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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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eQuIC detection of human PrPvCJD spiked into human plasma. Dilutions of human nonprion (tumor and Alzheimer’s disease) control or vCJD brain tissues were spiked into 500 µl of human plasma to give final dilutions of 4 × 10−7 (tumor and Alzheimer’s disease) and 4 × 10−12, 4 × 10−13, and 4 × 10−14 (vCJD; containing ~100, 10, and 1 ag PrPres, respectively). PrPvCJD was immunoprecipitated with 15B3-coated beads (a) or mock anti-IgM-coated beads (b), and a portion of the beads were used to seed replicate eQuIC reaction mixtures containing 400 mM NaCl. After 24 h, the substrate was replaced. The chimeric Ha-S rPrPC was used as a substrate in all reactions. The vertical axes indicate the average fluorescence from four replicate wells, and the fractions on the right indicate the positive/total replicate reactions associated with the adjacent traces.
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f4: eQuIC detection of human PrPvCJD spiked into human plasma. Dilutions of human nonprion (tumor and Alzheimer’s disease) control or vCJD brain tissues were spiked into 500 µl of human plasma to give final dilutions of 4 × 10−7 (tumor and Alzheimer’s disease) and 4 × 10−12, 4 × 10−13, and 4 × 10−14 (vCJD; containing ~100, 10, and 1 ag PrPres, respectively). PrPvCJD was immunoprecipitated with 15B3-coated beads (a) or mock anti-IgM-coated beads (b), and a portion of the beads were used to seed replicate eQuIC reaction mixtures containing 400 mM NaCl. After 24 h, the substrate was replaced. The chimeric Ha-S rPrPC was used as a substrate in all reactions. The vertical axes indicate the average fluorescence from four replicate wells, and the fractions on the right indicate the positive/total replicate reactions associated with the adjacent traces.

Mentions: To improve the sensitivity of IP-RT-QuIC we introduced a substrate replacement step after ~24 h of the RT-QuIC reaction. In IP-RT-QuIC reactions, the beads and associated prions or prion-induced RT-QuIC conversion products tended to adhere to the bottom of reaction wells. Thus, reaction fluid could be removed and fresh rPrPC added while retaining most of the beads and bead-bound reaction products in the well. This combination of IP and RT-QuIC with substrate replacement, which we call “enhanced RT-QuIC” (eQuIC), allowed detection of 4 × 10−14 dilutions of vCJD brain tissue (~1 attogram [ag] vCJD PrPres) within ~28 h in all replicate reactions (n = 4) in three independent experiments (e.g., Fig. 4A; see Fig. S4A in the supplemental material) performed using four different lots of human plasma. With a further 4 × 10−15 dilution, three of four replicate reactions were positive in a single experiment (data not shown). By comparison, Alzheimer’s and tumor brain negative control dilutions gave uniformly negative reactions in each of these eQuIC experiments. Mock beads lacking 15B3 gave much reduced sensitivity and consistency (Fig. 4b). Moreover, the 15B3-coupled beads provided for ≥106-fold more sensitive eQuIC detection than superparamagnetic nanoparticles that were reported recently to have prion-binding capacity (39) (Fig. S4). These results showed the ability of the 15B3-based eQuIC to detect extremely low concentrations of prions spiked into human plasma.


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)

eQuIC detection of human PrPvCJD spiked into human plasma. Dilutions of human nonprion (tumor and Alzheimer’s disease) control or vCJD brain tissues were spiked into 500 µl of human plasma to give final dilutions of 4 × 10−7 (tumor and Alzheimer’s disease) and 4 × 10−12, 4 × 10−13, and 4 × 10−14 (vCJD; containing ~100, 10, and 1 ag PrPres, respectively). PrPvCJD was immunoprecipitated with 15B3-coated beads (a) or mock anti-IgM-coated beads (b), and a portion of the beads were used to seed replicate eQuIC reaction mixtures containing 400 mM NaCl. After 24 h, the substrate was replaced. The chimeric Ha-S rPrPC was used as a substrate in all reactions. The vertical axes indicate the average fluorescence from four replicate wells, and the fractions on the right indicate the positive/total replicate reactions associated with the adjacent traces.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: eQuIC detection of human PrPvCJD spiked into human plasma. Dilutions of human nonprion (tumor and Alzheimer’s disease) control or vCJD brain tissues were spiked into 500 µl of human plasma to give final dilutions of 4 × 10−7 (tumor and Alzheimer’s disease) and 4 × 10−12, 4 × 10−13, and 4 × 10−14 (vCJD; containing ~100, 10, and 1 ag PrPres, respectively). PrPvCJD was immunoprecipitated with 15B3-coated beads (a) or mock anti-IgM-coated beads (b), and a portion of the beads were used to seed replicate eQuIC reaction mixtures containing 400 mM NaCl. After 24 h, the substrate was replaced. The chimeric Ha-S rPrPC was used as a substrate in all reactions. The vertical axes indicate the average fluorescence from four replicate wells, and the fractions on the right indicate the positive/total replicate reactions associated with the adjacent traces.
Mentions: To improve the sensitivity of IP-RT-QuIC we introduced a substrate replacement step after ~24 h of the RT-QuIC reaction. In IP-RT-QuIC reactions, the beads and associated prions or prion-induced RT-QuIC conversion products tended to adhere to the bottom of reaction wells. Thus, reaction fluid could be removed and fresh rPrPC added while retaining most of the beads and bead-bound reaction products in the well. This combination of IP and RT-QuIC with substrate replacement, which we call “enhanced RT-QuIC” (eQuIC), allowed detection of 4 × 10−14 dilutions of vCJD brain tissue (~1 attogram [ag] vCJD PrPres) within ~28 h in all replicate reactions (n = 4) in three independent experiments (e.g., Fig. 4A; see Fig. S4A in the supplemental material) performed using four different lots of human plasma. With a further 4 × 10−15 dilution, three of four replicate reactions were positive in a single experiment (data not shown). By comparison, Alzheimer’s and tumor brain negative control dilutions gave uniformly negative reactions in each of these eQuIC experiments. Mock beads lacking 15B3 gave much reduced sensitivity and consistency (Fig. 4b). Moreover, the 15B3-coupled beads provided for ≥106-fold more sensitive eQuIC detection than superparamagnetic nanoparticles that were reported recently to have prion-binding capacity (39) (Fig. S4). These results showed the ability of the 15B3-based eQuIC to detect extremely low concentrations of prions spiked into human plasma.

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