Establishment of Real-Time QUIC (RT-QUIC)
In the QUIC assays, soluble recombinant PrP (rPrP-sen) expressed in E. coli is used as a substrate to amplify the minute amounts of PrPSc. Using a dedicated shaker, the reaction is enhanced by vigorous intermittent shaking which induces rPrP-sen to aggregate and form fibrils.9 One of the advantages in QUIC is that shaking/agitation can be performed more easily and consistently than sonication, which has the problem of varied delivery of vibrational energy to the samples. On the other hand, further improvement in the rapidity and practicality of this method was required in order for it to become useful in the diagnosis of prion diseases, because the initial standard format of QUIC (S-QUIC) required a time-consuming western blot. Thus, we combined QUIC technology with thioflavin T (ThT) fluorescence dye, to monitor amyloid formation, in order to minimize the time necessary for the detection of protease-resistant rPrP fibrils (rPrP-res). We first determined if the shaking of our fluorescence microplate reader could induce PrPSc-dependent rPrP-res (rPrP-resSc) formation in a buffer containing 0.05–0.1% SDS, as in the S-QUIC. Human rPrP-sen (rHuPrP-sen) and CJD brain homogenate (BH) were used as a substrate and a seed, respectively, for the QUIC reaction. Unexpectedly, we did not observe rPrP-resSc formation or an elevation in the ThT fluorescence using our microplate reader (unpublished data). Our explanation for this is that the shaking power of the microplate reader was not strong enough to elicit the QUIC reactions in the presence of SDS. SDS tends to cause fibrils to stack and stabilize rPrP-res polymers as a result. In fact, we observed that fibrils formed in the presence of SDS were much larger and thicker than those formed in its absence. Taken together, sonication in PMCA or vigorous shaking in S-QUIC seems to be required as a means of fragmenting the rPrP-res polymers formed in the presence of SDS.

We then tested whether rPrP-resSc formation was induced when guanidine-HCl (GdnHCl) was added, because it has been thought that GdnHCl was required for the conversion of PrP-sen to PrP-res in a cell-free system.11 Somewhat unexpectedly, we observed rPrP-resSc formation even in the absence of GdnHCl. In contrast, the negative control reactions without seed and in the absence of GdnHCl exhibited a marked delay in spontaneous rPrP-res (rPrP-resspon) formation.12 For this reason, use of a GdnHCl-free buffer can dramatically reduce the risk of false-positive reactions and enhance the sensitivity of the method.

Shaking/agitation is considered to cause several facilitatory effects on the QUIC reaction. One is that a partial unfolding of a portion of the rPrP-sen is induced by increasing the air-water interface through which a denaturing boundary between the hydrophobic air and hydrophilic water is formed.13 Next, shaking/agitation enhances the interaction between rPrP-sen and PrPSc, and the fragmentation of rPrP-res polymers.14 The energetic barrier of spontaneous fibril formation is likely to be higher than that of seed-dependent fibril formation or elongation, because spontaneous formation initially necessitates nucleation as the rate-limiting step.15

Meanwhile, the extent of the partial unfolding of rPrP-sen by shaking alone is assumed to be more heterogeneous than that in the presence of GdnHCl, probably because the air-water interfaces are unequally distributed in the reaction mixture (Fig. 1). In contrast, the addition of GdnHCl accelerates the nucleation rate, resulting in an increase in the rate of spontaneous fibril formation. Of note, we observed that there was an inverse correlation between the rate of rPrP-res formation and the concentration of rPrP-sen substrate.12 It has been reported that the aggregation rate of several other proteins is inversely correlated with the concentration of substrate protein in a denaturant-free buffer with shaking.16,17 Conversely, previous studies using cell-free conversion18 and rPrP fibril formation,19–21 respectively, in the presence of denaturant or at low pH, have shown that the rate of PrP-res formation was directly proportional to the PrP-sen concentration. This seeming contradiction can be explained again by the difference in the denaturation status of PrP-sen under various conditions. We hypothesized that heterogenous denaturation of the substrate protein in a denaturant-free buffer with shaking is a major cause of the inverse correlation
We examined the effect of pH, and the concentrations of rHuPrP-sen and salt, on QUIC reactions in a GdnHCl-free buffer. We found that the presence of NaCl is essential for rPrP-res formation and the sensitivity of this method was maximal at 500 mM NaCl at pH 7.12 The requirement for NaCl in the formation of rPrP-res is compatible with previous studies, which have shown that salt is required for cell-free conversion in the absence of GdnHCl22 and the maintenance of a protease-resistant PrPSc conformation.23

We named this new assay “real-time QUIC (RT-QUIC)” by analogy with real-time PCR. The RT-QUIC enabled us to measure up to 96 replicates at a time, obtain the results immediately, and is potentially safer than S-QUIC or PMCA because the prions are sealed within a 96-well plate throughout the entire procedure.

Be careful of using corn. You very well could be hand feeding the herd CWD. Farmers in CWD positive states are sending corn and hay all across the country and are most likely spreading the prions right along with it.