Development of Diagnostic Platform for Human Cardiac Troponin I

• Main Authors: Tzu-HsunNi, 倪子訓
• Other Authors: Trai-Ming Yeh
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/639mqz

碩士 === 國立成功大學 === 醫學檢驗生物技術學系 === 104 === Development of Diagnostic Platforms for Human Cardiac Troponin I Tzu-Hsun Ni Trai-Ming Yeh College of Medicine Department of Medical Laboratory Science and Biotechnology SUMMARY Acute myocardial infarction (AMI) is a major leading cause of death and disability worldwide. In this study, we immunized the mice by human full length recombinant cardiac troponin I which was generated by our lab before. Using the technology of hybridoma to generate the anti-cTnI antibodies. We used one pairing of our monoclonal antibodies to establish our enzyme-linked immunosorbent assay (ELISA) system for AMI diagnosis and optimized the detection conditions to enhance the sensitivity. We established the stability of ELISA strips by batch production to confirm the stability and titer of our antibodies. We also developed the luminescence-based immunoassay to enhance the sensitivity of detection of cTnI. We hope cTnI can be detected in the clinics or even at home in the future; therefore, we tried to developed the lateral flow strips for point-of-care (POC). Key words: AMI, monoclonal antibody, enzyme-linked immunosorbent assay, troponin I INTRODUCTION AMI occurs when blood flow stops to part of the heart causing damage to the heart muscle. In Taiwan, cardiovascular disease is the second cause of death. The symptoms of AMI are chest pain, weakness, lightheadedness, dyspnea, diaphoresis, nausea, vomiting, and even sudden death. When the heart gets damage, it releases the cardiac troponin I (cTnI) which is a sensitive and specific biomarker for myocardial damage. Nowadays, the main diagnosis is using enzyme-linked immune-sorbent assay (ELISA) to detect this specific biomarker. Therefore, the sensitivity, specificity, affinity, and stability of the antibody to cTnI is very critical in this diagnosis. MATERIALS AND METHODS In the study, we used one pairing of our monoclonal antibodies (mAb) (3B5 as capture mAb and 3B as detection mAb) to detect human cTnI by ELISA system. We also set up the luminescence-based immunoassay to compare with the colorimetric ELISA system. And we developed the rapid test- lateral flow strip by using colloidal gold to shorten the time of AMI diagnosis. RESULTS AND DISCUSSION In our ELISA system, cTnI was measured with a limit of blank=0.0466 ng/mL, limit of detection=0.0862 ng/mL, and intra-CV in the test were all below 10%. We also found that the concentration of EDTA and serum matrix in the sample may affect the detection of cTnI in our ELISA system. Therefore, we used the new buffer- Buffer C to enhance the sensitivity of our ELISA system. In our assay, the sensitivity of our ELISA system with diluent Buffer C was much better than IVD-approval EIA kit (BioCheck). In our luminescence-based immunoassay, cTnI was measured with a limit of blank=0.06 ng/Ml, limit of detection=0.125 ng/Ml, and intra-CV in the test were between 9%-12%. We developed 2 different lateral flow strip, TABP-A and TABP-B. The performance of TABP-B was better than the commercial kit (Firstep). So far, the detection limit of cTnI-T-C was 0.5 ng/mL in serum sample and 5 ng/mL in plasma sample, and the detection limit of cTnI was only 10 ng/mL in both serum and plasma sample by TABP-B. CONCLUSION We have used our mAbs 3B5 as capture and 3B as detection to establish ELISA system, colorimetric ELISA system and luminescence-based immunoassay, for cTnI detection. In addition, the sensitivity of our ELISA system was enhanced when serum was used as our sample type and Buffer C as the diluent buffer. We also tried to develop lateral flow strip, TABP-B, as POC assay which can be used as early diagnosis for AMI patient.