Methods for analyzing blood to detect diseases associated with abnormal protein aggregation

Abstract

A method of detecting a disease associated with abnormal protein aggregation in a subject is provided, the method comprising (a) contacting non-plasma blood elements from the subject with a probe that binds to pathogenic protein aggregates, and (b) detecting the probe bound to the pathogenic protein aggregates, wherein the presence of pathogenic protein aggregates in the non-plasma blood elements is indicative that the subject has a disease associated with abnormal protein aggregation. In one embodiment, the disease is Alzheimer's disease, mild cognitive impairment or traumatic brain injury.

We claim: 
     
         1 . A method of detecting a disease associated with abnormal protein aggregation in a subject, comprising:
 contacting one or more non-plasma blood elements from the subject with a conformationally-sensitive probe that binds to pathogenic protein aggregates, and   generating a fluorescence emission spectrum or absorption spectrum of the probe bound to the pathogenic protein aggregates, wherein the fluorescence emission spectrum or absorption spectrum of the probe changes when the probe is bound to pathogenic protein aggregates,   comparing the fluorescence emission spectrum or absorption spectrum of the probe contacted with the non-plasma blood elements to one or more reference fluorescence emission spectrum or absorption spectrum, wherein correspondence between the fluorescence emission spectrum or absorption spectrum and the one or more reference fluorescence emission spectra or absorption spectra is indicative that the subject has a disease associated with abnormal protein aggregation.   
     
     
         2 . The method of  claim 1 , wherein the one or more non-plasma blood elements are selected from red blood cells, leukocytes and platelets. 
     
     
         3 . The method of  claim 1 , wherein the probe is K114, Congo Red, a Congo Red derivative, X34, BSB, FSB, IMSB, Chrysamine-G, methoxy-X34, methoxy-X04, thioflavin-T, thioflavin-S, Pittsburgh compound B, thiazine red R, auramine-O, p-FTAA or a luminescent conjugated polythiophene (LCP) or luminescent conjugated oligothiophene (LCO) related to p-FTAA. 
     
     
         4 . The method of  claim 1 , wherein generating the fluorescence emission spectrum of the probe comprises detecting the intensity of the fluorescence emission spectrum at two or more wavelengths, optionally elicited using 1- or multi-photon excitation at one or more excitation wavelengths. 
     
     
         5 . The method of  claim 1 , wherein at least one reference emission spectrum or reference absorption spectrum is a fluorescent emission spectrum or absorption spectrum representative of reference non-plasma blood elements from a reference subject who has a disease associated with abnormal protein aggregation, and correspondence between the fluorescence emission spectrum or absorption spectrum and the at least one reference fluorescence emission spectrum or reference absorption spectrum indicates that the subject has a disease associated with abnormal protein aggregation. 
     
     
         6 . The method of  claim 1 , wherein the method comprises:
 a. performing spectral unmixing to determine the weightings of individual basis spectra that contribute to the fluorescence emission spectrum or absorption spectrum; and   b. using the weightings to determine a probability that the subject has a disease associated with abnormal protein aggregation.   
     
     
         7 . The method of  claim 6 , wherein the individual basis spectra are determined from (a) samples of subjects known to have a disease associated with abnormal protein aggregation, (b) samples of healthy control subjects and/or (c) samples that have not been contacted with the probe. 
     
     
         8 . The method of  claim 6 , wherein the spectral unmixing is performed using linear algebraic methods. 
     
     
         9 . The method of  claim 6 , wherein the spectral unmixing is performed using non-linear algebraic methods. 
     
     
         10 . The method of  claim 9 , wherein the spectral unmixing is performed using the Levenberg-Marquardt algorithm. 
     
     
         11 . The method of  claim 1 , wherein comparing the fluorescence emission spectrum or absorption spectrum to one or more reference fluorescence emission spectra or reference absorption spectra comprises machine learning, genetic algorithms or principle components analysis. 
     
     
         12 . The method of  claim 1 , wherein the disease associated with abnormal protein aggregation is selected from the group consisting of: Alzheimer's disease, Parkinson's disease, Huntington's disease, mild cognitive impairment, cerebral amyloid angiopathy, myopathy, neuropathy, brain trauma, traumatic brain injury, frontotemporal dementia, Pick's disease, multiple sclerosis, prion disorders, Down's syndrome and Amyotrophic Lateral Sclerosis (ALS). 
     
     
         13 . The method of  claim 1 , wherein the pathogenic protein aggregates comprise amyloid proteins, optionally β-amyloid, α-synuclein, huntingtin, tau protein, hyperphosphorylated tau protein (pTau), prion protein, αB-crystallin (CRYAB), desmin, selenoproteins, actin, myosin and/or superoxide dismutase (SOD). 
     
     
         14 . The method of  claim 1 , wherein the pathogenic protein aggregates comprise β-amyloids and the disease is Alzheimer's disease. 
     
     
         15 . The method of  claim 1 , wherein the method comprises generating two or more fluorescence emission spectra at two or more excitation wavelengths. 
     
     
         16 . The method of  claim 15 , wherein at least one of the excitation wavelengths is in the near-ultraviolet, optionally about 200 nm to about 400 nm. 
     
     
         17 . The method of  claim 15 , wherein at least one of the excitation wavelengths is from about 300 nm to about 500 nm, optionally about 375 nm or about 445 nm. 
     
     
         18 . The method of  claim 1 , wherein generating the fluorescence emission spectrum comprises measuring the intensity of the fluorescence emission at one or more wavelengths between about 200 nm and about 2000 nm. 
     
     
         19 . The method of  claim 1 , wherein generating the absorption spectrum comprises measuring the absorbance of light at one or more wavelengths between about 200 nm and about 2000 nm. 
     
     
         20 . The method of  claim 1 , wherein the fluorescence or absorbance of the probe bound to the pathogenic protein aggregates is normalized to cell count. 
     
     
         21 . The method of  claim 1 , wherein generating the fluorescence emission spectrum or absorption spectrum comprises measuring the fluorescence or absorbance of the probe bound to the pathogenic protein aggregates in all or part of a 2-dimensional field comprising a sample of leukocytes. 
     
     
         22 . An in vitro method for detecting pathogenic protein aggregates in a blood sample from a subject, the method comprising:
 contacting non-plasma blood elements from the blood sample with a conformationally-sensitive probe that binds to pathogenic protein aggregates, wherein the non-plasma blood elements are selected from red blood cells, leukocytes and platelets, and   detecting the probe bound to the pathogenic protein aggregates.