Method of preparing cow brain homogenate
View Patent ↗A method of isolating and detecting the presence of a disease related conformation of a protein (e.g., PrP Sc ) is disclosed. The sample is treated such as by contacting it with a protease and then contacting the treated sample with a binding partner which binds the PrP Sc which can be isolated and separated from the sample.
1. A method of isolating a PrP Sc protein, comprising:
treating a sample suspected of containing PrP C protein and PrP Sc protein with a compound which hydrolyzes the PrP C protein but not PrP Sc protein to provide a treated sample; and
separating the PrP Sc protein away from the treated sample.
2. The method of claim 1 , wherein the compound which hydrolyzes PrP C but not PrP Sc is dispase.
3. The method of claim 1 , wherein the separating is carried out by centrifuging the treated sample.
4. An assay method, comprising:
treating a sample suspected of containing PrP C protein and PrP Sc protein with a compound which hydrolyzes the PrP C protein but not PrP Sc protein to provide a treated sample; and
separating the PrP Sc protein away from the treated sample;
concentrating the treated PrP Sc ;
contacting the concentrate of treated PrP Sc with a binding partner;
determining the presence of PrP Sc in the sample based on a level of binding to the binding partner.
5. The assay of claim 4 , wherein the separating is carried out by centrifuging the treated sample.
6. The assay method of claim 4 , wherein the compound which hydrolyzes PrP C but not PrP Sc is dispase.
7. The assay method of claim 4 , wherein the compound which hydrolyzes PrP C but not PrP Sc is a metalloendopeptidase.
8. The assay method of claim 4 , wherein the binding partner is an antibody.
9. The assay method of claim 4 , wherein the sample is obtained from a mammal.
10. The assay method of claim 4 , wherein the sample comprises brain tissue from a mammal selected from the group consisting of human, sheep and cow.
11. The method of claim 4 , wherein the binding partner is bound to a detectable label.
12. The method of claim 11 , wherein the detectable label is a fluorescent label and further wherein determining the presence of PrP Sc in the treated sample is carried out using time-resolved dissociation enhanced fluorescence.
13. The method of claim 11 , wherein the detectable label is a fluorescent label and further wherein determining the presence of PrP Sc in the treated sample is carried out using a dual wavelength, laser driven fluorometer.