Vegrandis proposes to develop a rapid, sensitive, high throughput, inexpensive, and easy to use system for the ante-mortem diagnosis of prion infection. The proposed system offers a fully automated equipment with a disposable microelectrochemical cartridge for diagnosis of ante- mortem levels as low as 1 x 10-15 molar (possibly 1 x 10-21molar) concentrations of PrPSc in a rapid assay at d 30 min, high throughput allowing >100 samples/h per fully automated instrument, high sensitivity with >95% reliability (eliminating false positives and false negatives), and with minimal potential hazard of handling patient biological fluid. Unlike existing diagnostic methods of prion infection which are usable only when patients already show acute signs of neurologic dysfunctions and are provisional until post-mortem tissue homogenate diagnosis rules out or confirms the diagnosis, the proposed system is anticipated to diagnose asymptomatic stage prion infection that can be used for at least three important purposes: 1) eliminate contaminated animals and animal products from entering the market;2) use in developing and monitoring the efficacy of drugs, vaccine, or other therapeutic molecules that are being developed;and 3) monitor changes in the PrPSc levels during medication. The proposed system will eliminate the use of post mortem tissue homogenates for diagnosis by allowing asymptomatic and ante-mortem diagnosis through the use of self-contained microelectro- chemistry in 50-micron diameter wells in a disposable cartridge (DC) format. The sample will be injected into the self-sealing inlet port on the DC, the DC will be inserted into the fully automated instrument, the reagents will be placed in the reagent module, and the proper program will be chosen. The instrument read out will display the concentration of PrPSc at the end of <30 min. During Phase I, Vegrandis will demonstrate the technology proof-of-concept by performing experiments using scrapie as the model analyte. Phase I is anticipated to:1) detect as low as femtomolar levels of PrPSc, 2) <45 min assay from capture to signal generation, 3) non- interference of debris from biological samples eliminating tedious sample preparation steps, and 4) low assay cost resulting from the use of small reagent volumes (d 10 Millions of cows are slaughtered annually for food in the United States. After the discovery of the first domestic case of mad cow disease in 2004, national US beef industry losses were recorded at $3.2 to $4.7 billion for that year alone. People have died worldwide from prion infection resulting from eating infected beef. Ante-mortem detection of prion infection will prevent the spread of this fatal disease that currently has no vaccine, antidote, or medicine to cure. Therefore, avoiding contamination is essential. Development of a fully automated, portable instrument that uses compact, disposable cartridges and requires no pre-analysis sample preparation for ante-mortem prion infection is proposed. Preliminary work indicates that the proposed approach will be able to meet the high sensitivity required to diagnose the asymptomatic stage of this fatal disease.Public Health Relevance
