关闭
 
读者在线:用户名 密码
首页 期刊简介 投稿须知 期刊目录 专家风采 编委会 特邀顾问 广告发行 联系我们 移动出版
  1. 1
  2. 2
  3. 3
  4. 4
  5. 5



刊物信息
期刊名称:药物分析杂志
主管单位:中国科学技术协会
主办单位:中国药学会
承办:中国食品药品检定研究院
主编:金少鸿
编辑部主任:粟晓黎
地址:北京天坛西里2号
邮政编码:100050
电话:010-67012819,67058427
电子邮箱:ywfx@nicpbp.org.cn
国际标准刊号:ISSN 0254-1793
国内统一刊号:CN 11-2224/R
邮发代号:2-237

 

访问统计
您是第  1 8 5 2 7 0 位浏览者
您当前的位置:首页 >> 正文

对斑点杂交方法检测治疗性单克隆抗体制品中残留宿主细胞DNA含量的影响因素研究

Research on factors affecting Dot-Blot detection of residual host cell DNA in therapeutic monoclonal antibodies

分类号:
出版年·卷·期(页码):2012,32 (1):0-0
DOI: 10.16155/j.0254-1793.2017.01.01
-----摘要:-------------------------------------------------------------------------------------------

目的: 对斑点杂交方法检测重组生物制品中残留宿主细胞DNA含量试验中的6个影响因素进行评价。方法: 对不同的样品稀释液,样品中是否含有蛋白,含蛋白样品是否采用蛋白酶K处理,采用不同方法预处理的模板DNA作为标记用模板,采用不同体积的离心管进行探针标记,分别采用不同的杂交温度,共计6个因素对检测结果的影响进行评价。结果: 在使用随机标记的探针通过斑点杂交方法检测重组生物制品中残留宿主细胞DNA含量试验中:标准品和样品稀释中使用水进行稀释时结果可信度较高;样品中含有蛋白会降低检测的灵敏度;在含蛋白样品中加入蛋白酶K处理可以在一定程度上提高检测的灵敏度;使用超声处理的模板DNA作为探针标记的模板时,检测灵敏度较高,同时背景较低;探针标记过程中应采用0.2 mL薄壁PCR管作为容器以保证试验成功率;最适的杂交温度为42 ℃左右。结论: 在斑点杂交方法检测治疗性单克隆抗体制品中残留宿主细胞DNA含量中,采用水作为稀释液,对含蛋白样品使用蛋白酶K消化,标记用模板DNA使用超声处理,使用0.2 mL薄壁PCR管作为标记反应管和在42 ℃杂交时,结果的稳定性和灵敏度有较大提高。

-----英文摘要:---------------------------------------------------------------------------------------

Objective: To evaluate six factors that possibly affect detection sensitivity of Dot-Blot applied to determinating the residual host cell DNA in recombinant bioproducts. Methods: Evaluated how 6 factors including different sample diluents,protein contained in samples,protease K treatment of protein-containing samples,differently pretreated template DNA for probe labeling,vials with different volume used for probe labeling and different temperature for hybridization,took effect on the detection results. Results: In the assays which used randomly labeled probe for detection of residual host cell DNA in recombinant bioproducts:the results of assays that took water for injection as diluent were more reliable than the results of assays that took fish sperm DNA as diluent;If there was protein in sample,the detection sensitivity would decrease;Protease K treatment would increase the detection sensitivity in the test of protein-containing samples;when used the supersonic treated template for probe labeling,the detection sensitivity and background of assay results would be greatly elevated;in the selection of vials for probe labeling,0.2 mL PCR tubes with thin wall were preferred;the preferred hybridization temperature was about 42 ℃. Conclusion: In the Dot-Blot test for determination of residual host cell DNA content in therapeutic monoclonal antibodies,taking water as diluents,digesting protein-containing sample with protease K,treating template DNA with ultrasonic,taking 0.2 mL PCR tube as labeling reaction tube and hybridization at 42 ℃ can improve the repeatability and sensitivity in some degree.

-----参考文献:---------------------------------------------------------------------------------------
1 ZHOU Zhang-fa(周长发),ZHANG Rui(张锐),ZHANG Xiao(张晓),et al.Technologicla improvement of Southern Blot using digoxigen-labeled probes with random-primed method(地高辛随机引物法标记探针的Southern杂交技术优化).J Agric Sci Technol(中国农业科技导报),2009,11(4):123
2 ZHU Hua-chen(朱华晨),XU Xin-ping(许新萍),LI Bao-jian(李宝健).A simple,rapid method of Southern Blot analysis(一种简捷的Southern印迹杂交方法).J Sun Yat-sen Univ(Nat Sci)(中山大学学报自然科学版),2004,43(4):128
3 Ausubel FM,Brent R,Kinston RE,et al.Short Protocols in Molecular Biology(精编分子生物学实验指南).Beijing(北京):Scince Press(科学出版社),1999.55,71
4 BAI Dong-ting(白东亭),ZHENG Qiu-jun(郑秋君),CHEN Bin(陈滨),et al.The detection of resodual DNA in purified rhIFN-β with labelled DIG-DNA(用地高辛配基标记探针检测基因工程人β干扰素制品中的外源DNA).Prog Microbil Immunol(微生物免疫学进展),1996,24(2):46
5 SHAO Xue-jun(邵雪君),WU Shi-liang(吴士良).Establishment and optimization of Dot Blot technique(基于尼龙膜的点杂交的建立及优化).Pract Med Tech(实用医技),2006,13(4):509
6 TIAN Tian(田甜),DONG Ying(董英),XUE Qiang(薛强),et al.Detection of norovirus by RT-PCR revere blot hybridization(诺如病毒RT-PCR反向斑点杂交检测方法的建立).Biotechnol Bull(生物技术通报),2010(3):173

欢迎阅读《药物分析杂志》!您是该文第 5207位读者!

药物分析杂志 © 2009
地址:北京天坛西里2号
邮政编码:100050; 技术支持:010-60213898

电子邮件:ywfx@nicpbp.org.cn