Abstract: A high-sensitivity chemiluminescent ELISA-based prion detection method is provided, the method comprising an ELISA plate coated with monoclonal antibody for the capture of PrPsc protein and treated with an optimized blocking reagent, and a set of labeling and detection reagents. The digestion cocktail is preferably a unique combination of proteolytic, lipolytic and nuclease enzymes for the liberation of the prion PrPsc antigen. The ELISA plate is preferably a multi-well, high-protein binding, microtiter plate coated with an anti-prion monoclonal antibody, used to capture the PrPsc antigen. The blocking agent is an optimized protein and buffer solution. The detection antibody is preferably a biotinylated monoclonal antibody. The chemiluminescent conjugate is preferably a conjugate of streptavidin and alkaline phosphatase enzyme. The final component is preferably an optimized chemiluminescent substrate for maximum sensitivity.