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Application of Phage-displayed Single Chain Antibodies in western blot

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Acta Biochimica et Biophysica Sinica 2005年第3期37卷 205-209页

作者： Yan-Li DING Mei-Yun LIU Wei HAN Sheng-Li YANG Hui LIU Yi GONG School of Pharmaceutical Engineering,Shenyang Pharmaceutical University,Shenyang 110015,China Shanghai Research Center of Biotechnology,Chinese Academy of Sciences,Shanghai 200233,China School of Pharmaceutical Engineering Shenyang Pharmaceutical UniversityShenyang 110015 China Shanghai Research Center of Biotechnology Chinese Academy of SciencesShanghai 200233 China

A phage display single chain fragment variable library constructed on pⅢ protein of M13filamentous phage was screened using B-lymphocyte stimulator and FP248 as selective molecules.After fourrounds of panning,there was a remarkable enrichment in the titer of bound phages.Twenty phage cloneswere selected from the last round and screened by means of phage-ELISA.With the antibody phages asprimary antibodies in western blot,we developed a method for detecting the specific antigen.The dilutions ofantibody phages depend on the affinity between antibody-displayed phage particles and antigens.

关键词： phage-display antibody western blot

A Study of the Technique of western blot for Diagnosis of Lyme Disease caused by Borrelia afzelii in China

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Biomedical and Environmental Sciences 2013年第3期26卷 190-200页

作者： LIU Zhi Yun HAO Qin HOU Xue Xia JIANG Yi GENG Zhen WU Yi Mou WAN Kang Lin State Key Laboratory for Infectious Disease Prevention and Control National Institute for Communicable Disease Control and PreventionChinese Center for Disease Control and Prevention Institute of Pathogenic Biology University of South China YuShui District Center for Disease Control and Prevention

Objective To study the technique of western blot for the diagnosis of Lyme disease caused by Borrelia afzelii in China and to establish the standard criteria by operational procedure. Methods FP1, which is the representative strain of B. afzelii in China, was analyzed by SDS-PAGE, electro transfer and irnmunoblotting assays. The molecular weights of the protein bands of FP1 were analyzed by Gel-Pro analysis software. In a study using 451 serum samples （159 patients with Lyme disease and 292 controls）, all observed bands were recorded. The accuracy of the WB as a diagnostic test was established by using the ROC curve and Youden index. Results Criteria for a positive diagnosis of Lyme disease were established as at least one band of P83/100, P58, P39, OspB, OspA, P30, P28, OspC, P17, and P14 in the IgG test and at least one band of P83/100, P58, P39, OspA, P30, P28, OspC, P17, and P41 in the IgM test. For IgG criteria, the sensitivity, specificity and Youden index were 69.8%, 98.3%, and 0.681, respectively; for IgM criteria, the sensitivity, specificity and Youden index were 47%, 94.2%, and 0.412, respectively.

关键词： Lyme disease western blot Diagnostic method Borrelia afzelii

Interpretation Criteria for Standardized western blot for the Predominant Species of Borrelia Burgdorferi Sensu Lato in China

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Biomedical and Environmental Sciences 2010年第5期23卷 341-349页

作者： YI JIANG XUE-XIA HOU ZHEN GENG QIN HAO KANG-LIN WAN State Key Laboratory for Infectious Diseases Prevention and Control National Institute of Communicable Disease Control & Prevention Chinese Center of Disease Control & Prevention Beijing 102206 China

Objective western blotting （WB;immunoblotting） is a widely used tool for the serodiagnosis of Lyme borreliosis （LB）,but so far,no generally accepted criteria for its performance and interpretation have been established in China.The present study was designed to determine the criteria for standardized western blot for the predominant species of Borrelia burgdorferi sensu lato in China,in which WB was produced with strain PD91 as the representative strain attributed to predominant genospecies Borrelia garinii of Borrelia burgdorferi sensu lato.Methods Approximately 13 bands between 14 and 100 kD were differentiated for strain PD91 by using Gel-Pro analysis software.In a study with 631 serum samples （taken from 127 patients with Lyme borreliosis and 504 controls）,all observed bands were documented.To establish criteria for a positive WB result for strain PD91,receiver operating characteristic （ROC） curves were used.Results The following interpretation criteria were recommended：for IgG,at least one band of P83/100,P58,P39,P30,OspC,P17,P66,and OspA;for IgM,at least one band of P83/100,P58,OspA,P30,OspC,P17 or P41.In addition,syphilis,leptospirosis and other related diseases should be excluded when the positive band is P41 in IgM.For IgG criteria,the sensitivity is 73.2%,the specificity is 99.4% and Youden index is 0.726;for IgM criteria,the sensitivity is 50.6%,the specificity is 93.1% and Youden index is 0.437.Conclusion Standardization of WB assays is necessary for comparison of results from different laboratories.Moreover,the criteria of other genospecies of Borrelia burgdorferi sensu lato should be determined in the future to complete the criteria of WB for the diagnosis of the Lyme disease in China.

关键词： Criteria western blot Borrelia burgdorferi sensu lato

Analysis of Protoscoleces-specific Antigens from Echinococcus Granulosus with Proteomics Combined with western blot

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Biomedical and Environmental Sciences 2012年第6期25卷 718-723页

作者： LI Zong Ji and ZHAO WeiDepartment of Medical Genetics and Cell Biology, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China Center of Scientific Technology of Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China The medical scientific institute of Ningxia, Yinchuan 750004, Ningxia Hui Autonomous Region, China Department of Medical Genetics and Cell Biology Ningxia Medical University Yinchuan 750004 Ningxia Hui Autonomous Region China Center of Scientific Technology of Ningxia Medical University Yinchuan 750004 Ningxia Hui Autonomous Region China The medical scientific institute of Ningxia Yinchuan 750004 Ningxia Hui Autonomous Region China

Objective To establish and optimize the proteomic analysis of protoscoleces-specific antigens from Echinococcus granulosus. To provide a foundation for identifying specific antigens in the soluble proteins of E. granulosus protoscoleces for further research. Methods Brood capsules were collected aseptically from fertile E. granulosus cysts from the livers of an infected patient. The fertile E. granulosus cysts were fractured, and protoscoleces were collected by centrifugation. The soluble proteins of protoscoleces were acquired using the 2D Quant kit according to the manufacturer＇s instructions. We employed two-dimensional electrophoresis （2-DE） combined with immunoblot assay （western blot） to analyze the soluble components of E. granulosus protoscoleces antigens. The 2-DE and immunoblot maps obtained were analyzed with PDQuest 8.0 image analysis software. Results About 233 soluble protein spots were identified with Coomassie-stained gels. Most of the proteins had a molecular weight of 16 000 Da to 117 000 Da, and an isoelectric point value of 3.0 to 10.0. 2-DE immunoblot was conducted and 57 specific antigen spots were observed, among which 23 spots were identified. Conclusion 2-DE combined with western blot is the key to successful proteomic analysis and presents a new possibility for searching the specific E. granulosus protoscoleces antigens.

关键词： Echinococcus granulosus protoscoleces Proteomics Specific antigen Two-dimensionalelectrophoresis （2-DE） western blot

Expression of Elk-1 in Non-Small Cell Lung Cancer Detected by western blot and Tissue Microarray

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Chinese Journal of Cancer Research 2007年第1期19卷 7-11页

作者：张曙光李放李文雅卢玮张林 Department of Thoracic Surgery the First Affiliated Hospital China Medical University Department of Thoracic Surgery Cancer Institute & Cancer Hospital China Academy of Medical Science & Peking Union Medical College

Objective： The aim of this study was to investigate the Elk-1 （Ets like transcription factor-1） expression in non-small cell lung cancer （NSCLC） and normal lung tissues and the relationship between its expression and clinicopathological characters. Methods： To observe Elk-1 expression, western blot and immunochemistry （IHC） on tissue microarray （TMA） containing 118 lung cancers and their corresponding normal tissues were used. Results： In western blot and IHC on TMA, Elk-1 was highly expressed in NSCLC, while its expression was almost undetectable in normal lung tissues. Elk- 1 expression in NSCLC had no relationship with the patients＇ age, gender, smoking status and histological type, but had relationship with the differentiation degree, clinical stages and lymphonode metastasis. The expression was lower in early stage group （Ⅰ＋Ⅱ） than in advanced stage group （Ⅲ）, and lower in well-moderately differentiated group than in poorly differentiated group. The same trend was seen with lymphonode metastasis. Conclusion： The progression of NSCLC may be related with the increased Elk-1 expression, and Elk-1 may be regarded as a prognostic factor for NSCLC tissues.

关键词： Elk-1 Non-small cell lung cancer western blot Tissue microarray

以结果验证为导向的中医学八年制western blot实践教学

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科教导刊 2023年第6期 29-31页

作者：林玩福梁淑芳杜娟翟笑枫程彬彬海军军医大学中医系上海200433

“实验中医学”是海军军区大学中医学八年制学员的主干课程。其中western blot是研究蛋白水平的基本方法,也是学员所必须掌握的实验研究方法。本课程以结果验证为导向的教学方式革新,最大限度压缩每个步骤的操作时间,聚焦于让学员完成... 详细信息

“实验中医学”是海军军区大学中医学八年制学员的主干课程。其中western blot是研究蛋白水平的基本方法,也是学员所必须掌握的实验研究方法。本课程以结果验证为导向的教学方式革新,最大限度压缩每个步骤的操作时间,聚焦于让学员完成最多的实验步骤,力求实验的完整性。该方法可在一堂课完成实验,展示结果并进行分析讨论。该教学方式有助于在最短时间内掌握完整的western blot实验技术,值得进一步推广应用。

关键词：实践教学 western blot 实验中医学

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基于特征多肽抗原的阿胶基原鉴定

中成药 2024年第4期46卷 1214-1219页

作者：袁陈婷杨容王立玮景凌洁陈璐阳加羊卓玛孙小祥孙锦秀杨欢如皋市人民医院药剂科江苏如皋226500 江苏大学药学院江苏镇江212013 南京中医药大学附属镇江医院药剂科江苏镇江212003

目的以特征多肽为半抗原并制备多克隆抗体,鉴定阿胶原材料基原。方法经文献调研及数据库比对筛选特征多肽,将多肽抗原与血蓝蛋白偶联并免疫Balb/c小鼠制备多克隆抗体,通过ELISA、dot blot和western blot分析与验证多克隆抗体的产生、... 详细信息

目的以特征多肽为半抗原并制备多克隆抗体,鉴定阿胶原材料基原。方法经文献调研及数据库比对筛选特征多肽,将多肽抗原与血蓝蛋白偶联并免疫Balb/c小鼠制备多克隆抗体,通过ELISA、dot blot和western blot分析与验证多克隆抗体的产生、效价及特异性。结果筛选所得5个特征多肽序列均具有T细胞表位和B细胞表位,亲水性-1~0,二级结构大多为无规则卷曲。多肽抗原ECS1、ECS3~ECS5在二次加强免疫后,效价分别为1∶6 400、1∶400、1∶12 800和1∶800。制备所得的多克隆抗体之间不产生交叉反应,而且多克隆抗体Ab-ECS3与驴皮及其伪品蛋白的结合显现出较大差异。结论以特征多肽为半抗原制备所得的多克隆抗体可通过western blot对驴皮进行真伪鉴别,为胶类中药的基原鉴定提供了参考。

关键词：阿胶特征多肽多肽抗原多克隆抗体 western blot 基原鉴定

Comparison of indirect immunofluorescence and western blot method in the diagnosis of hantavirus infections

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World Journal of Methodology 2021年第6期11卷 294-301页

作者： Tatjana Vilibic-Cavlek Ljubo Barbic Vladimir Stevanovic Vladimir Savic Anna Mrzljak Maja Bogdanic Irena Tabain School of Medicine University of ZagrebZagreb 10000Croatia Department of Virology Croatian Institute of Public HealthZagreb 10000Croatia Department of Microbiology and Infectious Diseases with Clinic Faculty of Veterinary MedicineUniversity of ZagrebZagreb 10000Croatia Poultry Centre Croatian Veterinary InstituteZagreb 10000Croatia Department of Gastroenterology and Hepatology University Hospital ZagrebZagreb 10000Croatia

BACKGROUND Serologic cross-reactivity between hantaviruses often complicates the interpretation of the results.AIM To analyze the diagnostic value of indirect immunofluorescence assay(IFA)and western blot(WB)in the diagnosis of hantavirus infections.METHODS One hundred eighty-eight serum samples from Puumala(PUUV)and Dobrava(DOBV)orthohantavirus infected patients were analyzed.Serology was performed using commercial tests(Euroimmun,Lübeck,Germany).RESULTS Using IFA,49.5%of acute-phase samples showed a monotypic response to PUUV,while 50.5% cross-reacted with other hantaviruses.The overall cross-reactivity was higher for immunoglobulin G(IgG)(50.0%)than for immunoglobulin M(IgM)(25.5%).PUUV IgM/IgG antibodies showed low/moderate reactivity with orthohantaviruses Hantaan(12.3%/31.5%),Seoul(7.5%/17.8%),DOBV(5.4%/28.1%),and Saaremaa(4.8%/15.7%).Both DOBV IgM and IgG antibodies were broadly reactive with Hantaan(76.2%/95.2%),Saaremaa(80.9%/83.3%),and Seoul(78.6%/85.7%)and moderate with PUUV(28.5%/38.1%).Using a WB,serotyping was successful in most cross-reactive samples(89.5%).CONCLUSION The presented results indicate that WB is more specific than IFA in the diagnosis of hantavirus infections,confirming serotype in most IFA cross-reactive samples.

关键词： Hantaviruses Serology Cross-reactivity Indirect immunofluorescence western blot

髓系特异性Spi1基因敲除小鼠的构建和基因鉴定

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安徽医科大学学报 2024年第3期59卷 413-417页

作者：吴旭铭王卉卉朱向玲周园园王安琪张慧茹刘崇涂佳杰安徽医科大学临床药理研究所合肥230032

目的构建髓系特异性Spi1基因敲除小鼠并分析其基因型,为疾病病理机制及药物靶点研究提供动物模型基础。方法根据CRISPR/Cas9技术原理和Cre/LoxP系统,设计并构建sgRNA和Donor载体,以第2号外显子(Exon 2)所在的转录本为敲除区域,在Exon 2... 详细信息

目的构建髓系特异性Spi1基因敲除小鼠并分析其基因型,为疾病病理机制及药物靶点研究提供动物模型基础。方法根据CRISPR/Cas9技术原理和Cre/LoxP系统,设计并构建sgRNA和Donor载体,以第2号外显子(Exon 2)所在的转录本为敲除区域,在Exon 2两侧各放置同向Loxp元件;将Cas9蛋白、sgRNA和Donor载体混合显微注射到C57BL/6J小鼠的受精卵中,移植受精卵到假孕的C57BL/6J母鼠的子宫中,19~20 d后获得F0代。将阳性F0代小鼠与C57BL/6J小鼠交配,得到稳定的F1代Spi1^(flox/+)小鼠。F1代Spi1^(flox/+)小鼠雌雄自交得到Spi1^(flox/flox)小鼠。Spi1^(flox/flox)与Lyz2-Cre^(+)小鼠交配得到Spi1^(flox/+)/Lyz2-Cre^(+)小鼠,再将其与Spi1^(flox/flox)交配,得到的Spi1^(flox/flox)/Lyz2-Cre^(+)小鼠为髓系特异性Spi1基因敲除(KO)小鼠;Spi1^(flox/flox)/Lyz2-Cre^(-)小鼠作为野生型(WT)小鼠。提取WT和KO小鼠DNA,PCR扩增后琼脂糖凝胶电泳鉴定基因型;western blot检测WT和KO小鼠免疫细胞中脾病灶形成病毒前病毒整合癌基因-1/富含嘌呤盒1(PU.1)表达。结果PCR鉴定结果显示,采用flox引物鉴定时仅扩增出220 bp条带的小鼠,即基因型为Spi1^(flox/flox)纯合子,采用Lyz2-Cre引物鉴定时扩增出700 bp的小鼠,基因型为Lyz2-Cre^(+);western blot结果显示,与WT组比较,KO组小鼠骨髓来源巨噬细胞(BMDMs)和腹腔原位巨噬细胞(PM)中的PU.1不表达(Pwestern blot结果均表明髓系特异性Spi1 KO小鼠构建成功。结论成功构建和鉴定髓系特异性Spi1 KO小鼠,为进一步揭示PU.1在免疫调节中的潜在机制研究提供动物模型基础。

关键词：髓系特异性 Spi1 基因敲除 CRISPR/Cas9 Cre/LoxP PCR western blot