Method of preparing cow brain homogenate
View Patent ↗An assay method is disclosed which isolates and detects the presence of a disease related conformation of a protein (e.g., PrP Sc ) present in a sample also containing the non-disease related conformation of the protein (e.g., PrP C ). The sample is treated (e.g., contacted with protease) in a manner which hydrolyzes the disease related conformation and not the non-disease related conformation. The treated sample is contacted with a binding partner (e.g., a labeled antibody which binds PrP Sc ) and the occurrence of binding provides and indication that PrP Sc is present. Alternatively the PrP Sc of the treated sample is denatured (e.g., contacted with guanadine) or unfolded. The unfolded PrP Sc is contacted with a binding partner and the occurrence of binding indicates the presence of PrP Sc in the sample. In another embodiment, PrP Sc and PrP C are reacted with a labeled antibody that binds both conformations and a conformation that binds only the disease related conformation, and the presence of the disease related conformation is determined by comparing the two.
1. A method of detecting PrP Sc , comprising the steps of:
removing a sample of brain tissue from an animal;
homogenizing the sample to produce a homogenate;
adding a dispase to the homogenate for a period of time and under conditions to allow for digestion of PrP C in the homogenate but leave PrP Sc in the homogenate substantially undigested thereby providing a treated homogenate;
contacting the treated homogenate with a binding partner that selectively binds PrP Sc in the treated homogenate;
detecting binding of the binding partner and thereby detecting PrP Sc in the treated sample.
2. The method of claim 1 , wherein the animal is bovine.
3. The method of claim 1 , wherein the dispase is a metalloendopeptidase dispase.
4. The method of claim 1 , wherein the binding partner is an antibody.
5. The method of claim 4 , wherein the antibody is 3F4.
6. The method of claim 1 , wherein the binding partner is attached to a detectable label.
7. The method of claim 1 , further comprising:
inhibiting action of the dispase by addition of an inhibitor.
8. The method of claim 7 , wherein the inhibitor is ethylene diaminetetracetic acid (EDTA).