Purified Antigen for Alzheimer's Disease and Methods of Obtaining and Using Same

Abstract

The invention relates, among other things, a preparation comprising Alzheimer's disease antigen (A68), as well as methods of obtaining this purified antigen, and methods of using this purified antigen, for instance, for diagnosing Alzheimer's disease and for detecting human autoantibodies to the Alzheimer disease antigen. The antigen preparation according to the invention is purified in that it is substantially free of immunoglobulin G. The invention further relates to methods of making Alzheimer disease antigens that can be used instead of or along with the A68 antigen preparation (e.g., for diagnosing AD), such as recombinant human tau, tau isolated from various species including human, and phosphorylated recombinant human tau or isolated tau, as well as A68 anti-idiotypic antibodies.

1 . A protein preparation consisting essentially of an antigen that is immunologically reactive with a monoclonal antibody produced by the hybridoma cell line identified as ATCC No. HB9205, said preparation being substantially free of immunoglobulin G. 
     
     
         2 . The protein preparation of  claim 1 , wherein said preparation has an amount of immunoglobulin G that is equal to or less than about 0.05% of the total protein of said preparation. 
     
     
         3 . The protein preparation of  claim 1 , wherein said preparation has an amount of immunoglobulin G that is equal to or less than about 0.0015% of the total protein of said preparation. 
     
     
         4 . The protein preparation of  claim 1 , wherein said preparation has less than about 500 pg of immunoglobulin G per μg of said antigen. 
     
     
         5 . The protein preparation of  claim 1 , wherein said preparation has less than about 15 pg of immunoglobulin G per μg of said antigen. 
     
     
         6 . A protein preparation consisting essentially of an antigen, which preparation is a diagnostic marker of Alzheimer's disease, wherein said antigen comprises a major polypeptide species that:
 (a) has an isoelectric point of about 6 in reduced or non-reduced form;   (b) binds to an affi-Blue column;   (c) is at least 50% soluble in a solution of 0.01 M sodium phosphate, 0.14 M sodium chloride and 1 mM phenyl methyl sulfonyl fluoride at pH 6.8, and precipitates in 50% saturated ammonium sulfate at 4° C.;   (d) is immunologically reactive with a monoclonal antibody produced by the hybridoma cell line identified as ATCC No. HB9205; and   (e) is substantially free of immunoglobulin G.   
     
     
         7 . The protein preparation of  claim 6 , wherein said preparation has an amount of immunoglobulin G that is equal to or less than about 0.05% of the total protein of said preparation. 
     
     
         8 . The protein preparation of  claim 6 , wherein said preparation has an amount of immunoglobulin G that is equal to or less than about 0.0015% of the total protein of said preparation. 
     
     
         9 . The protein preparation of  claim 6 , wherein said preparation has less than about 500 pg of immunoglobulin G per μg of said antigen. 
     
     
         10 . The protein preparation of  claim 6 , wherein said preparation has less than about 15 pg of immunoglobulin G per μg of said antigen. 
     
     
         11 . A process for obtaining the protein preparation of  claim 1 , said process comprising:
 (a) obtaining a sample of cortical brain tissue containing said antigen;   (b) homogenizing said sample in buffer to obtain a homogenate;   (c) removing particulate matter from said homogenate;   (d) removing said antigen from said homogenate by contacting the homogenate with an antibody under conditions wherein said antigen and said antibody form an antigen-antibody complex;   (e) eluting said antigen from said antigen-antibody complex; and   (f) removing immunoglobulin G from the eluent to obtain said protein preparation.   
     
     
         12 . The process of  claim 11 , wherein said immunoglobulin G is removed by incubation of said protein preparation with: (a) Protein A; (b) Protein G; (c) both Protein A and Protein G; or (d) an immunoglobulin G removal method that is substantially equivalent to (c). 
     
     
         13 . In an improvement of a process for obtaining a preparation consisting essentially of an antigen that is immunologically reactive with the monoclonal antibody produced by the hybridoma cell line identified as ATCC No. HB9205, said improvement comprising removing immunoglobulin G from the antigen preparation to obtain a preparation that is substantially free of immunoglobulin G. 
     
     
         14 - 37 . (canceled)