Detection of pathogenic abeta using an epitope protection assay
The invention relates to an epitope protection assay for use in diagnosis, prognosis and therapeutic intervention in diseases, for example, involving polypeptide aggregation, such as prion infections. The methods of the invention first block accessible polypeptide target epitope with a blocking agent. After denaturation of the polypeptide, a detecting agent is used to detect protein with target epitope that was inaccessible during contact with the blocking agent.
1. A method of detecting whether a sample contains a beta-amyloid polypeptide (Abeta) in an aggregated conformation, comprising: contacting the polypeptide in the sample with at least 2 mM concentration of peroxynitrite to block an accessible target epitope on the Abeta, wherein in the wildtype conformation, the target epitope is accessible and chemically reacts with the peroxynitrite, and wherein in the aggregated conformation, the target epitope is inaccessible and the target epitope cannot chemically react with the peroxynitrite; and wherein the target epitope is EFRHDS (SEQ ID NO:3) or amino acid residues at positions 18-22 of human Abeta;
removing unreacted peroxynitrite from contact with the Abeta;
disaggregating or denaturing the Abeta to convert any inaccessible target epitope to the accessible target epitope;
contacting the sample with an antibody that binds selectively to the target epitope that was converted from the inaccessible target epitope to the accessible target epitope, the antibody having at least a 2-fold increased specificity for the converted target epitope compared to the target epitope reacted with peroxynitrite; and
detecting the presence of binding between the antibody and the converted target epitope,
wherein binding between the antibody and the converted target epitope indicates that the Abeta was in an aggregated conformation.
2. The method of claim 1 , wherein the polypeptide is denatured by heat and/or detergent and/or chaotropic agents.
3. The method of claim 1 , wherein the polypeptide is modified by treatment with a disaggregation agent to disaggregate the polypeptide from the aggregated polypeptides.
4. The method of claim 1 , wherein the disaggregation agent is selected from at least one of the group consisting of chaotropic agents, detergent and heat.
5. The method of claim 1 , wherein the detergent comprises SDS.
6. The method of claim 1 , wherein prior to contacting the peroxynitrite with the Abeta polypeptide, the Abeta polypeptide is in a sample that is pretreated by one or more of the following methods: adsorption, precipitation, or centrifugation.
7. The method of claim 1 , wherein prior to contacting the peroxynitrite with the Abeta polypeptide, the target epitope is mapped.
8. The method of claim 1 , wherein the polypeptide is in a postmortem or antemortem sample selected from the group of: CSF, serum, blood, urine, biopsy sample or brain tissue.
9. The method of claim 1 , wherein the antibody comprises the antibody 6E10 or 4G8 and/or binds specifically to EFRHDS (SEQ ID NO:3) or amino acid residues at positions 18-22 of human Abeta.