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浙江大学学报（医学版）
浙江大学学报（医学版）  2015, Vol. 44 Issue (4): 423-428    DOI: 10.3785/j.issn.1008-9292.2015.07.12
原著     
环鸟苷酸兔单克隆抗体的制备和竞争酶联免疫吸附测定方法的建立
谭兴梅1,2, 胡海峰2, 陈枢青1
1. 浙江大学药学院药理毒理与生化药学研究所, 浙江 杭州 310058;
2. 宜康(杭州)生物技术有限公司, 浙江 杭州 310015
Preparation of rabbit monoclonal antibody against cGMP and development of competitive ELISA for cGMP
TAN Xing-mei1,2, HU Hai-feng2, CHEN Shu-qing1
1. Institute of Pharmacology, Toxicology and Biological Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China;
2. Abcam(Hangzhou)Biotechnology Co. Ltd., Hangzhou 310015, China
全文: PDF(685 KB)  
摘要: 

目的:研制环鸟苷酸(cGMP)兔单克隆抗体,并建立测定cGMP浓度的竞争ELISA方法.方法:用人工合成的环鸟苷酸—钥孔血蓝蛋白(cGMP-KLH)免疫新西兰大白兔,采用Epitomics公司的兔单克隆抗体技术制备针对cGMP的兔单克隆抗体;应用cGMP兔单克隆抗体建立竞争ELISA方法,并对该方法的特异性、精密度和回收率进行测定.结果:所制备的cGMP兔单克隆抗体的效价为3.1 ng/mL,50%抑制浓度(IC50)为12.57 ng/mL.与环磷酸肌苷的交叉反应率为33.1%,与其他结构类似的化合物无交叉反应.所建立的cGMP竞争ELISA方法的检测范围为0~120 ng/mL,最低检测限为1.95 ng/mL,回收率为89%~103%,批次内和批次间的精密度分别小于11.68%和13.85%.结论:成功制备高效价、高特异性的cGMP兔单克隆抗体,并建立了测定cGMP浓度的竞争ELISA方法.
关键词 ： 环GMP,  抗体,单克隆,  酶联免疫吸附测定,  兔    
Abstract：

Objective: To prepare rabbit monoclonal antibody (RabMab) against guanosine 3', 5'-cyclic monophosphate (cGMP) and to develop a competitive ELISA for the detection of cGMP. Methods: New Zealand white rabbits were immunized with synthesized cGMP-keyhole limpet hemoeyanin (cGMP-KLH) to prepared a RabMAb with monoclonal antibody technique of Epitomics. A competitive ELISA kit was produced with cGMP RabMAb. The specificity, the precision and the recoveries of the method were determined. Results: The RabMAb with high sensitivity towards cGMP were prepared with an antibody timer of 3.1 ng/mL and 50% inhibitive concentration (IC50) of 12.57 ng/mL. The cGMP RabMAb had 33% cross-reactivity to inosine 3', 5'-cyclic monophosphate (cIMP) and little or no cross-reactivity to other compounds. A competitive ELISA was developed for detection of cGMP. The range of detection was 0~120 ng/mL with a minimal limit of 1.95 ng/mL. The recovery of assay was 89%~103%. The inter-assay and intra-assay coefficient variations were below 11.68% and 13.85%, respectively. Conclusion: The RabMab against cGMP with high affinity and high specificity has been generated successfully, and a competitive ELISA for detection of cGMP has been developed with the prepared cGMP RabMAb.
Key words： Cyclic GMP    Antibodies, monoclonal    Enzyme-linked immunosorbent assay    Rabbits
收稿日期: 2014-10-20
CLC:  R392.11  
通讯作者: 陈枢青(1961-),男,博士,教授,博士生导师,从事生化毒理学研究;E-mail:chenshuqing@zju.edu.cn;http://orcid.org/0000-0002-0792-3735     E-mail: chenshuqing@zju.edu.cn
作者简介: 谭兴梅(1980-),女,硕士研究生,工程师,从事兔单克隆抗体的研究;E-mail:xingmei.tan@abcam.com;http://orcid.org/0000-0002-5726-7019
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引用本文:

谭兴梅, 胡海峰, 陈枢青. 环鸟苷酸兔单克隆抗体的制备和竞争酶联免疫吸附测定方法的建立[J]. 浙江大学学报（医学版）, 2015, 44(4): 423-428.
TAN Xing-mei, HU Hai-feng, CHEN Shu-qing. Preparation of rabbit monoclonal antibody against cGMP and development of competitive ELISA for cGMP. Journal of ZheJiang University(Medical Science), 2015, 44(4): 423-428.

链接本文:

http://www.zjujournals.com/xueshu/med/CN/10.3785/j.issn.1008-9292.2015.07.12      或      http://www.zjujournals.com/xueshu/med/CN/Y2015/V44/I4/423

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