双抗体夹心ELISA测定人广泛型线粒体肌酸激酶方法的建立  

Establishment of double antibody sandwich ELISA for the determination of human ubiquitous mitochondrial creatine kinase

作　　者：张健 简敏华 蒋玲丽 葛丹红 

机构地区：[1]上海市临床检验中心,上海200126

出　　处：《检验医学》2013年第10期913-916,共4页Laboratory Medicine

基　　金：上海市卫生局科研课题(2007068)

摘　　要：目的建立测定人血清广泛型线粒体肌酸激酶(uMtCK)浓度的双抗体夹心酶联免疫吸附试验(ELISA)。方法应用原核表达的uMtCK重组蛋白免疫小鼠获得7株单克隆抗体,采用棋盘格滴定法筛选出最佳双抗体组合,其中抗体19F11进行辣根过氧化物酶(HRP)标记作为检测抗体,抗体3C1作为俘获抗体,建立检测人uMtCK双抗体夹心ELISA方法,并进行方法学评价。同时采用该法检测50名健康体检者和85例肿瘤标志物[甲胎蛋白(AFP)或癌胚抗原(CEA)]阳性患者的血清。结果通过筛查获得配对抗体,建立检测人uMtCK的双抗体夹心ELISA方法。该法的检测限为28.8 ng/mL,批内变异系数(CV)为4.5%～7.7%,批间CV为8.2%～13.5%,平均回收率为97.1%。用该法检测50名健康体检者血清uMtCK浓度,结果均为阴性;85例肿瘤标志物阳性患者中有15例检出uMtCK,浓度为52.8～1 442.2 ng/mL。结论建立的双抗体夹心ELISA可以用于测定人血清uMtCK浓度。Objective To establish double antibody sandwich enzyme-linked immunosorbent assay （ELISA） for the determination of ubiquitous mitochondrial creatine kinase （uMtCK） in human serum. Methods Seven strains of monoclonal antibodies against human uMtCK were prepared with prokaryotic expression recombinant proteins from rats, and the best combination of double antibodies was selected by checkerboard titration method. Double antibody sandwich ELISA for the determination of human uMtCK was established by using HRP-labeled McAb 19Fll as a detection antibody and McAb 3C1 as a capture antibody. The methodology performance was evaluated. The serum concentrations of uMtCK in 50 healthy subjects and 85 patients with positive tumor markers [ alpha fetal protein （AFP） and carcinoembryonic antigen （CEA）] were determined. Results The double antibody sandwich ELISA for the determination of human uMtCK was established by selecting the combination of double antibodies. The detection limit was 28.8ng/mL, and the within-run and between-run coefficients of variation （CV） were 4.5% -7.7% and 8.2% - 13. 5% , respectively. The average recovery was 97. 1%. The serum concentrations of uMtCK in 50 healthy subjects were negative. The 15 of 85 patients with positive tumor markers had positive results of uMtCK, and the concentration was 52.8 - 1 442.2 ng/mL. Conclusions The established double antibody sandwich ELISA can be used to determine uMtCK in human serum.

关 键 词：线粒体肌酸激酶 广泛型亚型 酶联免疫吸附试验 

分 类 号：Q555[生物学—生物化学]