量子点流式微球技术检测抗胰岛素抗体方法学建立

目的将量子点荧光探针应用于流式微球技术,创建出量子点流式微球技术检测人抗胰岛素抗体的方法并对其偶联率、精密度、线性范围、灵敏度进行初步评价。方法将胰岛素与羧基化微球偶联制备免疫诊断微球,与血清或标准品中人抗胰岛素抗体结合后,加入兔抗人IgG抗体,最后加入生物素化的羊抗兔IgG抗体和链霉亲和素化的量子点,使微球表面呈现荧光,并通过流式细胞仪检测平均荧光强度(mean fluorescent intensity,MFI)并记录。结果羧基化微球偶联胰岛素的性能显著强于空白微球,偶联0.5～3 h为最佳偶联反应时间,偶联成功的免疫诊断微球放置4℃冰箱至少可稳定50 d。微球偶联微球表面所携带荧光MFI与所测抗胰岛素抗体浓度成正比,量子点流式微球技术检测同一标本的灵敏度(3.74 pg/ml)、精密度(8.24%)、线性范围(3.74～5 000 pg/ml)都优于酶联免疫吸附试验所测得灵敏度(24.53 pg/ml)、精密度(12.31%)、线性范围(24.53～2 500 pg/ml)。结论本实验创建的量子点流式微球技术可用于人血清抗胰岛素抗体测定,其检测性能良好。

Establishment of a quantum-dots cytometric bead assay for detecting human against insulin antibody

We aim to establish a quantum dots cytometric bead assay for detecting human against insulin antibody(AIAb) and study its coupling rate,precision,linearity,sensitivity.Firstly,microsphere surface were labeled by insulin to prepare immunodiagnosis microspheres.Then the diagnosis microspheres were combined with human against insulin monoclonal antibody and added with rabbit against human IgG antibody,biotinylated sleep against rabbit IgG antibody,and quantum dots streptavidin conjugate.At last,the mean fluorescent intensity(MFI) of microsphere was detected by flow cytometer.We found that the carboxyl microspheres coupling with insulin performance significantly better than non-carboxyl microspheres,and the best coupling time is 0.5-3 h.The immune diagnosis microspheres can keep tenable at least 50 d in freezer of 4 ℃.The MFI of immune diagnosis microspheres were in direct proportion to the concentrations of human AIAb.Moreover,both of the sensitivity(3.74 pg/ml),precision(8.24%),linearity(3.74-5 000 pg/ml) of quantum-dots cytometric bead assay were better than the sensitivity(24.53 pg/ml),precision(12.31%),Linearity(24.53-2 500 pg/ml) of ELISA.The result showed that quantum dots cytometric bead assay can be used for determination of human AIAb with well detection performance.