高浓度HBsAg检测化学发光微粒子免疫分析法的建立及性能评价  

Establishment and performance evaluation of chemiluminescent microparticle immunoassay for the determination of high concentration HBsAg

作　　者：刘杨 张志平 彭道荣 宋浏伟 葛胜祥 郝晓柯 刘家云 LIU Yang;ZHANG Zhiping;PENG Daorong;SONG Liuwei;GE Shengxiang;HAO Xiaoke;LIU Jiayun(The First Affiliated Hospital of Air Force Medical University,Xi'an 710032,Shaanxi,China)

机构地区：[1]空军军医大学第一附属医院,陕西西安710032

出　　处：《检验医学》2020年第10期1056-1061,共6页Laboratory Medicine

基　　金：国家高技术研究发展计划(863计划)重点项目(2011AA02A101)。

摘　　要：目的建立定量检测乙型肝炎表面抗原(HBsAg)的化学发光微粒子免疫分析法(CMIA),并进行性能评价。方法将小鼠特异性抗HBsAg抗体包被在磁微粒表面,结合相应HBsAg,加入吖啶酯标记的特异性抗HBsAg抗体,在磁微粒表面形成双抗体夹心免疫复合物,加入预激发液和激发液,激发复合物上的吖啶酯发出光子,光信号值与HBsAg浓度呈正比。筛选最佳反应条件,对自建CMIA检测HBsAg的性能(线性范围、检测限、精密度、干扰试验、交叉反应、稳定性及与电化学发光法的相关性和一致性)进行评价。结果磁微粒最适浓度为0.4 mg/mL,吖啶酯标记抗体的最适稀释比例为1∶200(浓度为250 ng/mL)。反应稀释液用量、最适加样量、试剂用量分别为100、20、50μL。自建CMIA检测HBsAg的线性范围为20~100000 IU/mL,最低检测限为9.9 IU/mL,批内和批间变异系数(CV)均<10.0%。血红蛋白<400 mg/L、胆红素<4 mg/L、类风湿因子<800 IU/mL、三酰甘油<2 mg/L、柠檬酸钠<22 mmol/L、乙二胺四乙酸(EDTA)<8 mmol/L对自建CMIA检测HBsAg无干扰。甲型肝炎病毒(HAV)、丙型肝炎病毒(HCV)、戊型肝炎病毒(HEV)、人类免疫缺陷病毒(HIV)、人类嗜T淋巴细胞病毒(HTLV)、巨细胞病毒(CMV)、EB病毒(EBV)阳性样本对自检CMIA未发生交叉反应。配制好的试剂在37℃可稳定6 d,开盖后上机(2~8℃)保存可稳定4周,2~8℃密封保存可稳定6个月。自建CMIA与电化学发光法的相关性(r=0.978,P<0.001)和一致性均良好。结论自建CMIA检测HBsAg的线性范围较宽,高浓度样本可直接检测,无需稀释,且检测性能优良,有较好的临床应用前景。Objective To establish a chemiluminescent microparticle immunoassay(CMIA)for the quantitative determination of hepatitis B surface antigen(HBsAg)and conduct performance evaluation.Methods Mouse-specific anti-HBsAg antibody was coated on the surface of magnetic particle to bind the corresponding HBsAg.The specific anti-HBsAg antibody labeled with acridinium ester was added to form a double antibody sandwich immune complex on the surface of magnetic particle.Pre-trigger solution and trigger solution were added.The acridinium ester on the complex was excited to emit photons,and the relative light unit was proportional to HBsAg concentration.The best reaction conditions were screened,and the performance of self-established CMIA for determining HBsAg(linear range,determination limit,precision,interference,cross-reaction,stability,correlation and consistency with electrochemiluminescence method)was evaluated.Results The optimal concentration of magnetic particles was 0.4 mg/mL,and the optimal dilution ratio of acridinium ester-labeled antibody was 1∶200(a concentration of 250 ng/mL).The amounts of reaction diluent,sample and reagent were 100,20 and 50μL,respectively.The linear range of self-established CMIA to determine HBsAg was 20-100000 IU/mL,the minimum determination limit was 9.9 IU/mL,and the within-run and between-run coefficents of variation(CV)were<10.0%.The self-established CMIA was not affected by the interference factors,such as hemoglobin<400 mg/L,bilirubin<4 mg/L,rheumatoid factor<800 IU/mL,triglyceride<2 mg/L,sodium citrate<22 mmol/L and ethylenediaminetetraacetic acid(EDTA)<8 mmol/L.Hepatitis A virus(HAV),hepatitis C virus(HCV),hepatitis E virus(HEV),human immunodeficiency virus(HIV),human T-lymphotropic virus(HTLV),cytomegalovirus(CMV)and Epstein-Barr virus(EBV)positive samples did not cross-react with this assay.The prepared reagent could be stable for 6 d at 37℃,it could be stable for 4 weeks after opening lid at 2-8℃,and it could be stable for 6 months when stored in a sealed container at 2-8

关 键 词：乙型肝炎表面抗原 乙型肝炎病毒 化学发光微粒子免疫分析法 性能评价 

分 类 号：R446.62[医药卫生—诊断学]