Method of concentrating proteins from serum
View Patent ↗A sample is prepared from blood in a manner which makes it possible to further analyze proteins in the sample, e.g. to detect prions in the sample. Blood is extracted, allowed to clot and subjected to separation processing (e.g. centrifugation) to obtain serum. The serum is treated with a complexing agent which agent binds prions in the sample forming an agent/protein complex which makes it possible to concentrate the complex. Concentration of the complex results in a sample which can be successfully analyzed, e.g. assayed using a range of different types of assay methodologies for detecting prions.
1. A method of concentrating a PrP protein present in a serum sample for the purpose of analysis, comprising the steps of:
allowing blood extracted from an animal to clot;
separating serum comprising PrP protein away from the clotted blood;
contacting the separated serum with a complexing agent which forms a complex with a pathogenic conformation of the PrP protein present in the serum;
concentrating the complex to thus form a sample having a concentrated pathogenic conformation of the PrP protein for use in analysis.
2. The method of claim 1 , wherein the complexing agent is chosen from a heteropoly acid and a salt thereof.
3. The method of claim 1 wherein the serum is separated using centrifugation.
4. The method of claim 1 , wherein the blood is obtained from a mammal selected from the group consisting of a human, a cow and a sheep not exhibiting symptoms of diseases and wherein the complexing agent is a salt of phosphotungstic acid.
5. The method of claim 1 , wherein the blood is human, the concentrated pathogenic conformation of the PrP protein is PrPSC and the complexing agent is sodium phosphotungstate.
6. The method of claim 1 , further comprising:
analyzing the pathogenic conformation of the PrP protein in the complex to determine characteristics.
7. The method of claim 6 , wherein the characteristics are selected from the group consisting of solubility, three-dimensional structure and infectivity.
8. The method of claim 1 , wherein the blood is extracted from a cow, the serum is separated using centrifugation and the complexing agent is a salt of phosphotungstic acid.
9. A method of claim 1 , further comprising:
treating the sample to convert the pathogenic conformation of the PrP protein into a binding conformation having an affinity for a labeled binding partner higher than the pathogenic conformation of the PrP protein prior to treating;
contacting the treated sample with the labeled binding partner to determine a concentration of the treated pathogenic conformation of PrP protein bound to the binding partner in the sample;
adjusting the concentration to provide an adjusted concentration which compensates for increased affinity of a non-pathogenic conformation of the PrP protein to the binding partner resulting from the treating; and
comparing said adjusted concentration to a known concentration determined from a treated non-infected control sample to determine the presence of the protein in the pathogenic conformation of the PrP protein in the sample.
10. The method of claim 9 , wherein said labeled binding partner comprises a labeled antibody, the complexing agent comprises a metal salt of phosphotungstic acid, and wherein the concentration is determined using flow cytometry.
11. The method of claim 9 , wherein the blood is extracted from a cow, the serum is separated using centrifugation and the complexing agent is a salt of phosphotungstic acid.
12. The method of claim 9 , wherein the protein in the pathogenic conformation of the PrP protein is present in the sample in a concentration of 1 x 10 3 protein molecules or less per ml and wherein the protein in the non-pathogenic conformation of PrP protein is present in the sample in a concentration of 1 x 10 6 protein molecules or more per ml.
13. The method of claim 10 , wherein the antibody is 3 F 4 and the complexing agent is sodium phosphotungstate.
14. A method of claim 1 , further comprising:
treating the sample to convert the pathogenic conformation of the PrP protein into a binding conformation having an affinity for a labeled binding partner higher than the pathogenic conformation of the PrP protein prior to treating;
contacting the treated sample with the labeled binding partner to determine a concentration of treated pathogenic conformation of PrP protein bound to the binding partner in the sample;
adjusting the concentration to provide an adjusted concentration which compensates for increased affinity a non-pathogenic conformation of the PrP protein to the binding partner resulting from the treating; and
comparing said adjusted concentration to a known concentration determined from a treated sample from a non-infected population of mammals selected from the group consisting of humans, cows and sheep to determine the presence of the protein in the pathogenic conformation of the PrP protein in the sample.
15. The method of claim 14 , wherein the blood is extracted from a cow, the serum is separated using centrifugation and the complexing agent is a salt of phosphotungstic acid.