根據統計，心肌梗塞在全世界是死亡率最高的疾病，在台灣則是全國第二高。心肌梗塞的發生是由於供應心臟氧氣及養份的血管受到阻塞，使得血液沒有辦法流過，造成心肌細胞受損。當心臟受損時，會釋放出心肌旋轉蛋白-I (cTnI)，是一種對心肌細胞受損時，具有非常優越的特異性和靈敏性的生物標記。現今檢測cTnI大多使用酵素免疫分析法(enzyme-linked immunosorbent assay, ELISA)，因此對於檢測cTnI的抗體其靈敏性、特異性和穩定性顯得非常重要。至今已有許多廠商發展出檢測試劑套組或是cTnI的抗體，而對心肌梗塞的病患而言，早期診斷並把握治療的黃金時間是相當重要的。在本篇研究中，由先前實驗室所製作出來的人類全長cTnI重組蛋白(human full-length rcTnI)來免疫小鼠，利用融合瘤技術來生產cTnI抗體，並經過篩選及配對，得到一對的具高靈敏性和特異性的單株抗體，3B5和3B分別作為捕捉抗體及偵測抗體，並發展cTnI的檢測平台，建立我們的ELISA系統，目前對於cTnI的檢測，最低空白極限為0.0466 ng/mL，最低偵測極限為0.0862 ng/mL，10%變異係數在我們的測試中也都在10%以下。建立好的系統，經過不同條件的優化測試，我們嘗試把此系統進行批量製造、產品化，模擬市售的檢測試劑套組，並且確認我們抗體的穩定性和效價。同時，我們也建立冷光的ELISA系統來做比較，目的是希望可以增加檢測的靈敏性。在冷光ELISA系統，目前cTnI的最低空白極限為0.06 ng/mL，最低偵測極限為0.125 ng/mL，而10%變異係數在測試中則介於9%-12%之間。除此之外，我們嘗試發展更快速、方便的檢測方式，將cTnI的診斷建立在point-of-care (POC)的概念，所以利用膠體金與抗體鍵結，建立免疫層析試紙(lateral flow strip)，目前在這方面，cTnI-T-C在血清及血漿檢體中分別可以偵測到0.5 ng/mL及5ng/mL，cTnI在兩種檢體中能偵測到的濃度皆為10 ng/mL。
關鍵詞：心肌梗塞、單株抗體、酵素免疫分析法、心肌旋轉蛋白I

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.

目錄
中文摘要...........................................................................................I
英文延伸摘要English Extended Abstract........................................III
致謝................................................................................................V
目錄...............................................................................................VI
表目錄.............................................................................................X
圖目錄............................................................................................XI
縮寫指引.......................................................................................XII
1. 緒論.............................................................................................1
1.1 心肌梗塞..................................................................................1
1.1.1 心肌梗塞現況.....................................................................1
1.1.2 心肌梗塞臨床症狀..............................................................1
1.1.3 心肌梗塞病理特徵..............................................................2
1.1.4 心肌梗塞診斷方式..............................................................3
1.2 生物標記.................................................................................3
1.2.1 心肌梗塞常見之生物標記...................................................4
1.2.2 心肌旋轉蛋白....................................................................5
1.2.3 心肌旋轉蛋白I於臨床診斷上的價值與評估..........................6
1.3 心肌旋轉蛋白I的偵測方法..............................…................……...6
1.3.1 酵素免疫分析法.................................................................7
1.3.2 化學冷光免疫分析法..........................................................7
1.3.3 螢光免疫分析法................................................................8
1.3.4 免疫層析試紙....................................................................8
2. 實驗動機與實驗目標...................................................................10
3. 實驗方法與材料..........................................................................12
3.1 表現cardiac troponin I (cTnI)重組蛋白....................................12
3.1.1重組蛋白的表現及純化......................................................12
3.1.2 SDS-PAGE分析重組蛋白.................................................13
3.1.3 西方墨點法分析重組蛋白.................................................13
3.2 單株抗體配對........................................................................14
3.3 抗體辨識之抗原決定位...........................................................15
3.3.1 噬菌體效價測試...............................................................15
3.3.2 噬菌體胜肽庫之結合篩選.................................................15
3.3.3 噬菌體結合能力測試........................................................16
3.3.4 噬菌體單股DNA萃取.......................................................17
3.4 抗體最低空白極限limit of blank (LoB)、最低偵測極限limit of detection (LoD)、10%變異係數值coefficient variation (CV)分析....18
3.5 臨床檢體測試與市售EIA檢測試劑套組平行比較.......................19
3.6 決定ELISA系統檢測檢體類型.................................................20
3.7 檢體血清含量濃度影響測試....................................................20
3.8 EDTA與EGTA含量濃度對檢體檢測的影響................................21
3.9 優化ELISA系統對血清檢體偵測的條件....................................21
3.10 實驗室ELISA系統與市售EIA檢測試劑套組比較......................22
3.11 抗體穩定性測試....................................................................23
3.12 建立新的ELISA系統以提升對cTnI-T-C檢測之靈敏度..............24
3.13 冷光ELISA系統之抗體LoB、LoD、10% CV分析....................24
3.14 優化冷光ELISA系統對血清檢體偵測的條件...........................25
3.15 免疫層析試紙檢測系統建立與測試並且和市售套組比較..........26
4. 結果..........................................................................................27
4.1 重組蛋白之分析.....................................................................27
4.2 單株抗體配對........................................................................27
4.3 抗體辨識之抗原決定位..........................................................28
4.4 抗體LoB、LoD、10% CV分析................................................28
4.5 臨床檢體測試與市售EIA檢測試劑套組平行比較.......................28
4.6 決定ELISA系統檢測檢體類型.................................................29
4.7 檢體血清含量濃度影響測試....................................................29
4.8 EDTA與EGTA含量濃度對檢體檢測的影響...............................29
4.9 優化ELISA系統對血清檢體偵測的條件....................................30
4.10 實驗室ELISA系統與市售EIA檢測試劑套組比較......................30
4.11 抗體穩定性測試...................................................................30
4.12 建立新的ELISA系統以提升對cTnI-T-C檢測之靈敏度..............31
4.13 冷光ELISA系統之抗體LoB、LoD、10% CV分析....................31
4.14 優化冷光ELISA系統對血清檢體偵測的條件............................31
4.15 免疫層析試紙檢測系統建立與測試並且和市售套組比較..........32
5. 討論..........................................................................................33
5.1 抗體辨識抗原決定位..............................................................33
5.2 血清以及EDTA對cTnI-T-C檢測的影響....................................33
5.3 肝素(heparin)對cTnI檢測的影響…………………….........…………...34
5.4 免疫層析試紙於檢測AMI的價值與改善...................................34
5.5 cTnI與cTnT檢測上的不同與比較……………......……………………….35
5.6 cTnI與hs-TnI的不同…………………………………………………………...36
5.7 冷光ELISA系統理論與實際實驗結果的差異…………………….......36
5.8 其他的生物標記………………………………………………………………….37
6. 結論..........................................................................................38
7. 參考文獻....................................................................................39
8. 表附錄.......................................................................................46
9. 圖附錄.......................................................................................52
附錄一、試劑配方..........................................................................68
附錄二、試劑及實驗耗材和抗體......................................................71
附錄三、儀器.................................................................................74

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