新型原子力显微镜基于生物淘选的形态特殊试剂对TDP-43变型肌萎缩侧索硬化症隔离
Summary
Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis.
Abstract
因为蛋白变体中的许多疾病，包括TDP-43在肌萎缩性脊髓侧索硬化症（ALS），α-突触核蛋白中发挥关键作用帕金森氏病和β-淀粉样蛋白和tau蛋白在阿尔茨海默氏病，这是极为重要的发展形态特异性试剂，可以选择性地靶向这些疾病特异性蛋白变体来研究疾病的病理和潜在的诊断和治疗应用这些变体的作用。我们已经开发出新颖的原子力显微镜（AFM）基于生物淘选技术，使试剂选择性识别特定疾病的蛋白质变体的分离。有参与的过程中，负和正淘选相位的两个主要阶段。在负相淘选，噬菌体是反应性的脱靶抗原，通过多轮利用一系列精心挑选的脱靶抗原消减淘选被消除。否定一个主要特征平移相是利用原子力显微镜成像以监测过程，并确认所有不希望的噬菌体颗粒被除去。用于正淘选阶段中，感兴趣的靶抗原被固定在云母表面和结合的噬菌体洗脱，并筛选以鉴定噬菌体选择性结合靶抗原。靶蛋白变体不需要进行纯化提供适当的阴性对照摇动已被使用。即使靶蛋白变体是唯一存在的非常低的浓度在复杂的生物材料可以在正淘选步骤中使用。通过应用这种技术，我们收购了抗体TDP-43是在人类ALS脑组织选择性地发现蛋白变体。我们预计，这个协议应适用于生成试剂选择性结合蛋白变体中存在的多种不同的生物过程和疾病。
Introduction
蛋白质变体的存在已被认为在许多疾病，包括神经变性疾病如阿尔茨海默氏症，帕金森氏，ALS和额颞痴呆（FTD）1,2,3,4,5,6,7,8,9的进展的一个因素，10,11。蛋白质的β-淀粉样蛋白和α-突触核蛋白的寡聚形式被认为是负责阿尔茨海默氏症和帕金森氏分别2,3,4,5有毒物种。在TAR DNA结合蛋白43（TDP-43）的聚集已与ALS和FTD 12,13,14。因此试剂如能够选择性靶向不同的蛋白质的变体可以是有力工具作为诊断标志物和潜在的治疗剂的抗体。在这项研究中，我们专注于开发的试剂选择性结合的TDP-43蛋白牵连在ALS的变体，但在本文中概述的技术应当适用于试剂的隔离针对广泛PROT的EIN变种。
TDP-43的细胞浆聚合已被确定为在ALS 15,16,17,18,19病理特征。典型地，TDP-43是在从正常个体的所有细胞的细胞核中，虽然它趋向于胞质溶胶和细胞核15,17之间移动。 TDP-43。然而，在ALS聚集形式中选择的神经元的细胞质和神经胶质与在细胞核中的疾病进展16,20暗示TDP-43的从细胞核向细胞质的移动发现低浓度的被检测到。而TDP-43的聚合中的大多数的ALS病例发现，它不自1％的总的ALS病例（或15％的家族性ALS例-20％）-2％被链接到在所述突变占所有病例超氧化物歧化酶1（SOD1）基因15,17。由于TDP-43在ALS的情况下，绝大多数的重要作用，在这里，我们专注于开发基于抗体的试剂，可以选择性结合TDP-43的变体是目前在利用我们的新型AFM基于生物淘洗技术人力ALS脑组织。
首先，我们需要的抗体结合结构域的不同剧目。我们结合三个不同的噬菌体展示单链可变区抗体片段（scFv的）库，（汤姆林森I和J和表库21）。所述平移过程分为阴性和阳性平移相。来自文库的噬菌体首先经受负淘选过程噬菌体其间反应到多个脱靶抗原被排除。在完成每一轮负淘选针对每个脱靶抗原后，该过程是通过原子力显微镜成像监测，以确保所有的噬菌体结合的脱靶抗原已被删除。只有原子力显微镜成像，所有反应噬菌体被删除核实后，我们进入到下一个目标。为了隔离对TDP-43的变种牵连ALS试剂，我们利用一系列的负面平移抗原： 1）的BSA以除去噬菌体结合弱的或非特异性的蛋白质; 2）聚合α-突触核蛋白除去噬菌体结合到聚集的蛋白质的一般结构元件; 3）人脑组织匀浆以除去噬菌体结合于任何蛋白或本健康人脑组织验尸样品中其它组分; 4）免疫TDP-43从健康人的大脑，除去噬菌体结合与健康人的大脑相关的所有TDP-43表格;和5）免疫沉淀TDP-43从FTD脑匀浆中分离除去噬菌体结合与非ALS的病理相关联的TDP-43变体。去除所有的噬菌体的反应性的所有脱靶抗原后，我们接着就在此期间，该结合目的抗原的抗体片段被隔离的正淘选相，在这种情况下，TDP-43从人类ALS的脑组织中免疫沉淀。这些分离的抗体可以是反应性的TDP-43的凝集或修饰的形式。
“>传统噬菌体生物淘选主要集中在正淘选阶段22,23。一般感兴趣的靶被固定，所述噬菌体文库并将结合的噬菌体洗脱。噬菌体，然后放大并再次加入到目标，这扩增和培养过程通常重复几次，以增加的阳性结合噬菌体的百分比。尽管这种方法的变体已被广泛使用，以隔离抗体试剂对多种靶抗原的，它们一般都需要大量的纯化的靶抗原24,25,26的， 27，而我们的过程只需要微量的靶抗原的量。这里所描述的协议可以用于分离选择性结合靶抗原存在于非常低的浓度，而不需要纯化和平移可以直接针对进行试剂目前复杂的组织样本中的抗原，利用详尽的负面平移协议的验证用AFM确保克隆中分离针对抗原阳性应选择性地结合靶即使在不进行纯化或富集。Kasturirangan和他的同事（2003年）都进行了类似的消极和积极的生物淘洗过程隔离的抗体反应来使用目标5纳克的浓度低聚β-淀粉样蛋白。在这里，我们在这个过程中展开，使试剂选择性结合疾病特异性蛋白变体直接从人组织样品的产生。在未来的研究中，我们打算不仅要进一步调查此隔离试剂的诊断价值，而且评估其治疗的相关治疗ALS。
总的来说，我们的新颖的AFM基于生物淘选技术应当适用于任何特定疾病的蛋白质变体在任何生物材料的分离而无需蛋白质纯化或修饰，即使当靶抗原concentratioNS是非常低的。
Protocol
Representative Results
Discussion
Protein variants have been shown to be involved in the progression of many neurodegenerative diseases such as Alzheimer’s, Parkinson’s, ALS and FTD1,2,3,4,5,6,7,8,9,10,11. Isolation of antibodies that can selectively recognize these different protein variant targets can be effective reagents to study, diagnose and potentially treat such ailments. To generate such variant specific antibodies we have developed a novel biopanning process that utilizes atomic force microscopy to monitor the progress …
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究得到了美国国立卫生研究院的拨款：R21AG042066。我们要感谢菲利普·舒尔茨，他在创建屏幕捕获视频的贡献。
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| Tomlinson I and J Libraries | MRC (Cambridge, England) | ||
| Sheets Library | MRC (Cambridge, England) | ||
| 2xYT | BD Sciences | 244020 | |
| Glucose | Amresco | 0188-2.5KG | |
| Ampicillin | Amresco | 0339-25G | Irritant |
| KM13 Helper Phage | MRC (Cambridge, England) | ||
| Kanamycin | OmniPur | 5880 | Irritant |
| Polyethylene Glycol 8000 | OmniPur | 6510 | Irritant |
| Sodium Chloride | Macron | 7647-14-5 | |
| Sodium Phosphate Dibasic | Amresco | 0404-1KG | Irritant |
| Potassium Chloride | EMD | PX1405-1 | Irritant |
| Potassium Phosphate Monobasic | Amresco | 0781-500G | Irritant |
| TG1 Cells | MRC (Cambridge, England) | ||
| Luria-Bertani Agar | EMD | 1.10283.0500 | |
| Bovine Serum Albumin | Amresco | 0332-100G | |
| STEN buffer | Crystalgen Inc. | 33429775 | |
| Immunotubes | Thermo Scientific | 470319 | |
| Mica | Spruce Pine Mica | 24365 | |
| Tween 20 | EMD | ||
| Trypsin | Sigma | T-0303 | Irritant |
| Triethylamine | Sigma | T-0886 | Flammable |
| Glycerol | Amresco | 0854-1L | Irritant |
| DNA Plasmid Prep Kit | qiagen | 27106 | Irritant |
| Non-Fat Milk Powder | Carnation | ||
| 96-Well High Binding ELISA Plate | Costar | 3590 | |
| Anti-M13 HRP | GE Healthcare Life Sciences | 27-9421-01 | |
| ELISA Femto Chemiluminescence Substrate Kit | Thermo Scientific | 37074 | |
| Anti-TDP 43 Polyclonal Antibody | ProteinTech | 10782-2-AP | |
| A/G Agarose Beads | Santa Cruz Biotechnology | sc-2003 | |
| HB 2151 Cells | MRC (Cambridge, England) | ||
| Isopropylthiogalactoside | Teknova | 13325 | |
| 9e10 HRP | Santa Cruz Biotechnology | sc-40 | |
| Nitrocellulose Membrane | Biorad | 162-0115 | Flammable |
| Centrifuge | Thermo Scientific | Sorvall RC 6+ | |
| Nanoscope IIIa Atomic Force Microscope | Veeco | ||
| AFM Probes | VistaProbes | T300R-10 |
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