Collectively, these results showed that prions can be captured from a complex inhibitor-laden biological fluid in a manner that is compatible with ultrasensitive detection by in vitro
prion amplification assays. The eQuIC assay in particular provides a practical, high-throughput, and rapid means of testing for amounts of PrPres
(e.g., 1 ag) that are several orders of magnitude below those typically required to cause prion disease by intracerebral inoculation into animals. The ability of eQuIC to detect prions in plasma samples raises the possibility that this assay could be used to improve prion disease diagnosis in humans and animals and to screen the blood supply for prion contamination. We have demonstrated discrimination of scrapie-infected and uninfected hamsters based on eQuIC analysis of their blood plasma samples; thus, the assay has at least some capability to detect natural blood-borne prion seeding activity. However, although we have also shown extreme sensitivity of the eQuIC in detecting brain-derived spikes of vCJD into human plasma, the sensitivity of eQuIC detection of vCJD seeding activity that is endogenous to plasma remains to be determined. We focused initially on plasma because it is a main target fluid for the development of an in vivo vCJD test. However, as 5- to 10-fold more CJD infectivity has been found in leukocyte fractions of blood (40
), it is possible that somewhat greater sensitivity could be obtained by eQuIC analysis of leukocytes. 15B3-based prion capture might also enhance the utility of other prion amplification assays. Ultimately, it will be important for TSE surveillance labs to have multiple assays at their disposal so that positive tests can be reevaluated by other types of tests.
The two-stage substrate addition that we describe here for the eQuIC differs from serial (multiple-round) amplification steps that have been described previously for protein misfolding cyclic amplification (PMCA) (5
), rPrP-PMCA (21
), and QuIC (20
) reactions because most of the bead-bound prions and prion-seeded products are retained in the reaction vessel so that the substrate can be replaced without removing most of the seed particles. In contrast, in serial PMCA and S-QuIC reactions, only a small proportion (typically ≤10%) of the total reaction mixture is transferred to a new vessel containing fresh substrate, so that much of the seeding activity from the first round is lost.
The mechanistic basis for the dramatic improvements in assay time and sensitivity due to the substrate replacement is not yet clear, but our results suggest that at least two processes are occurring during the initial lag phase of the eQuIC reaction, i.e., between the addition of seed and substrate replacement (). First, the rPrPC
must be moving into a pool that is less rapidly accessible to prion-seeded fibril assembly, such as an off-pathway oligomer (OO); otherwise, the addition of fresh rPrPC
after 20 h, but before the initial substrate is converted to detectable ThT-positive fibrillar products, would not accelerate the reaction. We suggest a “less rapidly accessible” pool rather than an inaccessible pool because even without substrate replenishment, the vast majority of the substrate can still be converted if given enough time. Second, the initial seed must be being altered and primed in some way to seed more rapid fibril assembly upon the addition of fresh substrate; otherwise, it would have been capable of seeding rapid ThT-positive rPrP-res(Sc)
assembly at the beginning of the reaction, when there was the same concentration of fresh substrate. This priming effect might be explained by secondary nucleation mechanisms (41
), such as those marked with red stars in . For example, during the lag phase, prion seeds may elongate by incorporating rPrPC
at a relatively slow and largely undetected rate determined in part by the concentration of seed particles. With continued elongation, the seeded rPrP fibrils would become long enough to be sheared by agitation, increasing the seed particle concentration and accelerating overall fibril assembly. Moreover, other types of fibril-dependent secondary nucleation might contribute to the acceleration of fibril assembly. For instance, fibril assembly might be hastened by the prealignment or scaffolding of rPrPC
substrate or amyloidogenic intermediate (AI) along the sides of an existing fibril, either with or without the need for a similarly aligned seed. In any case, further studies will be required to define the mechanistic underpinnings of the effects of two-phase substrate addition.
Potential mechanisms of substrate replacement effect.
As we have seen before with the S-QuIC assay (24
), the best rPrPC
substrate for the RT-QuIC is not always the one that is the most homologous with the type of prion/PrPres
being assayed. For example, we were surprised to find that the substrate that worked best for the detection of human vCJD was the chimeric hamster-sheep construct (Ha-S rPrPC
) rather than a human rPrPC
molecule. The reasons for this are not clear, but may relate to factors such as (i) the relative tendencies of the rPrPC
to be consumed by off-pathway changes that remove substrate from the assembly-competent pool or promote spontaneous, prion-independent fibril formation (i.e., false positives) or (ii) differences in propensities to form an amyloidogenic precursor or intermediate which interacts most favorably with prion seeds (). Such a precursor may be distinct from native rPrPC
, as has been described for other types of seeded polymerizations of PrPC
). These factors are likely to be condition dependent and, for the practical purposes of detecting vCJD PrPres
, appear to override problems associated with sequence mismatches between the vCJD seed and the Ha-S rPrPC
The eQuIC assay offers advantages compared to other currently established ultrasensitive prion/PrPSc
assays. Relative to the first-generation RT-QuIC assay, the eQuIC not only allows for prion detection in inhibitor-laden samples such as plasma, but also enhances the sensitivity for vCJD brain homogenate dilutions into human plasma by at least 10,000-fold. Compared to PMCA reactions that have been described, the eQuIC is more rapid for a given sensitivity level, more practical by using bacteria rather than brain as the source of PrPC
substrate, more easily replicated by using shaking rather than sonication, and more amenable to high-throughput analyses due to multiwell-plate-based reactions and fluorescence detection. Fluorescence plate readers should be more commonly available than the specialized instrumentation required for some other tests (25
). Recently, Edgeworth and colleagues described a new vCJD PrPres
detection assay which includes prion capture on stainless steel beads and an enzyme-linked immunosorbent assay (ELISA) detection method (30
). Whereas this capture ELISA detected 1010
-fold dilutions of vCJD brain in whole blood, our eQuIC assay detected 1014
-fold dilutions in plasma. The Edgeworth assay detected PrPvCJD
in blood from 15 symptomatic patients with ~70% sensitivity and 100% specificity, which is nearly as effective as the RT-QuIC in diagnosing sporadic CJD by using CSF samples (32
). However, the ~10,000-fold greater sensitivity of the eQuIC in detecting brain-derived vCJD seeding activity suggests that it has considerable potential to improve the sensitivity of vCJD and sCJD diagnosis using blood, plasma, CSF, or other samples. However, we have not yet gained access to vCJD patient plasma or CSF samples, and further studies will be required to assess the diagnostic utility of eQuIC based on the detection of vCJD seeding activity that is endogenous to these and other specimens. The remarkable resistance to inactivation of prions relative to other pathogens also makes it important to develop practical assays for prion contamination in a wide variety of materials, such as foods, feeds, transplanted tissues, medical devices, agricultural wastes and by-products, soils, water sources, and other environmental samples. The ability to immunoprecipitate prions from complex matrices and detect them with high sensitivity should foster the further development of such assays.