定量检测人超敏C-反应蛋白双抗体夹心ELISA 方法的建立及初步临床应用

中国临床药理学与治疗学 ›› 2009, Vol. 14 ›› Issue (1): 84-89.

定量检测人超敏C-反应蛋白双抗体夹心ELISA 方法的建立及初步临床应用

沈丹丹¹, 卞智萍², 何国平¹, 顾春荣², 徐晋², 杨笛², 张寄南²

¹江苏大学附属武进医院心内科, 常州 213002, 江苏;
²南京医科大学第一附属医院心血管病研究所, 南京 210029, 江苏

收稿日期:2008-10-31 修回日期:2008-12-15 出版日期:2009-01-26 发布日期:2020-10-27
通讯作者: 何国平,男, 主任医师, 教授, 硕士生导师, 研究方向:心血管疾病的介入治疗和分子生物学。Tel:1391306027 E-mail:guopinghe8@sina.com
作者简介:沈丹丹, 女, 在读硕士, 研究方向:心血管疾病分子诊断。Tel:15961103592 E-mail:sdd-600@163.com。卞智萍, 并列第一作者, 女, 副主任技师, 研究方向:心血管疾病分子诊断。Tel:13951935294
基金资助:
重大疾病分子诊断和生物治疗的高技术平台基金项目(XK200705)

Establishment of a sandwich ELISA for quantitative measurement of human supersensitivity C-reactive protein and primary clinical application study

SHEN Dan-dan¹, BIAN Zhi-ping², HE Guo-ping¹, GU Chun-rong², XU Jin-dan², YANG Di², ZHANG Ji-nan²

¹Department of Vasculocardiology, the Affiliated Wujin Hospital of JiangsuUniversity, Changzhou 213002, Jiangsu,China;
²Institute of Cardiovaslular Science, theFirst Affiliated Hospital of NJMU, Nanjing 210029, Jiangsu, China

Received:2008-10-31 Revised:2008-12-15 Online:2009-01-26 Published:2020-10-27

摘要/Abstract

摘要： 目的: 建立定量检测人超敏C-反应蛋白(CRP) 双抗体夹心ELISA 方法。方法: 采用Protein A 亲和层析法纯化本室制备的抗CRP 单克隆抗体(mAbs), 并行SDS-PAGE 和Western blotting 对纯化抗体特性进行鉴定;利用简易过碘酸钠法对抗CRP mAbs 进行标记HRP 后行抗体配对实验;通过方阵滴定法确定包被抗体和酶标抗体的最适工作浓度;以纯化的CRP 抗原为标准品建立标准曲线;以重复性、灵敏性和回收率实验评价ELISA 方法。初步对人血浆中的超敏CRP 水平进行检测。结果: 最佳配对组合为抗CRP mAb 1C10 和HRP标记的抗CRP mAb 2C11, 最适工作浓度分别为10 μg/mL 和1 ∶2000, 该方法的批内、批间变异系数分别为3.1 %~9.7 %和3.6 %~13.6 %, 灵敏度达8.3 ng/mL, 回收率为90 % ~ 109 %。用ELISA 法测定左右冠无明显狭窄者68 例, 狭窄小于50 %者59 例和狭窄大于50 %患者67 例的血浆hs-CRP 水平, 冠脉狭窄小于50 % 组(3.7±1.2)mg/L 与冠脉无狭窄组(1.8±0.7) mg/L 比较明显升高(P<0.05);狭窄大于50 %组(8.9±3.3) mg/L 与狭窄小于50 %组比较明显升高(P<0.05) 。结论: 建立了一种可用于检测人超敏CRP的双抗体夹心ELISA 方法。

关键词: 定量检测, 超敏C-反应蛋白, ELISA 双抗体夹心法

Abstract: AIM: To establish a sandwich ELISA for quantitative measurement of supersensitivity C-reactive protein(hs-CRP).METHODS: Anti-CRP monoclonal antibodies (mAbs) prepared by our laboratory were purified by Protein A affinity chromatography and analyzed by SDS-PAGE and Western-blotting to test their characteristics.All the mAbs were labeled with horseradish peroxidase by sodium oxidation method and antibody mating test were performed using anti-CRP mAb as coating antibody and HRP labeled anti-CRP mAb as labeled antibody, in which optimal concentrations were defined by square mateix titration.Standard curve was performed using purified CRP and the sensitivity, reproducibility and recovery rate test of ELISA was evaluated.The CRP levels in plasma were measured in this assay.RESULTS: The optimal paired antibodies were anti-CRP mAb 1C10 and HRP labeled anti-CRP mAb 2C11, and the optimal concentrations were 10 μg/mL and 1 ∶2000, respectively.The coefficient of variation were 3.1 % to 9.7 % within assay and 3.6 %to 13.6 % between assay.The sensitivity in this assay was 8.3 ng/mL.The recovery rate was 90 % to 109 %.According to the result, 68 normal persons' hs-CRP level in plasma of coronary arteriography were detected by ELISA, 59 with stenosis<50 % and 67 with stenosis ≥50 %.The results showed that the plasma hs-CRP level in patients with stenosis<50 % (3.65±1.15) mg/L was significantly higher (P<0.05) than hs-CRP in normol persons(1.75±0.74) mg/L, the plasma hs-CRP level in stenosis ≥ 50 % patients (8.93±3.29) mg/L was significantly higher (P<0.05) than stenosis<50 % patients.CONCLUSION: A sandwich ELISA for detecting hs-CRP was established.

Key words: quantitative measurement, hs-CRP, sandwich ELISA

中图分类号:

R969

沈丹丹, 卞智萍, 何国平, 顾春荣, 徐晋, 杨笛, 张寄南. 定量检测人超敏C-反应蛋白双抗体夹心ELISA 方法的建立及初步临床应用[J]. 中国临床药理学与治疗学, 2009, 14(1): 84-89.

SHEN Dan-dan, BIAN Zhi-ping, HE Guo-ping, GU Chun-rong, XU Jin-dan, YANG Di, ZHANG Ji-nan. Establishment of a sandwich ELISA for quantitative measurement of human supersensitivity C-reactive protein and primary clinical application study[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2009, 14(1): 84-89.

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