Optimization of the selective capture of PrPSc from brain samples using a prion specific antibody magnetic beads based immuno-precipitation.

N.E.L. Kadi, S. Dudas, S. Czub
CFIA, CA

Keywords: immuno-precipitation (IP), western blot (WB), Dynabeads, complex antibody-antigen: 15B3, mouse anti-IgM, PrPsc

Summary:

Prion diseases include scrapie of sheep and goats, bovine spongiform encephalopathy (BSE or mad cow disease) in cattle, Creutzfeldt-Jakob disease (CJD) in humans, and chronic wasting disease (CWD) in deer and elk. To diagnose prion disease, most methods employ an enzymatic treatment to digest PrPC and leave behind the characteristic disease associated protease-resistant core of PrPSc (designated PrP 27–30). In BSE, this partially protease resistant conformer accumulates in the brains of infected cattle. Immunoprecipitation can be used to purify a single antigen (PrPsc in our case) from a complex mixture using a specific antibody attached to a beaded support (ex. Dynabeads Rat anti-Mouse IgM). In this project, the IgM monoclonal antibody, 15B3, was incubated first with the beads, followed by addition of the antigen-containing sample (e.g., brain homogenate). After binding the antigen, antibody and support, the beads were washed extensively and then the antigen (PrPSC) was eluted from the support using an elution buffer. The goal was to potentially increase the level of sensitivity of common detection methods and in turn the potential for immuno-precipitation to be used as an ultra sensitive and specific method for PrPSc capture and prion disease diagnosis.