Wes/Jess Simple全自動定量western blot在病原微生物感染的應(yīng)用

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一、檢測病原微生物感染關(guān)鍵蛋白

美國NIH國家糖尿病和消化及腎臟疾病研究所科學(xué)家，闡述脂質(zhì)相關(guān)TM6SF2蛋白在調(diào)節(jié)丙肝病毒HCV脂質(zhì)體（LVPs）形成和HCV生命周期關(guān)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篩選

(診斷和疫苗開發(fā)靶點)

案例1. 結(jié)核桿菌高免疫原性蛋白篩選

高免疫原性蛋白質(zhì)是疫苗和診斷開發(fā)的靶蛋白。

案例：結(jié)核桿菌膜蛋白是結(jié)核桿菌高免疫原性蛋白來源，中國醫(yī)學(xué)科學(xué)院病原生物所金奇教授重組表達219種結(jié)核桿菌膜蛋白作為樣本，使用病人血清作為一抗，使用Wes進行化學(xué)發(fā)光定量檢測。

高免疫原性蛋白會引起病人引起產(chǎn)生抗體，病人血清和重組抗原結(jié)合信號越強，則可以提示該抗原具有強的免疫原性，可以用作重組疫苗開發(fā)的靶點。

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

案例2. 埃博拉病毒病毒特異性IgG和IgM檢測

埃博拉病毒感染后或者疫苗免疫接種后，病人血清中會存在IgG或者IgM抗體。因而可以通過病毒特異性IgG和IgM血清學(xué)檢測，可以診斷人體的過往感染以及疫苗的免疫效果。

美國陸軍傳染病醫(yī)學(xué)研究所將病毒多肽表位鏈接生物素，利用鏈霉親和素形成四聚體作為樣本，使用疫苗接種的非人靈長類動物血清作為一抗，再使用anti-IgM，anti-IgG，anti-IgA作為二抗，使用Wes進行定量檢測。可以研究表位的免疫原性，及其引起的抗體類別。

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、病原微生物抗體驗證實驗

在很多情況下，病原微生物感染后的血清抗體確證實驗是有價值的。

美國CDC艾滋病毒、肝炎、性病和結(jié)核病國家中心，基于Wes全自動定量western blot系統(tǒng)，建立HCV血清抗體確證研究，建立以Wes為基礎(chǔ)的全自動化檢測平臺。

評價：特異性：100%; 靈敏度：95%；自動化：全程自動化；單次運行時間：3小時；一天樣本量：100個

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、 病人樣本蛋白質(zhì)定量檢測

1. 支氣管-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系統(tǒng)可以生成標(biāo)準(zhǔn)曲線，檢測甲流感染C57BL/6J小鼠支氣管肺泡灌洗液，定量BPIFA1蛋白濃度。（IAVX31為流感病毒）An innate defense peptide BPIFA1/SPLUNC1 restricts influenza A virus infection, Mucosal Immunology volume 11, pages 71–81(2018).

2. 腦脊液

（肺炎鏈球菌腦炎病人）

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.

肺炎鏈球菌感染，容易引起腦炎。本文利物浦大學(xué)通過Wes定量病人腦脊液中腦炎相關(guān)病毒

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

Wes/Jess 全自動定量Western Blot特點