Epitomic Characterization of the Specificity of the Anti-Amyloid Aβ Monoclonal Antibodies 6E10 and 4G8

Abstract

The monoclonal antibodies 6E10 and 4G8 are among the first anti-amyloid monoclonal antibodies against Aβ and the most widely used antibodies in Alzheimer's disease research. Although the epitopes for 6E10 and 4G8 have been reported to correspond to residues 1-16 and 17-24, a more recent high-resolution mapping approach indicates that 6E10 maps to residues 4-10 while 4G8 maps to residues 18-23. To characterize the binding specificity of both antibodies in greater detail, we used immunoselection of random sequences from phage display library followed by deep sequencing and analysis of resulting patterns from thousands of immunoselected sequences. We found that the minimum sequence required for 6E10 binding is R-x-D with over half (53%) of the immunoselected sequences conforming to this pattern. The vast majority of these sequences contain an H at position x (R-H-D), corresponding to residues 5-7 of the Aβ target sequences, but Y is also permitted at this position in a minority of sequences. For 4G8 we found that the most frequent pattern is F-x-A contained in approximately 30% of the sequences, followed by F-A, L-x(3)-A, L-x-F, and F-F each accounting for approximately 18% of the sequences. The F-x-A motif also occurs in islet amyloid poly peptide which may explain why 4G8 also recognizes amyloid fibrils of this peptide. Immunoselection of random sequences and deep sequencing may also be a facile and efficient means of determining residues critical for antibody binding and validating the specificity of monoclonal antibodies and polyclonal antisera.

Keywords: Amyloid antibodies; epitope mapping; epitopes; informatics; specificity.

Fig. 1.

Top ranked 32 unique sequences for 4G8. The top 10 sequences in the first panning are color coded to make their recognition more readily apparent. Of the top 10 sequences in the first panning, all ten are found in the top 32 of the second panning and 9 out of 10 are found in the third round of panning, indicating that their relative abundance does not change much with subsequent pannings.

Fig. 2.

Top ranked 32 unique sequences for 6E10. The top 10 sequences in the first panning were color coded to make their recognition more readily apparent. Of the top 10 sequences in the first panning, 7 were found in the top 32 of the second panning and 4 out of 10 were found in the third round of panning, indicating that their relative abundance does not change much with subsequent pannings.