关键词: 心肌肌钙蛋白I, 自身抗体, 微阵列蛋白芯片, 化学发光免疫分析法, 急性冠状动脉综合征

Abstract:

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> 0.1ng/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 50 μg/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 40 000-55 000. 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.

Key words: Cardiac troponin I, Autoantibody, Microarray protein chip, Chemiluminescence immunoassay, Acute coronary syndrome