中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (5): 874-877.doi: 10.3969/j.issn.1673-8225.2011.05.027

• 器官移植基础实验 basic experiments of organ transplantation • 上一篇    下一篇

建立夹心ELISA检测RANTES方法及在小肠移植中的应用

康振华1，王春艳2，王为忠3，杜建军3，郑建勇3，王  磊1   

  1. 1吉林大学第一医院结直肠肛门外科,吉林省长春市  130031
    2吉林医药学院病原教研室，吉林省吉林市  132013
    3解放军第四军医大学西京医院消化病院，陕西省西安市  710032
  • 收稿日期:2010-07-01 修回日期:2010-09-16 出版日期:2011-01-29 发布日期:2011-01-29
  • 通讯作者: 王磊，硕士生导师，教授，吉林大学第一医院结直肠肛门外科,吉林省长春市,130031 Wanglei11@163.com
  • 作者简介:康振华☆，男，1975年生，吉林省长春市人，汉族，2008年解放军第四军医大学毕业，博士，主治医师，主要从事移植排斥方面的研究。 k_z_h75@163. com 并列第一作者：王春艳☆，女，1973年生，吉林省吉林市人，满族，2008年解放军第四军医大学毕业，博士，讲师，主要从事分子免疫学方面的研究。 c_cy66@163. com

Establishment of a sandwich ELISA for RANTES detection and its application in rat small bowel transplantation

Kang Zhen-hua1, Wang Chun-yan2, Wang Wei-zhong3, Du Jian-jun3, Zheng Jian-yong3, Wang Lei1   

  1. 1Department of Colorectal and Anus Surgery, First Hospital, Jilin University, Changchun  130021, Jilin Province, China
    2Department of Immunology, Jilin Medical College, Jilin  132013, Jilin Province, China
    3Department of Gastrointestinal Surgery, Xijing Hospital, Fourth Military Medical University of Chinese PLA, Xi’an  710032, Shaanxi Province, China
  • Received:2010-07-01 Revised:2010-09-16 Online:2011-01-29 Published:2011-01-29
  • Contact: Wang Lei, Master’s supervisor, Professor, Department of Colorectal and Anus Surgery, First Hospital, Jilin University, Changchun 130021, Jilin Province, China Wanglei11@163.com
  • About author:Kang Zhen-hua☆, Doctor, Attending physician, Department of Colorectal and Anus Surgery, First Hospital, Jilin University, Changchun 130021, Jilin Province, China k_z_h75@163.com Wang Chun-yan☆, Doctor, Lecturer, Department of Immunology, Jilin Medical College, Jilin 132013, Jilin Province, China c_cy66@163. com Kang Zhen-hua and Wang Chun-yan contributed equally to this paper.

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摘要：

背景：RANTES 及其受体介导的细胞免疫是同种异体小肠移植急性排斥反应发生的重要组成部分，小肠移植后血清中RANTES 水平有可能作为早期检测急性排斥反应发生的较敏感的免疫指标。
目的：建立夹心ELISA检测RANTES的方法，并将其应用于大鼠小肠移植模型，探讨其作为检测早期急性排斥反应指标的可能性。
方法：以辣根过氧化物酶标记RANTES mAbs，通过竞争ELISA检测抗RANTES mAb 识别的表位，建立双夹心ELISA试剂盒并检测其敏感性，然后将其应用于大鼠小肠移植模型中RANTES的检测。
结果与结论：以anti-RNATES mAb No.2 (5 mg/L) 为包被抗体，HRP-anti-RNATES mAb No.4(1∶800)为酶标抗体建立了双抗体夹心ELISA法，其敏感性达到0.5 μg/L。异基因大鼠小肠移植模型术后血清RANTES明显增高。提示成功建立了特异性强、灵敏度良好的检测RANTES的双抗体夹心ELISA法，为早期诊断小肠移植排斥反应提供了一种新的方法。

关键词: 夹心ELISA, 单克隆抗体, RANTES, 小肠, 移植

Abstract:

BACKGROUND: RANTES and its receptors mediated cellular immunity are the essential components of acute allograft rejection, the dynamic observation on expression of RANTES in serum after small bowel transplantation maybe act as a relatively sensitive index for diagnosing acute allograft rejection.
OBJECTIVE: To establish a Sandwich ELISA for RANTES, utilize it in rat small bowel transplantation model and to discuss the possibility of early diagnosis by using ELISA in acute rejection.
METHODS: RANTES mAb was labeled by horseradish peroxidase and its discriminating epitopes was measured by competed ELISA. The sandwich ELISA kit was established sensitivity was detected. After that, the kit was used to measure RANTES in rat small bowel transplantation models. 
RESULTS AND CONCLUSION: In antibody-sandwich ELISA, the optimal capture mAb was No2 (5 mg/L), and the optimal anzyme-conjugate mAb was No4 (1: 800). The minimal RANTES antigen which could be detected by this method was 0.5 μg/L. The level of RANTES in serum was heightened obviously in heterogen rat small bowel transplantation group. A sensitive and specific antibody-sandwich ELISA was established for RANTES detection, which offers a new method to early diagnose acute rejection in small bowel transplantation.

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