Cell tests and diagnostic index for alzheimer's disease

Abstract

The present invention provides methods for the diagnosis of Alzheimer's disease using human cells. Specifically, one method detects differences between potassium channels in cells from Alzheimer's patient and normal donors, and differences in intracellular calcium concentrations between Alzheimer's and normal cells in response to chemicals known to increase intracellular calcium levels. Other methods detect differences between the memory associated GTP binding Cp20 protein levels between Alzheimer's and normal cells. In addition a diagnostic index for improved assessment between Alzheimer's and non Alzheimer's cells is provided.

We claim:  
     
         1 . A method of diagnosing Alzheimer's disease in a patient, said method comprising the steps of: 
 a. obtaining a protein sample from said patient; and    b. detecting the level of Cp20 protein in said sample.    
     
     
         2 . The method of  claim 1 , wherein said protein sample is isolated from cells selected from the group consisting of fibroblasts, buccal mucosal cells, neurons, and blood cells.  
     
     
         3 . The method of  claim 2 , wherein said cells are fibroblasts.  
     
     
         4 . The method of  claim 1 , wherein said detecting step (b) is performed by immunoassay.  
     
     
         5 . The immunoassay of  claim 4  wherein said immunoassay comprises the following steps: 
 (a) contacting said protein sample from said patient with an antibody which recognizes Cp20 protein; and  
 (b) detecting the complex between said antibody and said Cp20 protein.  
 
     
     
         6 . The immunoassay of  claim 5  wherein said antibody is a monoclonal antibody.  
     
     
         7 . The immunoassay of  claim 5  wherein said antibody is a polyclonal antibody.  
     
     
         8 . The method of  claim 4  wherein said immunoassay selected from the group consisting of radioimmunoassay, Western blot assay, immunofluorescent assay, enzyme immunoassay, immuno-precipitation, chemiluminescent assay, immunohistochemical assay, dot and slot blot assay.  
     
     
         9 . The method of  claim 8  wherein said immunoassay is a Western Blot Assay.  
     
     
         10 . A Cp20 protein comprising the amino acid sequence shown in FIG. 12A or a substantially homologous sequence thereof.  
     
     
         11 . A Cp20 peptide comprising the amino acid sequence shown in FIG. 12A or a substantially homologous sequence thereof.  
     
     
         12 . Antibodies reactive with the Cp20 protein or portions thereof.  
     
     
         13 . The antibodies of  claim 12  wherein said antibodies are monoclonal.  
     
     
         14 . The antibodies of  claim 12  wherein said antibodies are polyclonal.  
     
     
         15 . An isolated and purified nucleic acid sequence encoding for the Cp20 amino acid sequence shown in FIG. 12A.  
     
     
         16 . A Cp20 peptide comprising the amino acid sequence ARLWTEYFVIIDDDC (single letter code).  
     
     
         17 . Antibodies reactive with the Cp20 peptide of  claim 16 .  
     
     
         18 . The antibodies of  claim 17  wherein said antibodies are polyclonal.  
     
     
         19 . The antibodies of  claim 17  wherein said antibodies are monoclonal.  
     
     
         20 . A kit for performing a diagnostic assay for Alzheimer's disease comprising antibodies which recognize the Cp20 protein.  
     
     
         21 . The kit of  claim 21  further comprising a Cp20 protein sample for use as a control sample.  
     
     
         22 . A scoring method for diagnosing Alzheimer's disease in a sample, said method comprising the steps of: 
 a) acquiring a first value from a first Alzheimer's disease diagnostic test applied to a portion of said sample;    b) acquiring a second value from a second Alzheimer's disease diagnostic test applied to a second portion of said sample; and    c) combining said first value and second value according to a preselected numerical scale for said first and said second Alzheimer's disease diagnostic tests to provide a diagnostic index.    
     
     
         23 . The method of  claim 22  further comprising the step of acquiring a third value from a third Alzheimer's disease diagnostic test applied to a third portion of said sample obtained from said patient, wherein said combining includes combining said third value with said first and second values according to a preselected numerical scale determined for said third Alzheimer's disease diagnostic test.  
     
     
         24 . The method according to  claim 22 , wherein each of said preselected numerical scales is determined by applying the respective diagnostic test to a sample population of Alzheimer's Disease cells to acquire a first response value and apply the respective diagnostic test to a sample population of non-Alzheimer's disease cells to acquire a second response value to classify the cells based on the first and second response signals to provide a score index.  
     
     
         25 . The method of  claim 22  wherein at least one of said Alzheimer's Disease diagnostic tests is selected from the group consisting of: 
 (i) a 113ps potassium ion channel test;  
 (ii) an intracellular calcium concentration test in the presence of a potassium channel blocker test;  
 (iii) an intracellular calcium concentration in the presence of an activator of calcium release test; and  
 (iv) a memory associated GTP binding protein (Cp20) test.  
 
     
     
         26 . The method of  claim 25 , wherein said activator is selected from bradykinin, bombesin, prostoglandin, F 2  and vasopression.  
     
     
         27 . The method of  claim 25 , wherein said potassium channel blocker is selected from the group consisting of TEA, charybdotoxin, apamin, dendratoxin, kalotoxin, mcd-peptide, soyiltoxin, borium, cesium, leivrotoxin, I and noxiustoxin.  
     
     
         28 . The method of  claim 27  wherein said potassium channel blocker is TEA.  
     
     
         29 . The method of  claim 26  wherein said activator is bradykinin.  
     
     
         30 . The method of  claim 26  wherein said activator is bombesin.  
     
     
         31 . The method of  claim 26 , wherein said first diagnostic test comprises contacting the sample portion with bradykinin for a time period not more than 135 seconds; and wherein said first value is indicative of cell response.  
     
     
         32 . The method of  claim 31 , wherein said preselected scale is a score assigned to ranges of percentage of cells responding, said numerical score being: 
 i) a first value when the percentage of responding cells is less than 1.5%;    ii) a second value when the percentage of responding cells is greater than or equal to 1.5%; and    iii) a third value when the percentage of cells responding is greater than or equal to 4%.    
     
     
         33 . The method of  claim 26  wherein said first diagnostic test comprises contacting the sample portion with bombesin for a time period not more than 40 seconds and wherein said first value is indicative of cell response and magnitude of the response represented as time integrated calcium ion concentration, thereby representing an area under a graph of the calcium ion concentration measured as a function of time.  
     
     
         34 . The method of  claim 33  wherein said preselected scale is a score assigned to ranges of percentage cells responding and the magnitude of response is in units of nanomolar-seconds, said score and magnitude being: 
 i) a first value when the percentage of responding cells is less than 50% or the area <23000 nM-sec;  
 ii) a second value when the percentage of responding cells is greater than or equal to 50% and an area greater than or equal to 23000 nM-sec; and  
 iii) a third value when the percentage of cells responding is greater than or equal to 50% and an area greater than or equal to 30000 nM-sec.  
 
     
     
         35 . The method of  claim 28  wherein said diagnostic test comprises contacting said sample portion with TEA for a time period not more than 60 seconds; and wherein said first signal is indicative of cell response.  
     
     
         36 . The method of  claim 35  wherein said preselected score is a score assigned to the percentage of cells responding, said numerical score being: 
 i) a first value when the percentage of responding cells is less than 5%;  
 ii) a second value when the percentage of responding cells is greater than or equal to 5%; and  
 iii) a third value when the percentage of cells responding is greater than or equal to 16%.  
 
     
     
         37 . A sample scoring system for Alzheimer's Disease comprising: 
 i) first diagnostic means for providing a signal corresponding to the first diagnostic test in said sample;    ii) second diagnostic test means for providing a signal corresponding to the second diagnostic test in said sample; and means for combining the said first and second signals into a composite score.