Epitomic Characterization of the Specificity of the Anti-Amyloid Aβ Monoclonal Antibodies 6E10 and 4G8.
Epitomic Characterization of the Specificity of the Anti-Amyloid Aβ Monoclonal Antibodies 6E10 and 4G8.
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DOI：
10.3233/jad-180582
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发表时间：
2018
期刊：
影响因子：
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通讯作者：
Glabe C
中科院分区：
文献类型：
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作者：
Baghallab I;Reyes-Ruiz JM;Abulnaja K;Huwait E;Glabe C
单抗6E10和4G8是首批抗Aβ的抗淀粉样蛋白单抗之一，也是阿尔茨海默病研究中使用最广泛的抗体。虽然6E10和4G8的表位已被报道对应于残基1-16和17-24，但最近的高分辨率作图方法表明，6E10映射到残基4-10，而4G8映射到残基18-23。为了更详细地表征这两种抗体的结合特异性，我们使用了从噬菌体展示文库中免疫选择随机序列，然后对数千个免疫选择序列进行深度测序和结果模式分析。我们发现，6E10结合所需的最小序列是R-x-D，超过一半(53%)的免疫选择序列符合这一模式。这些序列中的绝大多数在位置x(R-H-D)包含H，对应于Aβ靶序列的5-7个残基，但在少数序列中在该位置也允许Y。对于4G8，我们发现最频繁的模式是F-x-A，大约30%的序列包含F-x-A，其次是F-A，L-x(3)-A，L-x-F和F-F，每个大约占序列的18%。F-x-A基序也存在于胰岛淀粉样多肽中，这可能解释了为什么4G8也识别该多肽的淀粉样纤维。随机序列的免疫选择和深度测序也可能是确定抗体结合关键残基和验证单抗和多克隆抗血清特异性的一种简便而有效的手段。
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.