Methods, compositions, and kits for detecting protein aggregates
View Patent ↗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.
1. A method of detecting a protein aggregate in a sample of interest comprising:
providing a first soluble precursor molecule and a second soluble precursor molecule, wherein the first precursor molecule is attached to a first oligonucleotide, and wherein the second precursor molecule is attached to a second oligonucleotide, wherein the first soluble precursor molecule and the second soluble precursor molecule are capable of aggregating;
contacting a sample suspected of containing the protein aggregate with the first soluble precursor molecule and the second soluble precursor molecule;
aggregating the first soluble precursor molecule, and the second soluble precursor molecules and the protein aggregate, if present in the sample, together to form an oligonucleotide-labeled protein aggregate, wherein the first and second precursor molecules are incorporated into the labeled protein aggregate;
interacting the first oligonucleotide of the first precursor molecule with the second oligonucleotide of the second precursor molecule, to form an interaction composition; and,
detecting the interaction composition to detect the presence of the protein aggregate in the sample of interest; wherein the interacting comprises hybridizing the first oligonucleotide and the second oligonucleotide to a splint oligonucleotide and ligating the first oligonucleotide to the second oligonucleotide to form a ligation product.
2. The method according to claim 1 wherein the first precursor molecule and the second precursor molecule are Sup35.
3. The method according to claim 1 wherein the detecting comprises amplifying the ligation product in a PCR.
4. The method according to claim 3 wherein the PCR is a real-time PCR.