A Potential Blood Test for Transmissible Spongiform Encephalopathies by Detecting Carbohydrate‐Dependent Aggregates of PrPres‐Like Proteins in Scrapie‐Infected Hamster Plasma

Abstract

PrPres has rarely been detected in blood (except in leukocytes) even in diseased animal models that are known to contain a large amount of PrPres in infected tissues. It seems likely that PrPres detection in blood is difficult because of the low titer of infectious material within the blood. Here, we demonstrate the detection of proteinase K‐resistant 3F4‐reactive protein in the plasma of scrapie‐infected hamsters but not in the plasma of mock‐infected hamsters by partial purification using a novel method termed “acidic SDS precipitation,” in conjunction with a highly sensitive chemiluminescence detection system used to show the presence of PrP at a concentration equivalent to 1.4 ×10–9 g of brain homogenate or 1.5 × 10–12 g (6.5 ×10–17 mol) of rPrP by conventional Western blotting. The 3F4‐reactive proteins in scrapie‐infected hamster plasma often resulted in multiple Mw protein bands occurring at higher Mw positions than the position of the di‐glycosyl PrP molecule. Mixing scrapie‐infected hamster brain homogenate with mock‐infected hamster plasma resulted in the formation of similar Mw positions for multiple 3F4‐reactive proteins. Predigestion of carbohydrate side chains from the proteins in the plasma or brain homogenate before mixing resulted in failure to obtain these multiple 3F4‐reactive proteins. These observations indicate that PrPres aggregated with other proteins in the plasma through carbohydrate side chains and was successfully detected in the plasma of scrapie‐infected hamsters. Counterparts in these aggregates with PrPres‐like proteins in scHaPl are not known but any that exist should resist the PK digestion.

Affinity Association between Polynucleotide, Glycoprotein, or Sulfated Polysaccharides and Disease-Associated Prion Protein

“…26 As we experienced in our experiments, dilution of the scBrh with TBS-SDS resulted in almost diminished on the 30 kDa band and the smaller recovery of the PrPsc than expected even used the same volumes of original one. So, if we compared the observed patterns of PrPsc in standard lane of PK-scBrh on Figure 1 to that on Figures 2-5 electrophoresis patterns were quite different each other especially on their Mw and the intensity of the PrPsc signals on Figures 2-5 were unexpectedly low.…”

“…We previously reported that mixing of scBrh with mcPl resulted in the appearance of novel antiPrP mAb-reactive protein bands, in a manner similar to the scPl-derived bands yielded by ASP. 26 This observation suggested that the PrPres may be associated with additional partner substances in the blood of infected animals and hence can be precipitated. However, the substances that may become associated with the PrPres in the blood of infected animals have not yet been identified.…”

“…[22][23][24][25] We previously attempted to develop a blood test system using the plasma of scrapie-infected hamsters. 26 This system was designated as "acidic sodium dodecyl sulfate (SDS) precipitation (abbreviated to ASP)." This system was able to effectively discriminate between infected and mock-infected hamsters by analyzing blood samples.…”

Factors intrinsic and extrinsic to blood hamper the development of a routine blood test for human prion diseases

“…The acidic SDS precipitation method was H. Abdel-Haq proposed by Tsukui et al (2007) and is based on the detection of carbohydrate-dependent aggregates of PrPreslike proteins in the plasma of scrapie-infected hamsters. The immunocapillary electrophoresis technique consists of fluorescence-based detection of PrP TSE in a competitive immunoassay in which a fluorescently labelled synthetic prion peptide and a polyclonal antibody against this peptide are titrated (Lourenco et al, 2006).…”

Magnetic microparticle-based multimer detection system for the detection of prion oligomers in sheep

“…Platelets from scrapie-infected hamsters were not able to transmit scrapie; 31 however, PrP Sc in plateletfree hamster plasma samples were detected by PMCA. 32,33 Other studies also revealed that PrP Sc infectivity exists in the cell-free plasma of sheep. 34,35 These findings suggest the presence of varying infectivity in rodents and ruminants.…”