-specific signal could also be detected in a number of CSF samples from patients with CJD but not in control samples, providing the basis for a rapid and specific test for CJD and other prion diseases. Furthermore, this method could be adapted to the sensitive detection of other disease-associated amyloid aggregates such as in Alzheimer's disease.

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Figures

Figure 1
Figure 1
Evaluation of different probe molecules. Hamster rPrP (90–231) labeled with Oregon Green (A and B) and mAb 3F4 labeled with Alexa488 (C and D) were added to pooled CSF of control patients that was spiked with prion rods derived from scrapie-infected hamster brain. Measurements were performed in a single-color SIFT setup with a channel width set at 500 μs and a scanning speed of 1 mm/s for a measurement time of 600 s. The high-intensity signal seen in the cumulative intensity distribution analysis (A and C) was quantified by counting the number of channels with ≥500 photons per channel (B and D). Whereas the rPrP probe contains a significant amount of intrinsic high-intensity signal, virtually no background signal is found in the antibody probe, which allows quantitative target detection to be made with very high sensitivity.
Figure 2
Figure 2
Dual-color fluorescence intensity histogram. mAbs 3F4-Alexa488 and 12F10-Cy5 were added to pooled CSF of control patients that was spiked with prion rods derived from scrapie-infected hamster brain at various dilutions (A, 1:1,000; B, 1:100,000; C, no rods) or aggregates of Aβ (1–42) peptide at a concentration of 1 μM (D). (E) Unspecific antibody probes mAb(Aβ)-Alexa488 and mAb(IL-8)-Cy5 were added to prion rods diluted 1:1,000 in CSF. Samples were measured for 600 s with a channel time of 500 μs in an open volume element moving at 1 mm/s. Each dot represents a pair of fluorescent intensities. The number of channels is color-coded on a logarithmic scale. (F) Schematic representation of an intensity histogram. Low-intensity fluorescent signal of probe molecules (gray) is separated from unspecific aggregates incorporating only one type of label (red). Channels with a high-intensity signal in both colors above a linear cutoff (yellow) are summed for quantitative analysis (see Fig. 3). Fred, red fluorescence; Fgreen, green fluorescence.
Figure 3
Figure 3
Western blotting and dual-color SIFT measurement of prion rod dilution. (A) Brain homogenate of scrapie-infected hamster 263K (lanes a–f) and prion rod material (lanes g–o) in CSF was diluted as indicated. PrPSc was detected by Western blotting with 3F4 antibody after digestion with proteinase K (100 μg/ml) for 30 min at 37°C. (B) In parallel, aliquots of prion rod material were measured by dual-color SIFT, and the peak signal was quantified as in Fig. 2.
Figure 4
Figure 4
Dual-color SIFT measurements in CSF samples. Measurements were performed for a measurement time of 600 s with a channel time of 500 μs with mAbs 3F4-Alexa488 and 12F10-Cy5 and analyzed as described in Fig. 3. By using a cutoff level of one high-intensity channel, a specific dual-color, high-intensity signal was found in 5 of 24 patients with probable or definite CJD, whereas none of the CSF samples from 13 non-CJD cases with neurodegenerative disease contained a positive signal.

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