Wes/Jess Simple全自动定量western blot在病原微生物感染的应用

一、检测病原微生物感染关键蛋白

美国NIH国家糖尿病和消化及肾脏疾病研究所科学家，阐述脂质相关TM6SF2蛋白在调节丙肝病毒HCV脂质体（LVPs）形成和HCV生命周期关系。
 

TM6SF2 Promotes Lipidation and Secretion of Hepatitis C Virus in Infected Hepatocytes, Gastroenterology Volume 155, Issue 6, December 2018, Pages 1923-1935.e8.
 

二、病原微生物高免疫原性蛋白筛选
 

       (诊断和疫苗开发靶点)
 

案例1. 结核杆菌高免疫原性蛋白筛选

高免疫原性蛋白质是疫苗和诊断开发的靶蛋白。
 

案例：结核杆菌膜蛋白是结核杆菌高免疫原性蛋白来源，中国医学科学院病原生物所金奇教授重组表达219种结核杆菌膜蛋白作为样本，使用病人血清作为一抗，使用Wes进行化学发光定量检测。
 

高免疫原性蛋白会引起病人引起产生抗体，病人血清和重组抗原结合信号越强，则可以提示该抗原具有强的免疫原性，可以用作重组疫苗开发的靶点。
 

Analysis of the Antigenic Properties of Membrane Proteins of Mycobacterium tuberculosis, Scientific Reports | (2019) 9:3042.
 

案例2. 埃博拉病毒病毒特异性IgG和IgM检测
 

埃博拉病毒感染后或者疫苗免疫接种后，病人血清中会存在IgG或者IgM抗体。因而可以通过病毒特异性IgG和IgM血清学检测，可以诊断人体的过往感染以及疫苗的免疫效果。
 

美国陆军传染病医学研究所将病毒多肽表位链接生物素，利用链霉亲和素形成四聚体作为样本，使用疫苗接种的非人灵长类动物血清作为一抗，再使用anti-IgM，anti-IgG，anti-IgA作为二抗，使用Wes进行定量检测。可以研究表位的免疫原性，及其引起的抗体类别。
 

Qualitative Profiling of the Humoral Immune Response Elicited by rVSV-DG-EBOV-GP Using a Systems Serology Assay, Domain Programmable Arrays,Cell Reports 24, 1050–1059, July 24, 2018.
 

三、 病原微生物抗体验证实验

在很多情况下，病原微生物感染后的血清抗体确证实验是有价值的。
 

美国CDC艾滋病毒、肝炎、性病和结核病国家中心，基于Wes全自动定量western blot系统，建立HCV血清抗体确证研究，建立以Wes为基础的全自动化检测平台。
 

评价：特异性：100%; 灵敏度：95%；自动化：全程自动化；单次运行时间：3小时；一天样本量：100个
 

An Automated Immunoblot Method for Detection of IgG Antibodies to Hepatitis C Virus: a Potential Supplemental Antibody Confirmatory Assay, Journal of Clinical Microbiology, March 2019 Volume 57 Issue 3 e01567-18.
 

四、 病人样本蛋白质定量检测
 

1. 支气管-肺灌洗液（甲流）

Quantitative protein analysis
 

The Wes system was used to identify and quantify BPIFA1 protein in broncho-alveolar lavage and lung samples (ProteinSimple) and rabbit anti-mBPIFA1 as a primary antibody. The area of BPIFA1 peak, in relation to the standard curve, was determined and virtual blot-like images were generated using Compass software (ProteinSimple).
 

Wes系统可以生成标准曲线，检测甲流感染C57BL/6J小鼠支气管肺泡灌洗液，定量BPIFA1蛋白浓度。（IAVX31为流感病毒）An innate defense peptide BPIFA1/SPLUNC1 restricts influenza A virus infection, Mucosal Immunology volume 11, pages 71–81(2018).
 

2. 脑脊液
 

（肺炎链球菌脑炎病人）

Quantitative western blotting
 

Automatic western blots were performed using a Wes automated system (ProteinSimple, California, USA). Purified recombinant proteins were used as calibration standards. Serial dilutions of both sample and standard were used to determine the linear dynamic range of the assay. Additionally, the optimal concentration of each antibody for use in the Wes system was determined, as this can differ from that used in traditional western blot. Samples (20 SPP, 15 hospital controls and 5 healthy controls) were mixed with a 5x sample buffer containing SDS, DTT and fluorescent molecular weight standards and heated at 95 °C for 5 min and then, loaded onto a plate prefilled with stacking and separation matrices, along with blocking and wash buffers, antibody solutions and detection reagents. Default settings were used for the analysis.
 

肺炎链球菌感染，容易引起脑炎。本文利物浦大学通过Wes定量病人脑脊液中脑炎相关病毒

Quantitative Proteomics of Cerebrospinal Fluid in Paediatric Pneumococcal Meningitis, Scientific Reports volume 7, (2017).
 

Wes/Jess 全自动定量Western Blot特点
 

1. 全程自动化，无需检测人员值守

2. 从样本到结果，全程3小时

3. 标准曲线定量检测病人样本靶蛋白浓度

4. 微量样本

5. 数据可追溯

6. 自动数据计算存储