|本期目录/Table of Contents|

[1]李天宁,邵艳宏,朱浩,等.人-人嵌合抗丙肝抗体检测阳性对照品的研制及应用[J].天津医科大学学报,2021,27(02):180-184.
 LI Tian-ning,SHAO Yan-hong,ZHU Hao,et al.Preparation and characterization of human-human chimeric anti-HCV antibody detection positive quality control[J].Journal of Tianjin Medical University,2021,27(02):180-184.
点击复制

人-人嵌合抗丙肝抗体检测阳性对照品的研制及应用(PDF)
分享到:

《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:
27卷
期数:
2021年02期
页码:
180-184
栏目:
技术与方法
出版日期:
2021-03-15

文章信息/Info

Title:
Preparation and characterization of human-human chimeric anti-HCV antibody detection positive quality control
文章编号:
1006-8147(2021)02-0180-05
作者:
李天宁1邵艳宏2朱浩3王文皓1刘运德2袁玉华3
(1.天津医科大学总医院检验科,天津 300052;2.天津医科大学检验学院,天津 300302;3.天津医科大学总医院空港医院检验科,天津 300308)
Author(s):
LI Tian-ning1SHAO Yan-hong2ZHU Hao3WANG Wen-hao1LIU Yun-de2YUAN Yu-hua3
(1.Department of Clinical Laboratory,General Hospital, Tianjin Medical University,Tianjin 300052,China;2.School of Medicine Laboratory,Tianjin Medicine University,Tianjin 300203,China;3.Department of Clinical Laboratory,Tianjin Medical University General
关键词:
丙型肝炎病毒抗体噬菌体展示对照品
Keywords:
hepatitis C virus antibodyphage displayquality control
分类号:
R446
DOI:
-
文献标志码:
A
摘要:
目的:获得人-人嵌合型抗丙肝抗体的阳性对照品。方法:采集20例具有高抗体滴度的抗丙肝抗体阳性患者外周血,混合后分离外周血单个核细胞,TRIzol提取总RNA,逆转录合成cDNA。PCR分别扩增重链可变区和轻链可变区并以其为模板,获得ScFv,导入到PCANTAB 5E载体上构建噬菌体展示库。分别用NS3、NS4、NS5和core抗原对噬菌体库进行5轮的富集和淘洗;通过Phage ELISA的方法对单克隆菌落进行特异性鉴定,分别获得抗丙肝NS3、NS4、NS5抗体和两个抗丙肝core抗体。将ScFv的VH和VL分别克隆到pcDNA3.4载体上,构建人IgG全长抗体并通过Lipo3000转染293T细胞。收集细胞上清,经protein A纯化,保存于-80℃。结果:成功构建了抗丙肝抗体噬菌体库;筛选并表达了5个分别针对NS3、NS4、NS5和core抗原的阳性抗体,经验证表达的抗丙肝阳性抗体在-20°C~37°C表现稳定。多次重复冻融后不会影响抗体的使用效果。且5种抗体在国内外多种丙型肝炎检测试剂盒间测试均为阳性,具备一定的通用性。结论:获得5种人-人嵌合型抗丙肝抗体的阳性对照品且适用于多种丙型肝炎检测试剂盒。
Abstract:
Objective: To obtain positive quality control materials for human-human chimeric anti-hepatitis C antibody. Methods: Peripheral blood was collected from 20 hepatitis C positive patients with high antibody titer,and the peripheral blood mononuclear cells were separated after mixing. Total RNA was extracted by TRIzol,and cDNA was synthesized by reverse transcription. The heavy chain variable region and light chain variable region were amplified by PCR and used as templates to obtain ScFv,which was introduced into the PCANTAB 5E vector to construct a phage display library. This phage library was enriched and elutriated respectively with hepatitis C NS3,NS4,NS5,and core antigens for five rounds. The monoclonal colonies were specifically identified by means of Phage ELISA to obtain respectively anti-hepatitis C NS3,NS4,NS5 antibodies,and two anti-hepatitis C core antibodies. The VH and VL of ScFv were separately cloned into pcDNA3.4 vector to construct human IgG full-length antibody and transfected into 293T cells by Lipo3000. Cell supernatants were collected,purified by protein A,and stored at -80°C. Results: A phage library of anti-hepatitis C antibodies was successfully constructe and five hepatitis C positive antibodies were screened and expressed,targeting respectively hepatitis C NS3,NS4,NS5,and core antigens. The confirmed expression of hepatitis C positive antibody is stable at 37°C to -20°C. Repeated freezing and thawing will not affect the effect of antibody use. In addition,the five antibodies had tested positive in various hepatitis C test kits at home and abroad,and they had certain versatility. Conclusion: We successfully obtain five kinds of positive control materials for human-to-human chimeric anti-HCV antibodies that are suitable for multiple hepatitis C diagnostic kits.

参考文献/References:

[1] Martinez A D,Gish R G. HCV in at risk populations:who can be treated and how?[J]. Curr Hep Rep,2012,11(1):1
[2] Chevaliez S,Mélanie Wlassow,Johann V,et al. Assessing molecular point-of-care testing and dried blood spot for hepatitis C virus screening in people who inject drugs[J]. Open Forum Infect Dis,2020,7(6):196
[3] Chevaliez S. Strategies for the improvement of HCV testing and diagnosis[J]. Expert Rev Anti Infect Ther,2019,17:341
[4] 谷金莲,于洋,梁争论. 第6套丙型肝炎病毒抗体诊断试剂国家参考品的研制[J]. 中国生物制品学杂志,2016,29(7):720
[5] 谷金莲,于洋,梁争论. 新一代丙型肝炎病毒抗体试剂国家参考品研究的要点与思考[J]. 中国药事,2016,30(9):891
[6] Nouhin J,Bollore K,Castera-Guy J,et al. Analytical and field evaluation of the biocentric generic HCV assay on open polyvalent PCR platforms in France and Cambodia[J]. J Clin Virol,2018,108:53
[7] Fekete T. The Xpert HCV Viral load finger-stick point-of-care test was accurate for detecting HCV RNA[J]. Ann Intern Med, 2018, 169:35
[8] Zhou M,Qin B, Deng X S,et al. HNTCP?expressing primary pig hepatocytes are a valuable tool for investigating hepatitis B virus infection and antiviral drugs[J]. Mol Med Rep,2019,20(4):3820
[9] Listek M,H?觟now A,Gossen M,et al. A novel selection strategy for antibody producing hybridoma cells based on a new transgenic fusion cell line[J]. Sci Rep ,2020,10(1):1664
[10] Molek P,Strukelj B,Bratkovic T. Peptide phage display as a tool for drug discovery:targeting membrane receptors[J]. Molecules, 2011, 16(1):857
[11] Warkad S D,Song K S,Pal D,et al. Developments in the HCV screening technologies based on the detection of antigens and antibodies[J]. Sensors,2019,19(19):4257
[12] Thibodeaux B A,Panella A N,Roehrig J T. Development of human-murine chimeric immunoglobulin G for use in the serological detection of human flavivirus and alphavirus antibodies[J]. Clin Vacc Immunol CVI,2010,17(10):1617
[13] Thibodeaux B A,Liss N M,Panella A N,et al. Development of a human-murine chimeric immunoglobulin M for use in the serological detection of human alphavirus antibodies[J]. Clin vacc Immunol CVI,2011,18(12):2181
[14] Tu B,Ziemann R N,Tieman B C,et al. Generation and characterization of chimeric antibodies against NS3,NS4,NS5,and core antigens of hepatitis C virus[J]. Clini vacc Immunol CVI,2010,17(6):1040
[15] Mohammed G A,Mohammed E M. Comparison of 2 different antibody assay methods,Elecsys Anti-HCVII (Roche) and Vidas Anti-HCV (Biomerieux),for the detection of antibody to hepatitis C virus in Egypt[J]. Diagn Microbiol Infect Dis,2018,92(2):107
[16] 魏兰,魏超,李维,等. 5种ELISA试剂检测抗-HCV结果的差异分析[J]. 中国输血杂志,2018,31(2):113
[17] Pierpont T M,Limper C B,Richards K L. Past,present,and future of rituximab—the world′s first oncology monoclonal antibody therapy[J]. Front Oncol,2018,8:163
[18] Huang T J,Reid M E,Halverson G R,et al. Production of recombinant murine-human chimeric IgM and IgG anti-Js(b) for use in the clinical laboratory[J]. Transfusion,2010,43(6):758

相似文献/References:

[1]郭 睿 综述,刘全忠 审校.蛋白质相互作用研究技术的新进展[J].天津医科大学学报,2015,21(06):542.

备注/Memo

备注/Memo:
作者简介 李天宁(1996-),女,硕士在读,研究方向:临床检验诊断学;通信作者:袁玉华,E-mail:yyhxxx39@sina.com。
更新日期/Last Update: 2021-03-10