建立基于磁微粒化学发光免疫分析法检测血清可溶性fms样酪氨酸激酶-1的方法及初步性能评价

Establishment of A Method for Serum Soluble Fms-like Tyrosine Kinase-1 Based on Magnetic Nanoparticle Chemiluminescence Immunoassay and Its Preliminary Performance Evaluation

目的基于磁微粒化学发光免疫分析法(chemiluminescence immunoassay,CLIA)建立一种定量检测人血清中可溶性fms样酪氨酸激酶-1(soluble fms-like tyrosine kinase-1,sFlt-1)含量的方法,并对其性能进行初步评价。方法采用“三明治”夹心法,血清中的sFlt-1与生物素化sFlt-1单克隆抗体和吖啶酯标记的sFlt-1单克隆抗体反应,形成生物素化抗体-抗原-吖啶酯标记抗体的免疫复合物。通过生物素与链霉亲和素的反应,免疫复合物被链霉亲和素包被的磁微粒捕获,标记在免疫复合物上的吖啶酯在碱性H2O2条件下产生化学发光。利用免疫复合物的光强度计算血清中sFlt-1的浓度,优化抗体浓度,建立检测sFlt-1的磁微粒CLIA方法。对该方法的灵敏度、精密度、线性范围、抗干扰、准确度及方法学比对等性能进行评价。收集2022年2~8月宁波鄞州中医院孕妇血清样本100份,应用建立的方法进行检测,结果与罗氏电化学发光法检测结果进行比较。结果最终确定试剂中生物素化抗体的最佳浓度为2μg/ml,吖啶酯标记抗体的最佳浓度为100 ng/ml。该方法空白限为1.67 pg/ml;批内及批间精密度变异系数(variable coefficient,CV)均<5%;线性范围为8.9~81206 pg/ml;血红蛋白浓度不大于500mg/dl,胆红素浓度不大于20mg/dl,乳糜浓度不大于1450 FTU,生物素浓度不大于50μg/L时,对检测结果不产生明显干扰;添加回收率为98%;与罗氏电化学发光法比对的相关系数r2为0.9953,两组数据之间差异具有统计学意义(P=0.0003)。结论建立的方法具有良好的初步性能,进一步开发后可应用于子痫前期的辅助筛查。

Objective To establish a method for soluble fms-like tyrosine kinase-1(sFlt-1)in human serum based on magnetic nanoparticle chemiluminescence immunoassay and preliminarily evaluate its performance.Methods According to the principle of sandwich plate theory,sFlt-1 in serum reacted with biotinylated anti-sFlt-1 monoclonal antibody and acridinium ester labeled anti-sFlt-1 monoclonal antibody to form an immune complex of biotinylated antibody-antigen-acridinium ester labeled antibody.Through the reaction between biotin and streptavidin,the immune complex was captured by the magnetic nanoparticles coated with streptavidin.The acridinium ester labeled on the immune complex caused chemiluminescence in alkaline H2O2.The chemiluminescence intensity of the immune complex was used to calculate the concentration of sFlt-1 in serum,optimize the concentrations of antibodies,and establish a method for sFlt-1 based on magnetic nanoparticle CLIA.The sensitivity,precision,linear range,interference,accuracy and method comparison of the established method were evaluated.A total of 100 clinical serum specimens were collected from pregnant women in the Ningbo Yinzhou Hospital of Traditional Chinese Medicine from February to August 2022.The established CLIA was used for detection,and the results were compared with the results of Roche electrochemiluminescence assay.Results The optimal concentrations of biotinylated antibody and acridinium ester labeled antibody in the reagent were ultimately determined to be 2μg/ml and 100 ng/ml,respectively.The limit of blank of this method was 1.67 pg/ml,the coefficient of variation(CV)of precision within and between batches was lower than 5%,and the linear range was 8.9~81206pg/ml.When the concentrations of serum hemoglobin,bilirubin,fat emulsion and biotin were lower than 500 mg/dl,20 mg/dl,1450 FTU and 50μg/L,respectively,there would be no significant interference with the detected results.The recovery rate of the method was 98%.The correlation coefficient(r2)between the established method and Roche electrochemiluminescence assay was 0.9953.The difference between the two group was significant(P=0.0003).Conclusion The established method have good preliminary performance and can be further applied to the auxiliary screening of preeclampsia.