Antibody based reagent that specifically recognizes toxic oligomeric form of beta-amyloid

Abstract

The invention relates to antibodies, antibody fragments and binding agents that specifically recognizes oligomeric Aβ that is resistant to denaturation by SDS but does not bind monomeric, fibrillar or other oligomeric forms of Aβ that are generated in vitro.

1 - 2 . (canceled) 
     
     
         3 . The antibody fragment of  claim 5 , wherein said antibody fragment does not contain the constant domain region of an antibody. 
     
     
         4 . (canceled) 
     
     
         5 . An antibody fragment isolated according to a method comprising the steps of:
 (a) negative panning a scFV phage library comprising serially contacting phage with:
 (i) brain derived control samples that do not contain oligomeric Aβ, 
 (ii) brain-derived mononeric forms of Aβ, and 
 (iii) synthetic monomeric forms of Aβ, 
 and until less than 5% of the phage is observed binding to antigen, which produces an aliquot of phage that does not bind to monomeric or fibrillar Aβ or in vitro-generated oligomeric Aβ; 
   (b) positive panning of the aliquot from step (a) comprising contacting the aliquot of phage from step (a) with natural brain derived, SDS-stable oligomers of Aβ, and incubating for time sufficient to allow binding of phage to said SDS-stable oligomers of Aβ; and   (c) eluting the bound phage particles from step (b).   
     
     
         6 . The antibody fragment of  claim 5 , wherein the observing of the binding of the phage to the antigen is by using Atomic Force Microscope (AFM) Imaging. 
     
     
         7 . The antibody fragment of  claim 5 , wherein the negative panning is repeated until less than 0-10% phage was observed by AFM imaging as binding to antigen in step (a). 
     
     
         8 - 12 . (canceled) 
     
     
         13 . A method of inhibiting the aggregation of Aβ comprising contacting a composition that comprises Aβ monomers with an antibody fragment of  claim 5 . 
     
     
         14 . The method of  claim 13 , wherein said aggregation of Aβ is in a cell. 
     
     
         15 . The method of  claim 13 , wherein said aggregation of Aβ is in brain tissue. 
     
     
         16 . The method of  claim 13 , wherein said contacting with an antibody fragment decreases the rate of formation of Aβ aggregates as compared to said rate in the absence of the antibody fragment. 
     
     
         17 . A method of detecting the presence of Aβ in a physiological sample comprising contacting a sample with the antibody fragment of  claim 5  and determining the binding of the antibody fragment with said tissue sample wherein binding of the antibody fragment to said tissue sample is indicative of the presence of SDS-stable Aβ oligomers in said tissue sample wherein said presence of said SDS-stable Aβ oligomers is indicative of early stage AD. 
     
     
         18 . The method of  claim 17 , wherein the physiological sample is brain tissue, serum, cerebrospinal fluid (CSF), urine or saliva. 
     
     
         19 . A method of preventing or inhibiting the accumulation of Aβ in the brain of a mammal comprising administering to said mammal a composition comprising an antibody fragment of  claim 5 . 
     
     
         20 - 31 . (canceled) 
     
     
         32 . The antibody fragment of  claim 5 , wherein the antibody binds the 12-16 kDa species of Aβ. 
     
     
         33 . The antibody fragment of  claim 5 , wherein the brain derived control sample of step (i) is human-derived. 
     
     
         34 . The antibody fragment of  claim 5 , wherein the brain derived monomeric form of Aβ sample of step (ii) is human-derived. 
     
     
         35 . The antibody fragment of  claim 5 , wherein steps (b) and (c) are repeated one or more times. 
     
     
         36 . The antibody fragment of  claim 5 , wherein step (a) and/or step (b) is performed on mica. 
     
     
         37 . A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an antibody fragment isolated according to a method comprising the steps of:
 (a) negative panning a scFV phage library comprising serially contacting phage with:
 brain derived control samples that do not contain oligomeric Aβ, 
 (ii) brain-derived mononeric forms of Aβ, and 
 (iii) synthetic monomeric forms of Aβ, 
 and until less than 5% of the phage is observed binding to antigen, which produces an aliquot of phage that does not bind to monomeric or fibrillar Aβ or in vitro-generated oligomeric Aβ; 
   (b) positive panning of the aliquot from step (a) comprising contacting the aliquot of phage from step (a) with natural brain derived, SDS-stable oligomers of Aβ, and incubating for time sufficient to allow binding of phage to said SDS-stable oligomers of Aβ; and   (c) eluting the bound phage particles from step (b).   
     
     
         38 . A detection composition comprising a detecting reagent conjugated to an antibody fragment isolated according to a method comprising the steps of:
 (a) negative panning a scFV phage library comprising serially contacting phage with:
 (i) brain derived control samples that do not contain oligomeric Aβ, 
 (ii) brain-derived mononeric forms of Aβ, and 
 (iii) synthetic monomeric forms of Aβ, 
 and until less than 5% of the phage is observed binding to antigen, which produces an aliquot of phage that does not bind to monomeric or fibrillar Aβ or in vitro-generated oligomeric Aβ; 
   (b) positive panning of the aliquot from step (a) comprising contacting the aliquot of phage from step (a) with natural brain derived, SDS-stable oligomers of Aβ, and incubating for time sufficient to allow binding of phage to said SDS-stable oligomers of Aβ; and   (c) eluting the bound phage particles from step (b).   
     
     
         39 . The detection composition of  claim 38 , wherein the detecting reagent is conjugated to the antibody fragment by means of a chemical linker. 
     
     
         40 . The detection composition of  claim 38 , wherein the detecting reagent is directly conjugated to the antibody fragment.