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 . A method of making an antibody fragment, the method comprising the steps of:
 (a) negative panning a scFV phage library, wherein the negative panning comprises serially contacting phage with:
 (i) brain-derived control samples that do not contain oligomeric Aβ, 
 (ii) brain-derived monomeric forms of Aβ, and 
 (iii) synthetic monomeric forms of Aβ, 
 and until less than 10% 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), wherein the positive panning comprises contacting the aliquot of phage from step (a) with natural brain derived, SDS-stable oligomers of Aβ, and incubating for sufficient time to allow binding of phage to the SDS-stable oligomers of Aβ;   (c) eluting the bound phage particles from step (b),   (d) infecting bacteria with the phage, and   (e) isolating antibody fragments generated by the bacteria, wherein the antibody fragment does not contain the constant domain region of an antibody.   
     
     
         2 . The method of  claim 1 , wherein the observing of the binding of the phage to the antigen is by using Atomic Force Microscope (AFM) Imaging. 
     
     
         3 . The method of  claim 1 , wherein the negative panning is repeated until less than 5% phage is observed by AFM imaging as binding to antigen in step (a). 
     
     
         4 . The antibody fragment of  claim 1 , wherein the brain-derived control sample of step (a)(i) is human-derived. 
     
     
         5 . The antibody fragment of  claim 1 , wherein the brain-derived monomeric form of Aβ sample of step (a)(ii) is human-derived. 
     
     
         6 . The antibody fragment of  claim 1 , wherein steps (b) and (c) are repeated one or more times. 
     
     
         7 . The antibody fragment of  claim 1 , wherein step (a) or step (b) is performed on mica. 
     
     
         8 . The method of  claim 1 , wherein the bacteria is  E. coli.    
     
     
         9 . A method of making a detection composition comprising a detecting reagent conjugated to an antibody fragment isolated according to a method, the method comprising the steps of:
 (a) negative panning a scFV phage library, wherein the negative panning comprises serially contacting phage with:
 (i) brain-derived control samples that do not contain oligomeric Aβ, 
 (ii) brain-derived monomeric forms of Aβ, and 
 (iii) synthetic monomeric forms of Aβ, 
 and until less than 10% of the phage is observed binding to antigen, 
 which produces an aliquot of phage that does not bind to monomeric or fibrillar AO or in vitro-generated oligomeric Aβ; 
   (b) positive panning of the aliquot from step (a), wherein the positive panning comprises 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 the SDS-stable oligomers of Aβ;   (c) eluting the bound phage particles from step (b),   (d) infecting bacteria with the phage,   (e) isolating antibody fragments generated by the bacteria, wherein the antibody fragment does not contain the constant domain region of an antibody, and   (f) conjugating a detecting reagent to the antibody fragment.   
     
     
         10 . The method of  claim 9 , wherein the detecting reagent is conjugated to the antibody fragment by means of a chemical linker. 
     
     
         11 . The method of  claim 9 , wherein the detecting reagent is directly conjugated to the antibody fragment. 
     
     
         12 . The method of  claim 9 , wherein the bacteria is  E. coli.    
     
     
         13 . The method of  claim 9 , wherein the negative panning is repeated until less than 5% phage is observed by AFM imaging as binding to antigen in step (a). 
     
     
         14 . A method of making an scFv, the method comprising the steps of:
 (a) negative panning a scFv phage library, wherein the negative panning comprises serially contacting phage with:
 (i) brain-derived control samples that do not contain oligomeric Aβ, 
 (ii) brain-derived monomeric forms of Aβ, and 
 (iii) synthetic monomeric forms of Aβ, and until less than 10% 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 the SDS-stable oligomers of Aβ;   (c) eluting the bound phage particles from step (b),   (d) infecting bacteria with the phage, and   (e) isolating scFv generated by the bacteria, wherein the scFv does not contain the constant domain region of an antibody.   
     
     
         15 . The method of  claim 14 , wherein the bacteria is  E. coli.    
     
     
         16 . The method of  claim 14 , wherein the negative panning is repeated until less than 5% phage is observed by AFM imaging as binding to antigen in step (a).