Methods for estimating prion concentration in fluids and tissue by quantitative PMCA

1. A method for preparing a calibration curve useful for quantitatively estimating a concentration of PrP Sc in a sample, the method comprising:

preparing a plurality of stock solutions, each stock solution in the plurality of stock solutions having a known different concentration of PrP Sc ;

separately mixing each of the plurality of stock solutions with PrP C to form a plurality of separate stock reaction mixes;

forming a plurality of separate amplified portions of PrP Sc by:

performing a plurality of protein misfolding cyclic amplification (PMCA) cycles on each of the plurality of separate stock reaction mixes to form a plurality of separate amplified stock reaction mixes comprising the plurality of separate amplified portions of PrP Sc , each cycle in the plurality of PMCA cycles comprising:

incubating each stock reaction mix; and

disaggregating aggregates formed in each stock reaction mix;

subjecting each of the plurality of separate amplified stock reaction mixes to an assay for a number of cycles of the plurality of PMCA cycles until a PrP Sc signal is detected; and

determining the calibration curve according to the known different concentration of the PrP Sc in each stock solution with the number of PMCA cycles corresponding to detection of the PrP Sc signal, at least a portion of the known different concentrations of PrP Sc among the plurality of stock solutions being below a concentration detectable by the assay such that the calibration curve provides for quantitative estimation of PrP Sc concentration in the sample below the concentration detectable by the assay.

2. The method of claim 1 , further comprising plotting the calibration curve in the form of a standard calibration curve.

3. The method of claim 1 , wherein the PrP C is provided for mixing with each of the plurality of stock solutions in the form of a normal tissue homogenate.

4. The method of claim 1 , wherein the assay is a western blot assay.

5. A method for quantitatively estimating a concentration of prion in a sample, the method comprising:

mixing the sample with a non-pathogenic PrP C protein to form a reaction mix;

forming an amplified portion of PrP Sc by:

performing a plurality of protein misfolding cyclic amplification (PMCA) cycles on the reaction mix to form an amplified reaction mix comprising the amplified portion of PrP Sc , each cycle comprising:

incubating the reaction mix; and

disaggregating aggregates formed in the reaction mix;

subjecting the amplified reaction mix to an assay for a number of the plurality of PMCA cycles until a PrP Sc prion signal is detected; and

quantitatively estimating the concentration of the PrP Sc prion in the sample according to the number of PMCA cycles corresponding to detection of the PrP Sc prion signal by using a predetermined calibration curve for quantitatively estimating the concentration of the PrP Sc in the sample according to the assay, the predetermined calibration curve determined according to a plurality of known different concentrations of PrP Sc each corresponding to a calibrating number of PMCA cycles, each calibrating number of PMCA cycles being effective to amplify each corresponding known different concentration of PrP Sc in the presence of non-pathogenic PrP C to a concentration of PrP Sc detectable by the assay, at least a portion of the plurality of known different concentrations of PrP Sc being below the concentration detectable by the assay such that the predetermined calibration curve provides for quantitative estimation of PrP Sc concentration in the sample below the concentration detectable by the assay.

6. The method of claim 5 , wherein the disaggregating comprises subjecting the reaction mix to sonication.

7. The method of claim 5 , wherein the assay is a western blot assay.

8. The method of claim 5 , further comprising:

removing a portion of the reaction mix;

contacting the portion with additional non-pathogenic PrP C protein to form a second reaction mix;

performing a plurality of PMCA cycles on the second reaction mix, each cycle in the plurality of PMCA cycles comprising:

incubating the second reaction mix; and

disaggregating aggregates formed in the second reaction mix;

subjecting the disaggregated second reaction mix to an assay for a number of cycles of the plurality of PMCA cycles until the PrP Sc prion signal is detected; and

quantitatively estimating the concentration of the PrP Sc prion in the second reaction mix according to the number of cycles corresponding to detection of the PrP Sc prion signal by using the predetermined calibration curve.

9. The method of claim 5 , wherein the sample is selected from the group consisting of: spleen, brain, blood, and urine.

10. The method of claim 5 , quantitatively estimating the concentration of the PrP Sc prion in the sample comprising quantitatively estimating the concentration of the PrP Sc below the concentration detectable by the assay.