心肌肌钙蛋白I-C复合物酶联免疫检测方法的建立

为了制备抗人心肌肌钙蛋白I(cTnI)单克隆抗体、建立检测人cTnI链酶亲和素-生物素双抗体夹心酶联免疫吸附测定(ELISA)方法,用基因工程表达的人cTnI免疫雌性BALB/c小鼠,以杂交瘤技术制备得到三株特异性抗人cTnI的单克隆抗体记为6G3,6A6,3G9.三株杂交瘤细胞中6G3和6A6为IgG1,3G9为IgG2b,腹水效价分别为4×10-5,4×10-5和10-5.亲和常数为5.0×109,6.4×109和3.8×109mol-1.Westernblotting结果表明6G3和6A6能够识别cTnI-C复合物.把6G3抗体生物素化并制备抗cTnI、cTnC和cTnI-C的大鼠多克隆抗体.以多抗作为固相捕捉抗体,生物素化6G3抗体作为检测抗体,建立检测cTnI的双抗体夹心ELISA方法并初步应用于临床病人标本检测.以cTnI-C复合物多抗捕捉检测方法具有良好的灵敏度、特异性和准确性,灵敏度为0.9ng/mL,较包被其他抗体捕捉方法提高1~2倍.批内变异系数为5.03%,回收率为94.56%.测定17例AMI病人和18例血清样本临床诊断结果符合率为100%.

Development of Enzyme Linked Immunosorbent Assay for Detection of Cardiac Troponin I-C Complex in the Diagnosis of Cardiac Damage

To prepare monoclonal antibody against human cardiac troponin I(cTnI),and establish a sandwich streptavidin-biotin enzyme-linked immunosorbent assay of cTnI for cardiac damage detection,BALB/c mice were immunized with recombinant cTnI expressed by gene-engineering and three monoclonal antibodies,6G3,6A6 and 3G9,were obtained by hybridoma technique.The isotypes of 6G3 and 6A6 were IgG1 while 3G9 was IgG2b isotype.Ascites titers were 10~6 and the affinity constants(K) were 5.010~9,6.410~9 and 3.810~9 mol~(-1) respectively.Western blotting analysis showed that three antibodies except 3G9 can react with cTnI-C complex.Then 6G3 was labeled with biotin and rat polyclonal antibodies against cTnIcTnC and cTnI-C were obtained.The sandwich ELISA was established using biotinylated-6G3 and cTnI-C polyclonal antibodies,cTnI-C polyclonal antibodies serving as solid phase antibody and biotinylated-6G3 as the detector antibody.This method was sensitive,specific and accurate and was applied in clinical patients for cTnI measurement.The sensitivity of this assay is 0.9 ng/mL.The average intra-coefficients of variation (CV) was 5.03% and the recovery rate of added cTnI was 94.56%.17 patients with AMI and 18 healthy human were detected by this method.The coincidence rate of the assays result with clinical diagnostic results was nearly 100%.These results suggest that this sandwich ELISA method is useful in the diagnosis of cardiac damage.