Rapid prion-detection assay

Abstract

Assays are provided for rapid detection, with high specificity of the pathogenic form of prion protein responsible for neurodegenerative diseases affecting humans and animals, such as transmissible spongiform encephalopathy in bovine, sheep, and cats. Also provided are assays for testing animal feedstock, such as animal feed, for the presence or concentration of pathogenic prion protein. Results are available in from about 0.5 to about 20 minutes and preferably within from about 5 to about 10 minutes. The assays employ proteinase-K to remove normal prion protein from a biological sample, so that the sample may be analyzed by immunochromatography to determine the presence and concentration of pathogenic prion protein. Because the proteinase-K is immobilized on a solid support for in situ removal of interfering components, the present invention obviates the need for subsequent extraction of the desired analyte. All aspects of the present invention are suitable for quantifying the minimal detectable amount of pathogenic prion protein in a biological sample. Moreover, the simplicity of sample preparation makes the present invention suitable for use in the field.

1 . An assaying method for detecting a prion disease in an animal or a human suspected of containing a pathogenic prion protein, the method comprising the steps of: 
 (a) homogenizing a biological sample from an animal or a human suspected of containing a pathogenic prion protein with a buffer and removing nonpathogenic prion protein from the biological sample;    (b) providing a test device comprising: 
 (i) a pair of antibodies to the pathogenic prion protein, one of the antibodies being immobilized on a membrane and the other of the antibodies being labeled for forming a complex with the pathogenic prion protein such that the complex migrates toward the immobilized antibody;  
   (c) applying the homogenized biological sample to the test device wherein: 
 (i) the pathogenic prion protein in the sample binds with both antibodies to produce a response; and  
   (d) interpreting the response to indicate the presence or concentration of the pathogenic prion protein in the sample.    
   
   
       2 . The assay of  claim 1  wherein the prion being analyzed causes a condition selected from the group consisting of transmissible spongiform encephalopathy (TSE) in bovine, sheep, and goats; scrapie in sheep and goat; transmissible mink encephalopathy (TME) in mink; chronic wasting disease (CWD) in mule deer and elk; feline spongiform encephalopathy in cats; and kuru, Creutzfeld-Jakob-disease (CJD), German-Straussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI) in humans.  
   
   
       3 . The assay of  claim 1  wherein the buffer comprises: 
 (i) at least one surfactant or emulsifier;    (ii) at least one polysaccharide; and    (iii) casein; and    (iv) albumin.    
   
   
       4 . The assay of  claim 1  wherein the emulsifier or surfactant is selected from octoxynol, nonoxynol, polyglycol ether, polyoxyethylene isooctylphenyl ether, sodium dodecyl sulfate, and sodium deoxycholate.  
   
   
       5 . An assay for determining the presence or concentration of pathogenic prion protein in a biological sample, the assay comprising: 
 (a) preparing a sample for analysis by homogenizing the sample with a buffer and removing nonpathogenic prion protein from the biological sample;    (b) providing a test device having: 
 (i) a conjugate pad having a labeled first antibody of an antibody pair to the pathogenic prion protein; and,  
 (ii) a test strip having an immobilized second antibody of the antibody pair for producing a response indicative of the presence or concentration of the pathogenic prion protein;  
   (c) applying the prepared sample to the test device so the pathogenic prion protein in the sample binds with both antibodies to produce a response; and    (d) determining the presence or concentration of the pathogenic prion protein in the sample based on the response.    
   
   
       6 . An assay for detecting the presence or concentration of pathogenic prion protein in foodstuffs, comprising: 
 (a) preparing a sample of foodstuff for analysis homogenizing the sample with a buffer and removing nonpathogenic prion protein from the biological sample;    (b) providing a test device having: 
 (i) a support;  
 (ii) a membrane in fluid communication with the support through which the sample migrates; and  
 (iii) a pair of antibodies to the pathogenic prion protein including an antibody immobilized on the membrane and a labeled antibody.  
   (c) applying the prepared sample to the test device for immunochromatographic binding of the pathogenic prion protein to produce a response; and    (d) determining the presence or concentration of the pathogenic prion protein in the sample based on the response.    
   
   
       7 . An assay for pathogenic prion protein in foodstuffs, comprising: 
 (a) preparing a sample of foodstuff for analysis, wherein the sample preparing step comprises:    (a) homogenizing the sample with a buffer and removing nonpathogenic prion protein from the biological sample;    (b) removing interfering constituents from the foodstuff sample;    (c) applying the sample to a test device having 
 (i) an antibody immobilized on a membrane; and  
 (iii) a labeled antibody that complexes with pathogenic prion protein in the sample and migrates through the membrane toward the immobilized antibody; and  
   (d) analyzing the labeled antibody that complexes with the pathogenic prion for the presence or concentration of pathogenic prion protein in the foodstuff.