Test for Detection of Disease-Associated Prion Aggregate in the Blood of Infected but Asymptomatic Animals

Abstract

We have developed a sensitive in vitro assay for detecting disease-associated prion aggregates by combining an aggregation-specific enzyme-linked immunosorbent assay (AS-ELISA) with the fluorescent amplification catalyzed by T7 RNA polymerase technique (FACTT). The new assay, named aggregation-specific FACTT (AS-FACTT), is much more sensitive than AS-ELISA and could detect prion aggregates in the brain of mice as early as 7 days after an intraperitoneal inoculation of PrP Sc . However, AS-FACTT was still unable to detect prion aggregates in blood of infected mice. To further improve the detection limit of AS-FACTT, we added an additional prion amplification step (Am) and developed a third-generation assay, termed Am-A-FACTT. Am-A-FACTT has 100% sensitivity and specificity in detecting disease-associated prion aggregates in blood of infected mice at late but still asymptomatic stages of disease. At a very early stage, Am-A-FACTT had a sensitivity of 50% and a specificity of 100%. Most importantly, Am-A-FACTT also detects prion aggregates in blood of mule deer infected with the agent causing a naturally occurring prion disease, chronic wasting disease. Application of this assay to cattle, sheep, and humans could safeguard food supplies and prevent human contagion.

A prion disease of cervids: Chronic wasting disease

“…PrP Sc was recently reported as detectable in deer blood using an antibody ELISA with signal amplification of antibody conjugated DNA catalyzed by T7 RNA polymerase [15].…”

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

“…To overcome some of the risks associated with PMCA while taking advantage of its underlying principles, aggregation-specific ELISA (AS-ELISA), which was initially developed to detect brain PrP TSE aggregates (Pan et al, 2005b), was combined with an in vitro PrP TSE -amplification step, similar to that used in PMCA, catalysed by T7 RNA polymerase (FACTT) (Chang et al, 2007). This assay was called Am-A-FACCT and was able to detect aggregates of PrP TSE during the terminal and asymptomatic phases of the disease in the plasma of CD1 mice infected intraperitoneally with the ME7 prion strain of scrapie, and of mule deer orally infected with CWD.…”

Non-invasive, ultra-sensitive, high-throughput assays to quantify rare biomarkers in the blood

“…Recently, FACTT has been integrated with aggregate-specific ELISA to detect prion proteins in the blood of infected animals [40]. In this method (Am-A-FACTT), an in vitro PrP Sc amplification step (Am) is included to further improve the detection limit.…”

“…In addition, only one antibody is used as both the capture antibody and the detecting antibody since the protein of interest, PrP Sc , is known to form aggregates. The ultra-sensitive Am-A-FACTT assay had 100% sensitivity in detecting disease-associated prion aggregates in blood of infected mice at late but asymptomatic stages of disease (70 days postinoculation) [40]. At a very early stage (35 days postinoculation), Am-A-FACTT had a sensitivity of 50% and a specificity of 100%.…”

“…The FACTT-based assays are not limited only to the detection of antigens found in the blood, but can also be applied to antigens found in the saliva, urine, cerebrospinal fluid, etc [12]. Finally, the principle of the FACTT-based assays for prion may be applicable to other diseases caused by abnormal protein aggregation, such as Alzheimer's disease or Parkinson's disease [40].…”