流式微球分析技术检测人心肌钙蛋白T方法的建立

Development a method of cytoometric bead assay for cardiac troponin T

目的建立流式微球分析技术(cytoometric bead assay,CBA)检测人肌钙蛋白T(cTnT)方法。方法用鼠抗人的cTnT的单克隆抗体包被在已激活的羧基化聚苯乙烯微球上,然后再用包被好的微球与检测标本进行抗原抗体免疫反应,并加入羊抗人cTnT的多克隆抗体和异硫氰酸荧光素(FITC)标记的驴抗羊的IgG,室温避光免疫反应一定时间后洗涤一次,上流式细胞仪检测FITC的荧光强度,以此测定标本中cTnT含量。结果通过正交试验,在100μl反应体系中,微球含量大约为1.25×106,选择10μg的鼠抗人cTnT单克隆抗体为最佳加入量,加入标本和抗体后,选择避光反应时间为120 min,洗涤选择一次,选择8μg/ml羊抗人cTnT多克隆抗体和20μg/ml FITC标记驴抗羊IgG为最佳工作浓度;方法检测灵敏度为16.0 pg/mL,线性范围是16-5 000 pg/mL,批内重复性变异系数为5.2-11.3%,回收率是94.7-111.5%,高浓度的胆红素、胆固醇和甘油三脂无明显干扰,与Roche Elecsys的电化学发光法有较好的相关性。结论自建CBA检测cTnT技术性能较好,可在临床工作中推广使用。

Objective To development of cytoometric bead assay（CBA）for cardiac troponin T（cTnT）.Methods Activated carboxyl polystyrene microspheres are coated with mouse anti-human cTnT monoclonal antibody and the unconjuncted microspheres sites of monoclonal antibodies are blocked with blocking buffer.And then add specimens to the coupled microspheres for antigen-antibody immune response,and add goat anti-human cTnT polyclonal antibody and fluorescein isothiocyanate（FITC） labeled donkey anti-goat IgG,immune response in certain period of time at room temperature away from light,after washing,detect by flow cytometry on the FITC fluorescence intensity,as measure cTnT content in samples.Results By orthogonal test,in 100 μl reaction system,microspheres content of about 1.25×106,select 10 μg mouse anti-human cTnT monoclonal antibodies for the best dosage,adding samples and antibodies,choose immune response 120 min away from light,washing one time,select 8 μg/ml goat anti-human cTnT polyclonal antibody and 20 μg/ml FITC labeled donkey anti-goat IgG as the best working concentration.Methodological evaluation of this detection,this method of detection sensitivity is 16.0 pg/mL,the linear range is 16-5 000 pg / mL,intra-repeatability coefficient of variation is 5.2-11.3% and the recovery is 94.7-111.5%,high concentrations of bilirubin,cholesterol,and triglycerides has no significant interference.It has good correlation with the Roche Elecsys electrochemiluminescence.Conclusion Detection cTnT by self-development of CBA technical performance was better,could be used widely in clinical practice.