人心肌肌钙蛋白Ⅰ酶促化学发光免疫分析方法的建立

摘要

Objective To develop chemiluminescence immunoassay for human cardiac troponin I. Methods Twornmonoclonal antibodies against cTnl were applied. One of the antibodies was immobilized on the microtiter plate; the other was labeled with HRP. Chemiluminecence immunoassay was established by HRP-Luminol-Peroxydol system. Sensitivity, linear scope, recovery and precision for the method were evaluated. The value for normal people was determined by testing 80 serum samples from healthy subjects. Results The reaction was proceeded for 30 min at 37℃. The Linear calibrator range was 0.3~100μg/L. The detection limit of the assay was 0. 16 μg/L and the recovery was 76. 8% ~111.1%. The intra-and inter-assay CV were 3. 90% ~6. 71% and 7. 66% ~ 10. 34% respectively. The results of dilution test demonstrated good correlation between dilution times and values (r =0.999). No cross reactivity were observed with cTnT, TnC, sKTnl, sKTnT, h-FABP, CK-MB, Myoglobin and C- reactive protein. The value for healthy individuals was below 0. 304μg/L. Patient samples tested using this method showed good correlation with Beckman Access cTnl assay. Conclusion The chemiluminescence immunoassay for human cardiac troponin I developed in this study is rapid, specific, accurate and suitable to clinical application.%目的 建立检测人心肌肌钙蛋白Ⅰ的酶促化学发光免疫分析方法.方法 采用两株抗人心肌肌钙蛋白Ⅰ的单克隆抗体,一株包被聚苯乙烯化学发光板,一株标记辣根过氧化物酶,采用辣根过氧化物酶/鲁米诺/过硼酸钠体系,建立了测定人血清心肌肌钙蛋白Ⅰ的酶促化学发光免疫分析方法,并进行了方法学鉴定.用该法测定80例正常人血清,确定正常参考值范围；测定临床病理样品27例,与Beckman全自动化学发光试剂测定值进行相关性分析.结果 本方法测定范围0.3 ～ 100μg/L,分析灵敏度0.16μg/L；37℃反应时间30min；回收率76.8％ ～ 111.1％；高浓度cTnI血清样品经系列倍比稀释后测定,测定值与稀释度呈线性相关,相关系数0.999；批内、批间变异系数分别为3.90％～6.71％,7.66％～10.34％；与心肌肌钙蛋白T(cTnT)、肌钙蛋白C(TnC)、骨骼肌肌钙蛋白Ⅰ(sKTnI)、骨骼肌肌钙蛋白T(sKTnT)、人心肌脂肪酸结合蛋白(h-FABP)、人心肌肌酸激酶(CK-MB)、人心肌肌红蛋白、C-反应蛋白(CRP)均无交叉；确定临床正常参考值小于0.304μg/L；与Beckman全自动化学发光试剂比较,两者测定值线性相关系数为0.925.结论 该方法反应时间短、特异性强、重复性好,具有应用及推广价值.