间接ELISA定量检测抗IgE自身抗体方法的建立  

An indirect ELISA method for quantitative detection of anti-IgE autoantibodies

作　　者：尹悦[1] 党宏[2] 高闯 彭霞[1] 梁玉婷[1] 廖焕金 李莉[1] Yin Yue;Dang Hong;Gao Chuang;Peng Xia;Liang Yuting;Liao Huanjin;Li Li(Department of Laboratory Medicine,Shanghai General Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200080,China;Department of Dermatology,Shanghai General Hospital,Shanghai 200080,China)

机构地区：[1]上海交通大学附属第一人民医院检验科,上海200080 [2]上海交通大学附属第一人民医院皮肤科,上海200080

出　　处：《中华检验医学杂志》2019年第9期782-786,共5页Chinese Journal of Laboratory Medicine

基　　金：国家自然科学基金(81471593,81601395,81871267);上海申康医院发展中心临床科技创新项目(SHDC22014005);上海市科学技术委员会(14411963200);上海市青年科技英才扬帆计划资助(16YF1409200).

摘　　要：目的建立一种间接ELISA法定量检测IgG型抗人IgE自身抗体,并初步评估慢性荨麻疹(CU)患者血清抗IgE含量。方法选取2018年1至12月于上海市第一人民医院首诊的75例CU患者及120名健康体检者,收集相应血清样本及临床资料。以人IgE包被微孔板,加入人源化抗人IgE标准品(奥马珠单抗)或待检血清,再加入辣根过氧化物酶(HRP)标记的抗人IgG,建立间接酶联免疫法,并进行条件优化及方法学评价。采用Mann-WhitneyU检验、卡方检验分析CU患者及健康人血清IgE水平,采用受试者工作曲线(ROC)分析抗IgE对CU的诊断效能。结果IgE包被量5.0×10^-4mg/ml,血清稀释度1∶300,酶标抗体稀释度1∶100000;标准曲线R2=0.996;批内及批间变异系数3.9%~7.5%、6.0%~8.2%;血红蛋白≤1.3g/L、三酰甘油≤4.6mmol/L、胆红素≤171μmol/L时,对检测结果无明显影响;低、高浓度样品回收率95.9%、108.4%;空白限5.8×10^-4mg/ml,检出限1.8×10^-3mg/ml,定量限2.0×10^-3mg/ml;线性范围约2.0×10^-3~354.4mg/ml;抗IgE达354.4mg/ml时未见钩状效应;血清4℃过夜、反复冻融6次后抗IgE水平保持稳定。CU患者血清抗IgE水平[19.0(1.9,58.6)mg/ml]显著高于健康人血清抗IgE水平[0.7(0.002,11.1)mg/ml],P<0.001;当抗IgE截断值定为15.3mg/ml时,CU患者阳性率为54.7%,显著高于健康对照(18.3%),P<0.001,AUC达0.736。结论成功建立血清抗IgE的间接ELISA法,CU患者血清中存在抗IgE自身抗体。ObjectiveThe accurate measurement of anti-IgE levels in patients′serum helps for make diagnosis of chronic urticaria(CU).An indirect ELISA method for quantitative detection of IgG anti-IgE,were established.MethodsThe serum samples and the clinical data of 75 first-diagnosed CU patients and 120 healthy controls were collected at Shanghai General Hospital during the year of 2018.In the indirect ELISA system to measure the IgG(anti-human IgE),the antigen,Human IgE Myeloma,was coated on the plate;Omalizumab(a humanized anti-human IgE antibody)was the standard,and horse radish peroxidase(HRP)-labeled anti-human IgG was the tracer.The optimum concentrations of serum and HRP-labeled anti-human IgG were determined by chessboard titration,and method evaluation was conducted.The comparison of serum anti-IgE levels in CU patients and healthy people were analyzed by Mann-Whitney U test and chi-square test.Receiver operating characteristic(ROC)curves were applied to establish the diagnostic performance of serum anti-IgE for CU.ResultsThe coating concentration of IgE was 5.0×10^-4 mg/ml;serum dilution was 1∶300;enzyme-labeled antibody dilution was 1∶100 000.Standard curve was in good linearity with R2=0.996.The intra-and inter-assay coefficient of variation were 3.9%-7.5%and 6.0%-8.2%,and the recovery rate of low and high concentration samples were 95.9%and 108.4%,respectively.When hemoglobin≤1.3 g/L,triglyceride≤4.6 mmol/L,bilirubin≤171μmol/L,no interference were observed.The limit of blank,limit of detection,and limit of quantitation were 5.8×10^-4,1.8×10-^3,and 2.0×10^-3 mg/ml.The linearity range was from 2.0×10^-3 to 354.4 mg/ml.No Hook effect was found until anti-IgE reached 354.4 mg/ml.The anti-IgE remained stable after serum storage at 4℃overnight or treated with 6 repeated freeze-thaw cycles.The anti-IgE levels in CU patients[19.0(1.9,58.6)mg/ml]were significantly higher than those in healthy controls[0.7(0.002,11.1)mg/ml]with P<0.001.When cut-off value was set as 15.3 mg/ml,in this method,the posit

关 键 词：免疫球蛋白E 自身抗体 酶联免疫吸附测定 荨麻疹 

分 类 号：R446.6[医药卫生—诊断学]