MeCP2蛋白质变异的电化学发光测定
1Institute of Medical Genetics, Center for Pathobiochemistry and Genetics,Medical University of Vienna (MUV), 2Murdoch Children’s Research Institute and Department of Paediatrics, University of Melbourne, Discipline of Child & Adolescent Health,Sydney Medical School, 3Department of Medical Chemistry and Pathobiochemistry,Medical University of Vienna
Summary
电化学发光免疫测定（ECLIA）是一种定量检测内源和外源应用的MeCP2蛋白变异的新方法，在广泛的工作范围内产生高定量、准确和可重复的测量，检测内误差低。此处介绍了 MeCP2-ECLIA 的 96 井格式的协议。
Abstract
ECLIA 是一种多功能方法，能够以 96 孔格式量化内源和重组蛋白量。为了证明ECLIA的效率，该测定用于分析小鼠脑组织中MeCP2的内在水平和TAT-MeCP2在人类皮肤纤维细胞中的接受。MeCP2-ECLIA 可生成高精度和可重复的测量，具有较低的内部和内部测定误差。总之，我们开发了一种定量方法，用于评估MeCP2蛋白变异，可用于高通量屏幕。
Introduction
电化学发光免疫测定（ECLIA）基于一个过程，该工艺利用标签，设计在电化学刺激时发出发光。它是一种广泛适用的生物分析物定量检测技术，用于基础工业和学术研究、食品工业以及临床诊断1。通常，使用带有碳墨水电极的一次性 96 孔板。这些电极充当免疫测定的固相载体。二次抗体与电化学发光标签结合，当电应用于系统时，触发化学标签的光发射。超低噪声电荷耦合装置（CCD）记录光强度与与捕获抗体结合的抗原成正比，从而对样品2的目标解解物进行定量。与酶相关的免疫吸附剂测定（ELISA）相比，ECLIA被认为是有利的，因为它提供了更高的灵敏度和可重复性，以及更好的自动化和一致性3。
在这里，我们分析了人类和鼠源样品中的甲基-CpG结合蛋白2（MeCP2）水平，以及使用新开发的ECLIA系统对重组蛋白的不同变异。MecP2是一种与X相关的核酸结合蛋白，已知与甲基化DNA序列相互作用。这种蛋白质已经牵连到基因表达的调节44，5。5编码这种蛋白质的基因功能丧失突变是导致Rett综合征（RTT）的罪魁祸首，一种严重的神经发育障碍6。另一种MeCP2相关疾病，MECP2重复综合征，也导致神经症状，可以重叠的RTT7。值得注意的是，女性大多受RTT影响，而男性则大多受MECP2重复综合征66、77的影响。
这些疾病分别与中枢神经系统（CNS）中MeCP2水平不足或过量有关。因此，RTT的治疗方案，涉及增加MECP2水平在CNS将需要避免与过量的MeCP27相关的有害影响。由于这一事实，ECLIA系统对MeCP2蛋白水平进行高度敏感和准确的定量，对于推进RTT以及MECP2重复综合征研究至关重要。从人体细胞系和小鼠组织样本中精确测量内源性和外源性MeCP2水平，以及由人类MeCP2等型B（也称为异体e1）组成的重组蛋白，以及具有跨越血脑屏障88、99的最小N-终端HIV-TAT转导域（TAT-MeCP2）。
Protocol
澳大利亚韦斯特米德儿童医院人体研究伦理委员会批准了为研究目的进行皮肤活检采购。动物实验的同意得到了奥地利联邦科学、研究和经济部的同意，这些实验是根据当地动物福利条例（GZ：66.009/0218-II/3b/2015）进行的。 注：图1中描述了ECLIA系统的原理。 1. 抗体选择 评估各种 MeCP2 抗体的信号噪声比，并在主小鼠单克隆 MeCP2…
Representative Results
图1描述了ECLIA系统的原理。图 2显示了两个 MeCP2 变体的标准曲线。在广泛的浓度（1~1，800纳克/mL）上可以精确定量。在图3中，分析了从小鼠大脑和HDF中提取的MeCP2水平裂灰。图3A对来自异质细胞、野生型和敲空小鼠的脑核结扎液中的MeCP2表达进行了对比，而图3B中，使用ECLIA的MECP2缺乏人…
Discussion
为了测量内源性MeCP2、重组MeCP2和TAT-MeCP2水平，开发了96孔板ECLIA。已经表明，MeCP2蛋白功能的丧失导致RTT综合征6，目前治疗仅限于症状管理和物理治疗。一个有前途的治疗途径是所谓的蛋白质替代疗法，其中MeCP2水平可以打分到他们所需的浓度12，13，14，15。12,13,14,15过去二十年来，TA…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们非常感谢布里吉特·斯特姆博士对ECLIA文书的支持。
Materials
| 1,4-Dithiothreitol (DTT) | Sigma-Aldrich | D9779 | Hypotonic lysis reagent, Extraction Buffer |
| Bio-Rad Protein Assay Dye Reagent Concentrate | Bio-Rad Laboratories Inc. | 500-0006 | Sample preperation |
| Detection AB SULFO-TAG labeled anti-rabbit | Meso Scale Diagnostics | R32AB-1 | Antibody |
| Discovery workbench 4.0 | Meso Scale Discovery | Software | |
| DMEM (1X) | gibco by Life Technologies | 41966-029 | Sample preperation |
| Dulbecco’s PBS (sterile) | Sigma-Aldrich | D8537-500ML | Sample preperation, Washing solution, Coating solution |
| EDTA | Sigma-Aldrich | EDS | Extraction Buffer |
| Fetal Bovine Serum | Sigma-Aldrich | F9665 | Sample preperation |
| Glycerol | Sigma-Aldrich | G2025 | Extraction Buffer |
| Gold Read Buffer T (1x) with surfactant | Meso Scale Diagnostics | R92TG | MeCP2 ECLIA protocol |
| HEPES | Sigma-Aldrich | H3375 | Hypotonic lysis reagent, Extraction Buffer |
| KIMBLE Dounce tissue grinder set | Sigma-Aldrich | D8938 | Sample preperation |
| Laboratory Shaker, rocking motion (low speed) | GFL | 3014 | MeCP2 ECLIA protocol |
| Magnesium chloride hexahydrate (MgCl*6H20) | Sigma-Aldrich | M2670 | Hypotonic lysis reagent, Extraction Buffer |
| MeCP2 (Human) Recombinant Protein (P01) | Abnova Corporation | H00004204-P01 | Cell treatment |
| Microseal B seal | Bio-Rad Laboratories Inc. | MSB1001 | for plate sealing |
| Monoclonal Anti-MeCP2, produced in mouse, clone Mec-168, purified immunoglobulin | Sigma-Aldrich | M6818-100UL; RRID:AB_262075 | Antibody, Coating solution |
| MSD Blocker A | Meso Scale Diagnostics | R93BA-4 | Blocker |
| MSD SECTOR Imager 2400 | Meso Scale Diagnostics | I30AA-0 | MeCP2 ECLIA protocol |
| Multi-Array 96-well Plate | Meso Scale Diagnostics | L15XB-3/L11BX-3 | MeCP2 ECLIA protocol |
| Penicillin-Streptomycin | gibco by Life Technologies | 15140122 | Sample preperation |
| Polyclonal Anti-MeCP2, produced in rabbit | Eurogentec S.A. | custom-designed | Antibody |
| Potassium chloride (KCl) | Merck KGaA | 1049361000 | Hypotonic lysis reagent |
| Primary AB Mouse, anti-MeCP2 (1B11) | Sigma-Aldrich | SAB1404063; RRID:AB_10737296 | Antibody |
| Primary AB Mouse, anti-MeCP2 (4B6) | Sigma-Aldrich | WH0004204M1; RRID:AB_1842411 | Antibody |
| Primary AB Mouse, anti-MeCP2 (Mec-168) | Sigma-Aldrich | M6818; RRID:AB_262075 | Antibody |
| Primary AB Mouse, anti-MeCP2 (Men-8) | Sigma-Aldrich | M7443; RRID:AB_477235 | Antibody |
| Primary AB Rabbit, anti-MeCP2 (D4F3) | Cell Signaling Technology | 3456S; RRID:AB_2143849 | Antibody |
| Protease Inhibitor Cocktail (100X) | Sigma-Aldrich | 8340 | Hypotonic lysis reagent, Extraction Buffer |
| Secondary AB, Rabbit, anti-MeCP2 | Eurogentec S.A. | custom | Antibody |
| Secondary AB, Rabbit, anti-MeCP2 | Merck | 07-013 | Antibody |
| Sodium chloride (NaCl) | Sigma-Aldrich | S3014 | Extraction Buffer |
| SULFO-TAG Labeled Anti-Rabbit Antibody (goat) | Meso Scale Diagnostics | W0015528S | Antibody |
| TAT-MeCP2 fusion protein | in-house production | Cell treatment | |
| Trypsin EDTA 0.25% (1X) | gibco by Life Technologies | 25200-056 | Cell treatment |
| Tween 20 | Sigma-Aldrich | P9416 | Washing solution |
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Citer Cet Article
Steinkellner, H., Beribisky, A. V., Mausberg, P., Christodoulou, J., Scheiber-Mojdehkar, B., Huber, A., Sarne, V., Laccone, F. An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants. J. Vis. Exp. (159), e61054, doi:10.3791/61054 (2020).