Immunoprecipitation of amyloid fibrils by the use of an antibody that recognizes a generic epitope common to amyloid fibrils.
Immunoprecipitation of amyloid fibrils by the use of an antibody that recognizes a generic epitope common to amyloid fibrils.
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DOI：
10.1371/journal.pone.0105433
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Palhano FL
中科院分区：
文献类型：
--
作者：
Greiner ER;Kelly JW;Palhano FL
淀粉样原纤维与许多疾病有关，包括阿尔茨海默病（AD）。从天然材料中分离淀粉样蛋白非常具有挑战性，因为淀粉样蛋白原纤维的极端结构稳定性使得很难将传统的蛋白质科学方案应用于其纯化。需要一种分离和检测淀粉样蛋白的方案来诊断淀粉样蛋白疾病和鉴定新的功能性淀粉样蛋白。我们的目标是根据淀粉样蛋白折叠的特殊特征，例如其对蛋白水解的抵抗力及其被特定构象抗体识别的能力，开发一种从生物体中纯化淀粉样蛋白的方案。我们采用两步策略，第一步是蛋白水解消化，然后是使用淀粉样蛋白构象抗体 LOC 进行免疫沉淀。我们使用体外产生的淀粉样原纤维、过度表达 Aβ 肽的线虫组织提取物以及诊断为 AD 的患者的脑脊液 (CSF) 作为模型，测试了该方法的功效。我们能够免疫沉淀体外产生的 Aβ1-40 淀粉样蛋白原纤维，然后将其添加到复杂的生物提取物中，但不能免疫沉淀 α-突触核蛋白和凝溶胶蛋白原纤维。该方法可用于从秀丽隐杆线虫 AD 模型的组织匀浆中分离淀粉样蛋白原纤维，特别是从老年蠕虫中。尽管当添加到复杂的生物溶液中时，我们能够捕获体外产生的皮克量的 Aβ1-40 淀粉样蛋白原纤维，但我们无法在 AD 患者的脑脊液中检测到任何 Aβ 淀粉样蛋白聚集体。我们的结果表明，尽管使用 LOC 抗体进行免疫沉淀可用于分离 Aβ1-40 淀粉样蛋白原纤维，但它无法捕获其他淀粉样蛋白原纤维，例如 α-突触核蛋白和凝溶胶蛋白。在将该方案应用于淀粉样蛋白的分离之前，可能需要进行额外的研究来提高这些淀粉样蛋白构象抗体对一系列淀粉样蛋白原纤维的亲和力，而不影响其选择性。
Amyloid fibrils are associated with many maladies, including Alzheimer’s disease (AD). The isolation of amyloids from natural materials is very challenging because the extreme structural stability of amyloid fibrils makes it difficult to apply conventional protein science protocols to their purification. A protocol to isolate and detect amyloids is desired for the diagnosis of amyloid diseases and for the identification of new functional amyloids. Our aim was to develop a protocol to purify amyloid from organisms, based on the particular characteristics of the amyloid fold, such as its resistance to proteolysis and its capacity to be recognized by specific conformational antibodies. We used a two-step strategy with proteolytic digestion as the first step followed by immunoprecipitation using the amyloid conformational antibody LOC. We tested the efficacy of this method using as models amyloid fibrils produced in vitro, tissue extracts from C. elegans that overexpress Aβ peptide, and cerebrospinal fluid (CSF) from patients diagnosed with AD. We were able to immunoprecipitate Aβ1–40 amyloid fibrils, produced in vitro and then added to complex biological extracts, but not α-synuclein and gelsolin fibrils. This method was useful for isolating amyloid fibrils from tissue homogenates from a C. elegans AD model, especially from aged worms. Although we were able to capture picogram quantities of Aβ1–40 amyloid fibrils produced in vitro when added to complex biological solutions, we could not detect any Aβ amyloid aggregates in CSF from AD patients. Our results show that although immunoprecipitation using the LOC antibody is useful for isolating Aβ1–40 amyloid fibrils, it fails to capture fibrils of other amyloidogenic proteins, such as α-synuclein and gelsolin. Additional research might be needed to improve the affinity of these amyloid conformational antibodies for an array of amyloid fibrils without compromising their selectivity before application of this protocol to the isolation of amyloids.
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影响因子：
5.6
作者：
de Laureto, PP;Taddei, N;Fontana, A
通讯作者：
Fontana, A
影响因子：
14.8
作者：
Bosco, DA;Fowler, DM;Kelly, JW
通讯作者：
Kelly, JW
DOI：
10.1073/pnas.0703793104
发表时间：
2007-12-04
影响因子：
11.1
作者：
Habicht, Gernot;Haupt, Christian;Faendrich, Marcus
通讯作者：
Faendrich, Marcus
影响因子：
2.9
作者：
Azevedo, Estefania P. C.;Pereira, Humberto M.;Palhano, Fernando L.
通讯作者：
Palhano, Fernando L.
DOI：
10.1007/978-1-61779-968-6_34
发表时间：
2012-01-01
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
De Genst, Erwin;Dobson, Christopher M
通讯作者：
Dobson, Christopher M