Standards for the assay of Creutzfeldt–Jakob disease specimens

Abstract

Assays for the agent of Creutzfeldt-Jakob disease (CJD) include measurement of infectivity in different animal systems, such as wild-type or transgenic mice, and detection of PrP Sc by different methods and formats. The various assays could be best calibrated against each other by use of uniform readily available materials, and samples of four human brains, two from sporadic CJD patients, one from a variant CJD patient and one from a non-CJD patient, have been prepared as 10 % homogenates dispensed in 2000 vials each for this purpose. Results of in vitro methods, particularly immunoblot assays, were compared in the first collaborative study described here. While dilution end-points varied, the minimum detectable volume was surprisingly uniform for most assays and differences in technical procedure, other than the sample volume tested, had no detectable systematic effect. The two specimens from sporadic CJD cases contained both type 1 and type 2 prion proteins in approximately equal proportions.

Characterization of prion protein (PrP)-derived peptides that discriminate full-length PrP Sc from PrP C

“…Also, our measurement for hamster PrP C plasma levels (8.7 ng/ml) was within the same range as those reported by MacGregor and Drummond (5.0-17 ng/ml) (15). Given that 0.18 pg of PrP Sc was detected in 70 l of plasma containing Ͼ700 pg of PrP C , these data suggest that PrP [23][24][25][26][27][28][29][30] binds PrP Sc over PrP C with a specificity of Ͼ3,800-fold.…”

Detection of Type 1 Prion Protein in Variant Creutzfeldt-Jakob Disease

“…6,11 The means by which such conformational difference is detected is somewhat indirect; relying on the action of proteases, primarily proteinase K, to degrade the normal A complication has recently arisen with the finding that both type 1 and type 2 can co-exist in the brains of patients with sCJD. 2,[5][6][7][8] More recently this same phenomenon has been demonstrated in patients with iatrogenically acquired and familial forms of human prion disease. 9,10 The existence of this phenomenon is now beyond doubt but its prevalence and its biological significance remain a matter of debate.…”

“…4 Recent reports show that certain cases of sCJD contain both type 1 and 2 in the same brain. [5][6][7][8] Regional variation in PrP Sc type has subsequently been reported in iatrogenic CJD 9 and in familial CJD, 10 suggesting that co-occurrence of different PrP Sc types is perhaps the rule in CJD. In contrast, all of the available evidence so far has suggested that the variant CJD (vCJD) brain contains a single type, which has been interpreted to reflect infection of susceptible individuals by a single defined pathogen, namely bovine spongiform encephalopathy (BSE).…”

Diagnosis of human prion disease

“…We chose not to perform Western blots on most of the samples used in this study because such immunoblots require denaturation of the sample, which eliminates measurement of the native signal corresponding to PrP C (Table 5). Moreover, a comparison between the CDI and Western blotting on brain samples from sCJD and variant CJD patients showed that the CDI was 50-to 100-fold more sensitive (15). Additionally, Western blots combined with densitometry are linear over a 10-to 100-fold range of concentrations, whereas the CDI is linear over a Ͼ10 4 -fold range.…”

“…1). The high sensitivity achieved by the CDI is due to several factors (8,10,11,15). First, both sPrP Sc and rPrP Sc conformers are specifically precipitated by PTA (Table 5) (8,9).…”