自建微阵列化学发光免疫分析法定量检测心肌肌钙蛋白I自身抗体及其初步临床应用

Microarray chemiluminescence immunoassay for quantitative detection of cardiac troponin I autoantibodies and preliminary clinical applications

目的基于微阵列蛋白质芯片技术建立定量检测心肌肌钙蛋白I自身抗体(cTnIAAb)的微阵列化学发光免疫分析法,并评价该方法的检测性能。方法以心肌肌钙蛋白I(cTnI)-C融合蛋白为检测抗原,对抗原点样浓度、封闭液、封闭时间和二抗进行筛选,以检测结果信噪比最大为标准优化条件。以100名健康体检者(正常对照组)cTnIAAb检测灰度值的第95百分位数为临界值,建立cTnIAAb检测的标准曲线,并评价微阵列化学发光免疫分析法的检测性能(稳定性、精密度、最低检测限、线性范围等)。采用微阵列化学发光免疫分析法检测500例血清cTnI>0.1 ng/mL的急性冠状动脉综合征(ACS)患者(ACS组)的血清cTnIAAb,采用免疫印迹法验证其特异性。结果最优实验条件:抗原点样浓度为50μg/mL,封闭液为2%人乳,最佳封闭时间为12 h,二抗为辣根过氧化物酶(HRP)-羊抗人IgG抗体。微阵列化学发光免疫分析法检测高、低值cTnIAAb的精密度良好,变异系数(CV)均<15%;空白限(Lo B)、检测限(LoD)分别为0.12、0.23 AU/mL;线性范围为0.23~815.00 AU/mL。免疫印迹法结果显示,cTnIAAb阳性样本和阳性对照在相对分子质量40000~55000处均出现特异性条带。ACS组血清cTnIAAb阳性率为8.4%(42/500),明显高于正常对照组[2%(2/100)](χ^(2)=5.023,P<0.05);但血清cTnIAAb浓度2个组之间差异无统计学意义(P>0.05)。Spearman相关性分析结果显示,ACS组cTnIAAb与cTnI无相关性(r^(2)=0.1,P>0.05)。结论自建的定量检测cTnIAAb的微阵列化学发光免疫分析法具有良好的检测性能,可为cTnIAAb的临床应用提供较好的方法学基础。

Objective To establish a microarray chemiluminescence immunoassay for the quantitative detection of cardiac troponin I autoantibody(cTnIAAb)based on microarray protein chip technology,and to evaluate the detection performance of this method.Methods The cardiac troponin I(cTnI)-C fusion protein was used as the detection antigen.The antigen spotting concentration,blocking solution,blocking time and secondary antibody were screened.The maximum signal-to-noise ratio of the detection results was taken as optimum condition.Taking the 95 th percentile of cTnIAAb gray value of 100 healthy subjects as the critical value,the standard curve of cTnIAAb detection was established.The detection performance(stability,precision,minimum detection limit,linear range,etc.)of microarray chemiluminescence immunoassay was evaluated.This method was used to detect cTnIAAb in 500 acute coronary syndrome(ACS)samples with serum cTnI>0.1 ng/mL.Western blotting was used to verify the specificity of microarray chemiluminescence immunoassay for cTnIAAb detection.Results The optimal experimental conditions were as follows:the concentration of antigen spot sample was 50μg/mL,the blocking solution was 2%human milk,the optimal blocking time was 12 h,and the secondary antibody was horseradish peroxidase(HRP)-sheep anti-human IgG antibody.The precision of microarray chemiluminescence immunoassay for detecting high and low values of cTnIAb was good,with CV<15%.The limits of blank(LoB)and detection(LoD)were 0.12 and 0.23 AU/mL,respectively.The linear range was 0.23-815.00 AU/mL.Western blotting showed that specific bands were found in both cTnIAAb positive samples and positive controls with relative molecular weights of 40000-55000.The positive rate of cTnIAb in ACS group was 8.4%(42/500),which was significantly higher than 2%(2/100)in normal control group(χ^(2)=5.023,P<0.05).However,there was no statistical significance in serum cTnIAAb concentration between the 2 groups(P>0.05).Spearman correlation analysis showed that there was no correlation between cTnIAAb and cTnI in ACS group(r^(2)=0.1,P>0.05).Conclusions The self-developed microarray chemiluminescence immunoassay has a good performance for quantitative detection of cTnIAAb,which can provide a good methodological basis for clinical application of cTnIAAb.