髓过氧化物酶双抗体夹心光激化学发光检测法的建立

检验医学 ›› 2012, Vol. 27 ›› Issue (4): 291-295.

髓过氧化物酶双抗体夹心光激化学发光检测法的建立

韩海荣

1. 第二军医大学附属长海医院实验诊断科，上海 200433；2. 第二军医大学附属长海医院心内科，上海 200433；3. 博阳生物科技（上海）有限公司，上海 201210

收稿日期:2012-03-29 出版日期:2012-04-30 发布日期:2012-04-09
通讯作者: 沈茜，联系电话：021-81873611。
作者简介:韩海荣，女，1980年生，学士，技师，主要从事自身免疫性疾病研究。

The establishment of double-antibody sandwich light initiated chemiluminescence assay for the determination of plasma myeloperoxidase

1. Experiment Diagnosis Department, Changhai Hospital, Second Military Medical University, Shanghai 200433, China ; 2. Department of Cardiology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China ; 3.Beyond Diagnostics (Shanghai) Company Limited, Shanghai 201210, China

Received:2012-03-29 Online:2012-04-30 Published:2012-04-09

摘要/Abstract

摘要： 目的建立一种定量检测血浆中髓过氧化物酶（MPO）浓度的双抗体夹心光激化学发光检测法（LiCA），用于心血管疾病的辅助诊断。方法采用棋盘格滴定法，从针对MPO抗原的12株单克隆抗体（McAb）中筛选出最佳双抗体组合（1株McAb包被发光微粒，另1株McAb进行生物素化），进而探索2种抗体最适反应浓度及MPO LiCA的最适反应条件，评价该法的重复性、敏感性、抗干扰性及回收率，并与经典的CardioMPO^TM酶联免疫吸附试验(ELISA)进行平行比较分析。结果筛选到2株最适McAb，并与链霉亲和素包被的感光微粒连接，建立用于MPO定量测定的LiCA。该法敏感性为1.76 ng/mL，批内变异系数（CV）和天间CV分别为2.73％、3.76％，平均回收率为102.5%±8.6%，与CardioMPO^TMELISA具有良好的相关性（r=0.961，P<0.01），正常参考范围为13.16～128.79 ng/mL。结论成功建立了一种用于MPO定量检测的LiCA，该法具有高的敏感性和好的稳定性，试剂有效期长，并且操作流程简便，耗时短，在心血管疾病的辅助诊断中具有很好的应用前景。

关键词: 髓过氧化物酶, 光激化学发光检测法, 酶联免疫吸附试验, 单克隆抗体

Abstract: Objective To establish a quantitative double-antibody sandwich light initiated chemiluminescence assay (LiCA) for the determination of plasma myeloperoxidase (MPO) concentration in order to assist to diagnose cardiovascular diseases. Methods From 12 anti-MPO monoclonal antibody (McAb), the optimum double-antibody combination (one McAb coated on luminescence particles and the other McAb conjugated with biotin) was picked out by the checkerboard titration. The suitable reaction concentration and conditions were analyzed. Consequently, the repeatability, sensitivity, anti-interference and recovery rate of the assay were evaluated and compared with the classic CardioMPO^TMenzyme-linked immunosorbent assay (ELISA). Results Two McAbs were selected successfully. The quantitative MPO LiCA system was constructed by connecting the sensitizer particles with streptavidin. The results showed that the sensitivity was 1.76 ng/mL. The within-run and interday-run coefficients of variation (CV) of MPO LiCA were 2.73％ and 3.76％ respectively, and the mean recovery rate was 102.5%±8.6%. The correlation with CardioMPO^TMELISA was high (r=0.961，P<0.01). The normal reference range was 13.16-128.79 ng/mL. Conclusions A quantitative LiCA for the determination of MPO is successfully established. MPO LiCA with good sensitivity, stability, long reagent validity, simple and convenience shows a better application prospect in assisting to diagnose cardiovascular diseases.

Key words: Myeloperoxidase, Light initiated chemiluminescence assay, Enzyme-linked immunosorbent assay, Monoclonal antibody

韩海荣;梁颖;赵卫国;沈茜. 髓过氧化物酶双抗体夹心光激化学发光检测法的建立[J]. 检验医学, 2012, 27(4): 291-295.

HAN Hairong;LIANG Ying;ZHAO Weiguo;SHEN Qian. The establishment of double-antibody sandwich light initiated chemiluminescence assay for the determination of plasma myeloperoxidase[J]. , 2012, 27(4): 291-295.

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