|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.
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人-人嵌合抗丙肝抗体检测阳性对照品的研制及应用(PDF)
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《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:
27
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.

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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