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Prion disease blood test using immunoprecipitation and improved quaking-induced conversion.

Orrú CD, Wilham JM, Raymond LD, Kuhn F, Schroeder B, Raeber AJ, Caughey B - MBio (2011)

Bottom Line: We focused initially on blood plasma because a practical blood test for prions would be especially valuable for TSE diagnostics and risk reduction.Variant Creutzfeldt-Jakob disease (vCJD) in particular has been transmitted between humans via blood transfusions.Enhanced real-time quaking-induced conversion (eRTQ) provides by far the most sensitive detection of vCJD to date.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.

ABSTRACT

Unlabelled: A key challenge in managing transmissible spongiform encephalopathies (TSEs) or prion diseases in medicine, agriculture, and wildlife biology is the development of practical tests for prions that are at or below infectious levels. Of particular interest are tests capable of detecting prions in blood components such as plasma, but blood typically has extremely low prion concentrations and contains inhibitors of the most sensitive prion tests. One of the latter tests is quaking-induced conversion (QuIC), which can be as sensitive as in vivo bioassays, but much more rapid, higher throughput, and less expensive. Now we have integrated antibody 15B3-based immunoprecipitation with QuIC reactions to increase sensitivity and isolate prions from inhibitors such as those in plasma samples. Coupling of immunoprecipitation and an improved real-time QuIC reaction dramatically enhanced detection of variant Creutzfeldt-Jakob disease (vCJD) brain tissue diluted into human plasma. Dilutions of 10(14)-fold, containing ~2 attogram (ag) per ml of proteinase K-resistant prion protein, were readily detected, indicating ~10,000-fold greater sensitivity for vCJD brain than has previously been reported. We also discriminated between plasma and serum samples from scrapie-infected and uninfected hamsters, even in early preclinical stages. This combined assay, which we call "enhanced QuIC" (eQuIC), markedly improves prospects for routine detection of low levels of prions in tissues, fluids, or environmental samples.

Importance: Transmissible spongiform encephalopathies (TSEs) are largely untreatable and are difficult to diagnose definitively prior to irreversible clinical decline or death. The transmissibility of TSEs within and between species highlights the need for practical tests for even the smallest amounts of infectivity. A few sufficiently sensitive in vitro methods have been reported, but most have major limitations that would preclude their use in routine diagnostic or screening applications. Our new assay improves the outlook for such critical applications. We focused initially on blood plasma because a practical blood test for prions would be especially valuable for TSE diagnostics and risk reduction. Variant Creutzfeldt-Jakob disease (vCJD) in particular has been transmitted between humans via blood transfusions. Enhanced real-time quaking-induced conversion (eRTQ) provides by far the most sensitive detection of vCJD to date. The 15B3 antibody binds prions of multiple species, suggesting that our assay may be useful for clinical and fundamental studies of a variety of TSEs of humans and animals.

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Detection of endogenous PrPSc in plasma of scrapie-infected hamsters by IP-S-QuIC. (a) Plasma samples from scrapie 263K-infected and uninfected (N) hamsters (500 µl) were subjected to IP-SQ as described in Materials and Methods with the first round of S-QuIC at 50°C for 10 h and the second round (b) at 50°C for 8 h, except for lanes marked with asterisks, which show the first-round products seeded with sample no. 6 for comparison. Plasma-free positive and negative control reactions (Rxn ctrls), rPrPC23–231 substrate, and analysis of PK-digested products were as described for Fig. 1. Open circles mark 17-kDa fragments, and brackets indicate the lower-molecular-mass bands (10 to 13 kDa).
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f2: Detection of endogenous PrPSc in plasma of scrapie-infected hamsters by IP-S-QuIC. (a) Plasma samples from scrapie 263K-infected and uninfected (N) hamsters (500 µl) were subjected to IP-SQ as described in Materials and Methods with the first round of S-QuIC at 50°C for 10 h and the second round (b) at 50°C for 8 h, except for lanes marked with asterisks, which show the first-round products seeded with sample no. 6 for comparison. Plasma-free positive and negative control reactions (Rxn ctrls), rPrPC23–231 substrate, and analysis of PK-digested products were as described for Fig. 1. Open circles mark 17-kDa fragments, and brackets indicate the lower-molecular-mass bands (10 to 13 kDa).

Mentions: To develop a blood test for prions, we initially attempted to detect prions spiked into human and sheep plasma samples by directly adding spiked plasma to S-QuIC and RT-QuIC reaction mixtures. However, plasma components strongly inhibited both assays (data not shown), consistent with previously reported inhibition of protein misfolding cyclic amplification (PMCA) (38) and flow cytometric assays (23). Accordingly, we sought methods to capture and concentrate prions in a detectable form from plasma. Prion immunoaffinity beads were prepared by coupling monoclonal antibody 15B3 (34) to magnetic beads. This antibody selectively binds PrPres and other PrP oligomers but not monomeric PrPC (34, 36). The ability of 15B3-coupled beads to immunoprecipitate prion activity from plasma was first tested with the S-QuIC assay. We spiked 0.5 ml of human plasma with hamster scrapie and human vCJD brain homogenate dilutions or comparable TSE-negative brain homogenates and incubated them with the beads. The beads were then washed, added directly to the S-QuIC reaction, and subjected to cycles of shaking and rest. As described previously (20, 21, 24), positive prion-seeded S-QuIC reactions were indicated by the characteristic pattern of 17-, 13-, 12, and 11-kDa proteinase K-resistant products [called rPrP-res(Sc)] in immunoblots. We performed two-round reactions by seeding aliquots of first-round reaction products into fresh rPrPC substrate. Control (mock) beads coated only with anti-IgM antibodies (without 15B3) had some affinity for prions, as indicated, for example, by the positive rPrP-res(Sc) products generated in one of the two replicate single-round reaction mixtures seeded with beads incubated with plasma spiked with a 1 × 10−9 dilution of scrapie brain homogenate containing ~100 fg PrPres (see the lane marked by the asterisk in Fig. S1a in the supplemental material). However, 15B3-coated beads were ~100-fold more efficient at capturing lower levels of prions from plasma, enabling detection of dilutions containing ≥1 fg PrPres (Fig. S1a and b). This IP-S-QuIC protocol gave positive reactions from as little as 4 × 10−10 dilutions of vCJD brain homogenate containing ~10 fg of human PrPres (Fig. 1) and 2 × 10−11 dilutions of scrapie hamster brain containing ~1 fg of PrPres (Fig. S1). In contrast, no positive rPrP-res(Sc) reaction products were obtained in reactions seeded with non-TSE human or hamster brain homogenates. Moreover, 15B3 IP-S-QuIC detected prion activity naturally present in 0.5 ml of plasma from nine near-terminal scrapie-infected hamsters, while no positive S-QuIC reactions were seeded by plasma from a negative control hamster in two-round reactions (Fig. 2).


Prion disease blood test using immunoprecipitation and improved quaking-induced conversion.

Orrú CD, Wilham JM, Raymond LD, Kuhn F, Schroeder B, Raeber AJ, Caughey B - MBio (2011)

Detection of endogenous PrPSc in plasma of scrapie-infected hamsters by IP-S-QuIC. (a) Plasma samples from scrapie 263K-infected and uninfected (N) hamsters (500 µl) were subjected to IP-SQ as described in Materials and Methods with the first round of S-QuIC at 50°C for 10 h and the second round (b) at 50°C for 8 h, except for lanes marked with asterisks, which show the first-round products seeded with sample no. 6 for comparison. Plasma-free positive and negative control reactions (Rxn ctrls), rPrPC23–231 substrate, and analysis of PK-digested products were as described for Fig. 1. Open circles mark 17-kDa fragments, and brackets indicate the lower-molecular-mass bands (10 to 13 kDa).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3101782&req=5

f2: Detection of endogenous PrPSc in plasma of scrapie-infected hamsters by IP-S-QuIC. (a) Plasma samples from scrapie 263K-infected and uninfected (N) hamsters (500 µl) were subjected to IP-SQ as described in Materials and Methods with the first round of S-QuIC at 50°C for 10 h and the second round (b) at 50°C for 8 h, except for lanes marked with asterisks, which show the first-round products seeded with sample no. 6 for comparison. Plasma-free positive and negative control reactions (Rxn ctrls), rPrPC23–231 substrate, and analysis of PK-digested products were as described for Fig. 1. Open circles mark 17-kDa fragments, and brackets indicate the lower-molecular-mass bands (10 to 13 kDa).
Mentions: To develop a blood test for prions, we initially attempted to detect prions spiked into human and sheep plasma samples by directly adding spiked plasma to S-QuIC and RT-QuIC reaction mixtures. However, plasma components strongly inhibited both assays (data not shown), consistent with previously reported inhibition of protein misfolding cyclic amplification (PMCA) (38) and flow cytometric assays (23). Accordingly, we sought methods to capture and concentrate prions in a detectable form from plasma. Prion immunoaffinity beads were prepared by coupling monoclonal antibody 15B3 (34) to magnetic beads. This antibody selectively binds PrPres and other PrP oligomers but not monomeric PrPC (34, 36). The ability of 15B3-coupled beads to immunoprecipitate prion activity from plasma was first tested with the S-QuIC assay. We spiked 0.5 ml of human plasma with hamster scrapie and human vCJD brain homogenate dilutions or comparable TSE-negative brain homogenates and incubated them with the beads. The beads were then washed, added directly to the S-QuIC reaction, and subjected to cycles of shaking and rest. As described previously (20, 21, 24), positive prion-seeded S-QuIC reactions were indicated by the characteristic pattern of 17-, 13-, 12, and 11-kDa proteinase K-resistant products [called rPrP-res(Sc)] in immunoblots. We performed two-round reactions by seeding aliquots of first-round reaction products into fresh rPrPC substrate. Control (mock) beads coated only with anti-IgM antibodies (without 15B3) had some affinity for prions, as indicated, for example, by the positive rPrP-res(Sc) products generated in one of the two replicate single-round reaction mixtures seeded with beads incubated with plasma spiked with a 1 × 10−9 dilution of scrapie brain homogenate containing ~100 fg PrPres (see the lane marked by the asterisk in Fig. S1a in the supplemental material). However, 15B3-coated beads were ~100-fold more efficient at capturing lower levels of prions from plasma, enabling detection of dilutions containing ≥1 fg PrPres (Fig. S1a and b). This IP-S-QuIC protocol gave positive reactions from as little as 4 × 10−10 dilutions of vCJD brain homogenate containing ~10 fg of human PrPres (Fig. 1) and 2 × 10−11 dilutions of scrapie hamster brain containing ~1 fg of PrPres (Fig. S1). In contrast, no positive rPrP-res(Sc) reaction products were obtained in reactions seeded with non-TSE human or hamster brain homogenates. Moreover, 15B3 IP-S-QuIC detected prion activity naturally present in 0.5 ml of plasma from nine near-terminal scrapie-infected hamsters, while no positive S-QuIC reactions were seeded by plasma from a negative control hamster in two-round reactions (Fig. 2).

Bottom Line: We focused initially on blood plasma because a practical blood test for prions would be especially valuable for TSE diagnostics and risk reduction.Variant Creutzfeldt-Jakob disease (vCJD) in particular has been transmitted between humans via blood transfusions.Enhanced real-time quaking-induced conversion (eRTQ) provides by far the most sensitive detection of vCJD to date.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.

ABSTRACT

Unlabelled: A key challenge in managing transmissible spongiform encephalopathies (TSEs) or prion diseases in medicine, agriculture, and wildlife biology is the development of practical tests for prions that are at or below infectious levels. Of particular interest are tests capable of detecting prions in blood components such as plasma, but blood typically has extremely low prion concentrations and contains inhibitors of the most sensitive prion tests. One of the latter tests is quaking-induced conversion (QuIC), which can be as sensitive as in vivo bioassays, but much more rapid, higher throughput, and less expensive. Now we have integrated antibody 15B3-based immunoprecipitation with QuIC reactions to increase sensitivity and isolate prions from inhibitors such as those in plasma samples. Coupling of immunoprecipitation and an improved real-time QuIC reaction dramatically enhanced detection of variant Creutzfeldt-Jakob disease (vCJD) brain tissue diluted into human plasma. Dilutions of 10(14)-fold, containing ~2 attogram (ag) per ml of proteinase K-resistant prion protein, were readily detected, indicating ~10,000-fold greater sensitivity for vCJD brain than has previously been reported. We also discriminated between plasma and serum samples from scrapie-infected and uninfected hamsters, even in early preclinical stages. This combined assay, which we call "enhanced QuIC" (eQuIC), markedly improves prospects for routine detection of low levels of prions in tissues, fluids, or environmental samples.

Importance: Transmissible spongiform encephalopathies (TSEs) are largely untreatable and are difficult to diagnose definitively prior to irreversible clinical decline or death. The transmissibility of TSEs within and between species highlights the need for practical tests for even the smallest amounts of infectivity. A few sufficiently sensitive in vitro methods have been reported, but most have major limitations that would preclude their use in routine diagnostic or screening applications. Our new assay improves the outlook for such critical applications. We focused initially on blood plasma because a practical blood test for prions would be especially valuable for TSE diagnostics and risk reduction. Variant Creutzfeldt-Jakob disease (vCJD) in particular has been transmitted between humans via blood transfusions. Enhanced real-time quaking-induced conversion (eRTQ) provides by far the most sensitive detection of vCJD to date. The 15B3 antibody binds prions of multiple species, suggesting that our assay may be useful for clinical and fundamental studies of a variety of TSEs of humans and animals.

Show MeSH
Related in: MedlinePlus