Digital Protein Misfolding Assays

A method of detecting, and optionally quantifying a level of,

• pathological protein aggregates of two or more different disease-associated proteins in a sample from a subject, preferably wherein the sample comprises cerebrospinal fluid, plasma, or serum, the method comprising:
• providing a sample from a subject, optionally a subject who is known or suspected to have a condition associated with aggregation of disease-associated proteins;
• optionally enriching the sample for extracellular vesicles;
• mixing the sample with a plurality of fluorescently labeled beads, wherein the beads are coated with antibodies that bind specifically to each of the two or more co-aggregates, preferably α-synuclein, PrP, amyloid beta, TDP-43, and/or tau co-aggregates, wherein each fluorescent label is associated with antibodies that bind specifically to a specific protein aggregate;
• diluting the mixture of sample and beads by addition of a diluent comprising reaction reagents to form a reaction mixture, wherein the reaction reagents comprise monomers of disease-associated proteins and a detection reagent;
• compartmentalizing the reaction mixture into separate confined volumes, preferably wherein each of the confined volumes has a volume of 1 pL to 1000 nL, and wherein each confined volume has zero or one pathological protein aggregate;
• incubating the confined volumes under conditions to allow co-aggregation of the monomers of disease-associated protein and any pathological protein aggregates present in the sample;
• detecting the presence or absence of co-aggregates of the disease-associated protein monomers and pathological protein aggregates in each confined volume, wherein the formation of co-aggregates indicates the presence of pathological protein aggregates in the confined volume;
• determining the identity of the co-aggregates based on the identity of fluorescent labels, and optionally quantifying the level of each of the pathological protein aggregates in the sample by counting the number of confined volumes comprising co-aggregates.