6H4 Methods
ELISA detection
Luminescence immunoassay (LIA)
Brain samples were homogenized Prionics®-Check Homogenization buffer using the PrioGENIZER homogenization system for 45 sec. at 24’000 rpm. 100 μl of brain homogenate was digested with 25 U/ml of proteinase K for 60 min. Fifteen microlitres of digested brain homogenate was then pretreated with assay buffer before the addition of the horseradish peroxidase conjugated mixtures detection antibody in a total volume of 240 μl. After 60 min incubation at room temperature on a shaking platform, 200 μl of the reaction was transferred to the ELISA plate precoated with the capture antibody 6H4, and incubated 90 min on a shaking platform. Subsequent washing was done using an automated washer. The plate was then incubated with the detection antibody. This anti-PrP antibody is covalently linked to horse radish peroxidase (POD). 100 μl of chemiluminescence substrate for peroxidase was then added to each well and the emitted light measured in a luminometer. The whole assay can be performed with the Prionics®-Check LIA kit that contains all the necessary ingredients to carry out 200 tests in duplicate.
Adapted from:
Biffiger K et al., (2002) Validation of a luminescence immunoassay for the detection of PrPSc in brain homogenate. J Virol Methods. 101:79-84
