Assay for rapid protein multimer detection, characterization and quantification
AssigneeWestern Michigan University Homer Stryker M.D. School of Medicine
InventorsMichael Gutknecht, Thomas L. Rothstein
Life of this patent
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Abstract
A number of protein aggregation diseases are associated with accumulation of misfolded proteins, which are known as protein aggregates, including, but not limited to, neurodegenerative and non-degenerative diseases and disorders. The present disclosure provides an assay, compositions and kits for the qualitative and quantitative assessment of aggregated proteins in solution using a microparticle immunocapture assay that combines the advantages inherent to a specific first and second capture moiety that binds specifically to an aggregated protein which can reveal at the same time the amount and the size of aggregates measured in a sample, fluid, tissue, cavity, or pharmacological product.
Claims56
A method for determining the presence and at least one of, the size and amount of an aggregated protein in a test sample, the method comprising:
- a. providing a capture substrate comprising a microparticle conjugated to a first capture moiety;
- b. blocking the capture substrate non-specific binding sites;
- c. incubating the capture substrate with a test sample suspected of having the aggregated protein for a period of time sufficient for the aggregated protein to specifically bind to the first capture moiety;
- wherein the first capture moiety binds specifically to the aggregate protein if present, thereby forming an capture complex on the capture substrate,
- d. incubating the capture substrate with a second capture moiety that specifically binds to the aggregated protein, wherein the second capture moiety is coupled to a signalling moiety, and wherein the signaling moiety comprises a detectable label;
- and
- e. determining the amount of detectable label present on the surface of the capture substrate.
A composition, comprising
- (a) a first component comprising a plurality of capture substrate microparticles, each microparticle conjugated to a first capture moiety antibody or antigen binding fragment thereof, or receptor, or ligand, or aptamer, an amino acid sequence, or lipid, or DNA nucleotide sequence, or RNA nucleotide sequence;
- the first capture moiety antibody or antigen binding fragment thereof, or receptor, or ligand, or aptamer, an amino acid sequence, or lipid, or DNA nucleoside sequence, or RNA nucleoside sequence, operable to bind to a single epitope or antigen, or sequence of amino acids of an aggregated protein involved in a protein aggregation mediated disease, or other aggregating species involved in disease.
- (b) a second component comprising a second capture moiety that comprises an antibody or antigen binding fragment thereof, a receptor, a ligand, an aptamer, an amino acid sequence, a lipid, or DNA nucleotide sequence, or RNA nucleotide sequence, as the first capture moiety, wherein the second capture moiety is conjugated to a signalling moiety that comprises at least one of:
- a detectable label and a first binding partner that is operable to bind specifically to a detectable label that is coupled to a second binding partner, and
- (c) optionally, a third component a detectable label coupled to a second binding partner, wherein the first and second binding partners bind specifically.
A system, comprising:
- a processor;
- and
- a non-transitory computer readable medium comprising instructions that cause the processor to:
- count a number of positive aggregate protein capture complexes present in a test sample, wherein each of the positive aggregate protein capture complexes comprise a first capture moiety, an aggregate protein, a second capture moiety, a signalling moiety and a fluorescently labeled detectable label;
- determine the total number of aggregate protein capture complexes acquired by a detection device;
- calculate the percentage of positive aggregate protein capture complexes among the total number of aggregate protein capture complexes;
- and
- determine the number and/or proportion of positive aggregate protein capture complexes that comprised the test sample.
A kit, comprising:
- a plurality of microparticles comprising a first capture moiety that specifically binds an aggregate protein of interest;
- and
- a composition comprising a second capture moiety that specifically binds to the aggregate protein, wherein the second capture moiety is coupled to a signalling moiety, wherein the first and second capture moieties bind to the same epitope or antigen present on the aggregated protein.
Dependencies are inferred from the claim text.