Methods, compositions, and kits for detecting protein aggregates

Patent metadata

Field	Value
Publication number	US-9518296-B2
Application number	US-201313975056-A
Country	US
Kind code	B2
Filing date	Aug 23, 2013
Priority date	May 3, 2006
Publication date	Dec 13, 2016
Grant date	Dec 13, 2016

Abstract

The present teachings provide methods, compositions, and kits for detecting the presence of protein aggregates. In some embodiments, the protein aggregate is treated with a labeled precursor, and the labeled precursor is incorporated into the protein aggregate to form a labeled protein aggregate. The labeled protein aggregate is then measured, thus detecting the presence of the protein aggregate. In some embodiments, the labeled protein aggregate is detected by interaction of labeled precursors, for example by a proximity ligation assay.

First claim

We claim:

1. A kit for detecting a protein aggregate in a sample comprising:
a first soluble precursor molecule;
a second soluble precursor molecule; and
an oligonucleotide splint,
wherein the first soluble precursor molecule is attached to a first oligonucleotide, and
wherein the second soluble precursor molecule is attached to a second oligonucleotide, and
wherein the first and second soluble precursor molecules are protein moieties of the protein aggregate prior to aggregation.

2. The kit according to claim 1 further comprising a ligase.

3. The kit according to claim 1 further comprising reagents for a polymerase chain reaction (PCR), wherein the reagents for the PCR comprise a first primer and a second primer, wherein the first primer corresponds to the first oligonucleotide of the first soluble precursor molecule and wherein the second primer corresponds to the second oligonucleotide of the second soluble precursor molecule.

4. The kit of claim 1 , where the first soluble precursor molecule and the second soluble precursor molecule are Sup35.

5. The kit of claim 1 , where the first soluble precursor molecule and the second soluble precursor molecule are beta amyloid protein.

6. The kit of claim 1 , where the first soluble precursor molecule and the second soluble precursor molecule are huntingtin.

7. The kit of claim 1 , where the first soluble precursor molecule and the second soluble precursor molecule are alpha-synuclein.

8. The kit of claim 1 , where the first soluble precursor molecule and the second soluble precursor molecule are bovine prion protein.

9. The kit of claim 1 , where the reagents for PCR further comprise one or more of deoxynucleotide phosphates, thermostable DNA polymerase, one or more PCR buffers.

10. The kit of claim 1 , wherein the first and second soluble precursor molecules are correctly folded proteins that are not yet in the conformation present in a protein aggregate.

11. The kit of claim 1 , wherein the protein aggregate is a naturally occurring homo-multimeric protein complex.

12. The kit of claim 1 , wherein the protein aggregate comprises abnormal mis-folded proteins.

13. The kit of claim 1 , wherein the protein aggregate comprises aggregated prion particles.

14. The kit of claim 13 , wherein the aggregated prion particles comprise yeast prion proteins selected from Sup35, Ure2 and Rnq1, human prion proteins, bovine prion proteins, or sheep prion proteins.

15. The kit of claim 1 , wherein the protein aggregate detected by the kit is selected from aggregated yeast prion proteins, human prion proteins, beta amyloid protein, tau proteins, huntingtin protein, alpha-synuclein protein, or bovine prion protein.

Assignees

• Applied Biosystems Llc

Inventors

• Ruff David
• Shannon Mark
• Livak Kenneth
• Guegler Karl
• Hennessy Kevin

Classifications

• C07K14/47 Primary

  from mammals · CPC title

• C12Q1/6816

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• C07K14/4711

  Alzheimer's disease; Amyloid plaque core protein · CPC title

• C12Q1/6876 Primary

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C12Q2533/107

  Probe or oligonucleotide ligation · CPC title