An antibody with high reactivity for disease-associated α-synuclein reveals extensive brain pathology
An antibody with high reactivity for disease-associated α-synuclein reveals extensive brain pathology
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DOI：
10.1007/s00401-012-0964-x
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发表时间：
2012-07-01
影响因子：
12.7
通讯作者：
Lachmann, Ingolf
中科院分区：
文献类型：
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作者：
Kovacs, Gabor G.;Wagner, Uta;Lachmann, Ingolf
-突触核蛋白是与路易体痴呆、帕金森病和多系统萎缩相关的主要蛋白。由于α -突触核蛋白在生理状态下作为突触前蛋白存在于大脑中，因此表征疾病相关修饰以开发体内生物标志物至关重要。为了制备对疾病相关α -突触核蛋白具有高特异性和敏感性的抗体，采用不同氨基酸序列的合成肽对小鼠进行免疫。产生α -突触核蛋白聚集体后，采用ELISA和免疫印迹法检测抗体的特异性。来自不同人类α -突触核蛋白病的组织微阵列切片用于比较免疫染色与其他市售抗体。用肽TKEGVVHGVATVAE （α -synuclein的氨基酸44-57）免疫小鼠，产生单克隆抗体（5G4），该单克隆抗体能够与聚集的α -synuclein制备物结合在夹心ELISA中或包被在磁珠上。通过揭示更广泛和独特的α -突触核蛋白病理，5G4在比较免疫组织化学研究中证明优于其他抗体。人脑组织的免疫印迹显示，在路易体痴呆中有一个额外的条带，而代表单体α -突触核蛋白的条带非常弱或缺乏。综上所述，5G4抗体最有希望用于档案材料的重新评估，并可能为开发检测体液中疾病相关的α -突触核蛋白的体内诊断方法提供新的视角。
alpha-Synuclein is the major protein associated with Lewy body dementia, Parkinson's disease and multiple system atrophy. Since alpha-synuclein is present in the brain in physiological conditions as a presynaptic protein, it is crucial to characterize disease-associated modifications to develop an in vivo biomarker. With the aim to develop antibodies showing high specificity and sensitivity for disease-associated alpha-synuclein, synthetic peptides containing different amino acid sequences were used for immunization of mice. After generation of alpha-synuclein aggregates, ELISA and immunoblotting were used to test the specificity of antibodies. Tissue microarray sections originating from different human alpha-synucleinopathies were used to compare immunostaining with other, commercially available antibodies. Immunization of mice with the peptide TKEGVVHGVATVAE (amino acid 44-57 of alpha-synuclein) resulted in the generation of a monoclonal antibody (5G4), which was able to bind aggregated alpha-synuclein preparation in sandwich ELISA or coated on magnetic beads. 5G4 proved to be superior to other antibodies in comparative immunohistochemical studies by revealing more widespread and distinct alpha-synuclein pathology. Immunoblotting of human brain tissue revealed an additional band seen in dementia with Lewy bodies, whereas the band representing monomeric alpha-synuclein was very weak or lacking. In summary, the 5G4 antibody is most promising for re-evaluation of archival material and may offer new perspective for the development of in vivo diagnostic assays for detecting disease-associated alpha-synuclein in body fluids.