人附睾蛋白4化学发光定量测定法的建立与评价  

Establishment and evaluation of a chemiluminescence quantitative immunoassay for detecting human epididymis protein 4

作　　者：罗湘宇 刘宇思[1] 曾令斌[1] 赵家宁[1] 何敏[2] 杨卫平 尹一兵[1] 胥文春[1] 

机构地区：[1]重庆医科大学检验医学院,重庆400016 [2]重庆医科大学附属第一医院,重庆400016 [3]四川迈克生物科技股份有限公司,成都611731

出　　处：《临床检验杂志》2014年第5期354-357,共4页Chinese Journal of Clinical Laboratory Science

基　　金：重庆市教委科学技术研究项目(KJ120318)

摘　　要：目的建立人附睾蛋白4（HE4）的化学发光定量检测方法,并进行方法学评价。方法血清中HE4与包被抗HE4单克隆抗体的微磁珠和辣根过氧化物酶（HRP）标记的抗HE4单克隆抗体反应形成双抗体夹心结构,HRP可催化发光底物液（含鲁米诺-H2O2-增强剂）产生大量光信号,根据光信号的量计算血清中HE4的浓度。依据国家体外诊断试剂性能评价指南性文件和美国临床和实验室标准化协会（CLSI）指南性文件,对本方法进行系统评价。结果建立的方法空白限（LoB）为1.014pmol/L、检测限（LoD）为5.252 pmol/L、定量检测限（LoQ）为10.568 pmol/L;甲胎蛋白（α-fetoprotein,AFP）≤400 IU/mL、癌胚抗原（carcinoembryonic antigen,CEA）≤1 777μg/L、糖类抗原（carbohydrate antigen,CA）125≤3 500 U/mL、CA19-9≤3 500U/mL对本方法无明显交叉反应;线性范围为20-1 700 pmol/L;在100 000 pmol/L时未出现明显钩状（Hook）效应;批内变异系数（CV）为1.5%-3.6%;日间CV为3.8%-6.4%;回收率为98.36%-99.14%;血红蛋白≤4.8 g/L、胆红素≤1 077.5μmol/L、乳糜微粒≤6 000浊度、生物素≤50μg/L、类风湿因子≤1 000 U/L对测定结果无明显影响;37℃保存7 d后相对偏差为-6.63%-10.23%;建立的方法（Y）与Fujirebio公司的ELISA试剂（X）进行方法学比对,相关曲线为Y=1.023X-12.280,r=0.989 7（P〈0.01）。结论本研究建立的HE4磁微粒化学发光免疫测定法各项性能符合临床实验室需求,为卵巢癌的辅助诊断提供了有效工具。Objective To establish a chemiluminescence quantitative method for detecting human epididymis protein 4（ HE4）,and evaluate its performance. Methods Serum HE4 can bind with both the anti-HE4 monoclonal antibody coated on micro-beads and the anti-HE4 monoclonal antibody labeled with horseradish peroxidase（ HRP） to form so-called sandwich structure. Then,the luminescent substrate solution containing luminol,H2O2and enhancer was catalyzed by HRP to produce light signals. According to light signals, the concentration of HE4 could be calculated. Next,the performance of the established method was evaluated by the Clinical and Laboratory Standards Institute（ CLSI） standards and National guidelines for Performance Testing for In Vitro Diagnostic Reagents. Results The limit of blank（ LoB）,the limit of detection（ LoD） and the limit of quantitation（ LoQ） of the established method were 1. 014 pmol / L,5. 252 pmol / L and 10. 568 pmol / L,respectively. Under the conditions of AFP≤400 IU / mL,CEA≤1 777 μg / L,CA125≤ 3 500 U / mL and CA19-9≤3 500 U / mL,the detection of HE-4 was not affected by their cross-reactivity. The linearity range of the established method was from 20 pmol / L to 1 700 pmol / L,and there was no Hook effect at 100 000 pmol / L of HE4. The intraassay and interassay coefficients of variation were 1. 5% - 3. 6% and 3. 8% - 6. 4%,respectively,and the recovery rate was 98. 36% - 99. 14%. Under the conditions of hemoglobin≤4. 8 g / L,bilirubin≤1 077. 5 μmol / L,chylomicrons≤6 000 turbidity,biotin≤50 μg/L and rheumatoid factor≤1 000 U/L,the detection of HE-4 was not affected by their interference. After serum samples were stored at 37 ℃ for 7 days,the relative deviation of HE-4 levels in serum was- 6. 63% - 10. 23%. In addition,there was a steady linear relationship with the equation Y = 1. 023X- 12. 280 between the established method（ Y） and ELISA kit（ X） from Fujirebio company（ r =0. 989 7,P〈0. 01）. Conclusion A chemiluminescence quantitative method f

关 键 词：人附睾蛋白4 化学发光法 定量检测 卵巢癌 

分 类 号：R446.6[医药卫生—诊断学]