Human variant Creutzfeldt-Jakob disease and sheep scrapie PrP(res) detection using seeded conversion of recombinant prion protein

Abstract

The pathological isoform of the prion protein (PrP(res)) can serve as a marker for prion diseases, but more practical tests are needed for preclinical diagnosis and sensitive detection of many prion infections. Previously we showed that the quaking-induced conversion (QuIC) assay can detect sub-femtogram levels of PrP(res) in scrapie-infected hamster brain tissue and distinguish cerebral spinal fluid (CSF) samples from normal and scrapie-infected hamsters. We now report the adaptation of the QuIC reaction to prion diseases of medical and agricultural interest: human variant Creutzfeldt-Jakob disease (vCJD) and sheep scrapie. PrP(res)-positive and -negative brain homogenates from humans and sheep were discriminated within 1-2 days with a sensitivity of 10-100 fg PrP(res). More importantly, in as little as 22 h we were able to distinguish CSF samples from scrapie-infected and uninfected sheep. These results suggest the presence of prions in CSF from scrapie-infected sheep. This new method enables the relatively rapid and sensitive detection of human CJD and sheep scrapie PrP(res) and may facilitate the development of practical preclinical diagnostic and high-throughput interference tests.

Fig. 1

Detection limit of vCJD-seeded QuIC reactions using human and hamster rPrP^sen as substrates. Reactions were seeded with vCJD BH dilutions containing the indicated approximate amounts of PrP^res or, as a negative control, AD BH at a dilution equivalent to the most concentrated vCJD BH dilution. PK-digested reaction products were analyzed by immunoblotting. (A) Second-round reaction products (see text) using human rPrP^sen (23–231) as a substrate and antibody 3F4 (epitope: residues ∼109–112) to probe the immunoblot. (B) Single-round reaction products using hamster rPrP^sen (23–231) as substrate and antiserum R20 (C-terminal epitope). Upper panels show 12 h reactions at 45°C and lower panels show 8 h reactions at 50°C. (C) Ten-hour reactions at 45°C using hamster rPrP^sen (23–231) as substrate and antiserum R20. Open circles mark 17 kDa fragments and brackets indicate the lower molecular weight bands (10–13 kDa) that are detected only by the R20.

Fig. 2

Sensitivity of single and multiple round reactions in the sheep scrapie-seeded/ha-rPrP^sen QuIC assay. Reactions were seeded with scrapie BH dilutions containing the indicated amounts of PrP^res or control BH at a dilution equivalent to the most concentrated scrapie BH dilution. The PrP genotype of the BHs is designated by the three letter code as described in the text. PK-digested reaction products were analyzed by immunoblotting with antiserum R20. Hamster (90–231) (A) or (23–231) (B) rPrP^sen served as the substrate in single-round reactions. (C) A two-round QuIC reaction was performed with hamster (23–231) rPrP^sen: the first round at 42°C for 14 h (not shown) followed by a second round at 45°C for 6 h. Open circles mark 17 kDa fragments and brackets indicate the lower molecular weight bands (10–13 kDa).

Fig. 3

Sheep scrapie CSF-seeded/ha-rPrP^sen QuIC assay. First-round QuIC reactions (45°C, 12 h) were seeded with 5 or 10 µl of CSF from a normal (VRQ/ARQ) or scrapie-positive (ARQ/ARQ) sheep. Hamster (23–231) rPrP^sen was used as a substrate. (A and B) Second- and (C and D) third-round reactions were initiated with 10% of the reaction volume from the previous round. PK-digested products were analyzed by immunoblot using the polyclonal R20 antibody. Open circles mark 17 kDa fragments and brackets indicate the lower molecular weight bands (10–13 kDa).

Fig. 4

Sheep CSF-seeded/ha-rPrP^sen QuIC assays on a panel of non-scrapie and scrapie-positive sheep. First-round QuIC reactions (45°C, 12 h) were seeded with 10 µl of CSF from three non-scrapie (two VRQ/ARR and one VRQ/ARQ genotype) or nine scrapie-positive (two ARQ/ARQ, four ARQ/VRQ, two ARH/VRQ and one ARQ/ARH genotype) sheep. Hamster (23–231) rPrP^sen was used as a substrate. Second- and third-round reactions were incubated at 50°C for 10 h. (A) Second- and (B) third-round reactions were initiated with 10% of the reaction volume from the previous round. An asterisk designates a scrapie CSF sample that showed no scrapie-seeding activity in this experiment but did elicit rPrP^res(Sc) formation in two out of four replicate reactions in a previous experiment (not shown). PK-digested products were analyzed by immunoblot using the polyclonal R20 antibody. Open circles mark 17 kDa fragments and brackets indicate the lower molecular weight bands (10–13 kDa).