心肌肌钙蛋白Ⅰ(cTnⅠ)提纯及ELISA的建立

Development of enzyme-linked-immunosorbent assay for cardiac troponin Ⅰ(cTnⅠ)

目的心肌肌钙蛋白Ⅰ(cardiactroponinⅠ,cTnⅠ)的提纯及酶联免疫分析方法的建立。方法从猝死健康人心脏中,提取人心肌肌钙蛋白Ⅰ作为抗原,用其免疫新西兰雌性大耳白家兔,制备出抗人心肌肌钙蛋白Ⅰ抗体,采用夹心方法,建立cTnⅠ酶联免疫吸附分析法。结果从人心肌中提取出11.7mgcTnⅠ,心肌肌钙蛋白Ⅰ抗血清滴度达1∶100000,Ka=2.38×109Lm/ol。方法学指标:灵敏度为0.2ngm/l;精密度,批内、批间变异系数分别为CV=3.8%和CV=8.2%;准确度,平均回收率99.7%;特异性,与心肌肌钙蛋白T、心肌肌钙蛋白C、肌酸磷酸激酶(CK)、肌酸磷酸激酶同工酶(CK-MB)均无交叉反应。用该方法和进口试剂盒对32例急性心肌梗死(AMI)患者和40例健康人血中cTnⅠ水平进行检测,两者符合率分别为93.7%、95.0%。结论该方法适于临床常规检验及科研工作需要,具有广泛应用前景。

Objective The development of Enzyme-Linked-Immunosorbent Assay（ELISA） for cardiac Tropinin Ⅰ （cTn Ⅰ ）. Methods New Zealand rabbits were immunized with human cardiac Troponin I as immunogens. The antibody of cTn Ⅰ was prepared. An enzyme-linked- immunosorbent assay for cTn Ⅰ was established. Results 11.7 mg of cTn Ⅰ from human cardiac muscle was obtained, the tiler of antisera for cTn ⅠI was 1 ： 100 000, Ka = 2.38 × 10^9 L/tool. Methodoloical parameters： sensitivity for the minimum detectable amount was 0.2 ng/ml ;precision of the inter- and intra-assay coeffient of variation was 3.8 % and 8.2 % respectively;accuracy in recovery was 99.7 % ;specificity,the cross reactivity of the antibody towards cTnT,cTnC,CK, CK-Mli was not significant. The cTn Ⅰ level of 32 patients with acute myocardial infarction （AMI） and 40 normal donors was detected in sera by this method with the kit of PBM co USA. The coincidence rate in the assays for patients and healthy controls were 93.7 % and 95.0 % respectively. Condusion This assay approach is appropriate for routine clinical test and scientific research with broad application prospects.