- 學位論文
退化性膝關節炎尿液試片研製及臨床評估
Development and Evaluation of Urine Strips for Knee Osteoarthritis
摘要
退化性關節炎為是一種常見的關節退化性疾病，但依目前現有非侵入式的檢驗方式只有退化嚴重的關節軟骨才易檢測出。利用X光照射顯影或核磁造影 (MRI) 的方式，觀測關節軟骨間的空隙大小可以推定軟骨退化的程度。然而當以 X光照射顯影或核磁造影檢測關節空隙縮小至與正常人有明顯差異時，已是嚴重退化一至兩年的時間。因此 X光的檢測或核磁造影並無法即時偵測關節軟骨開始退化的情形。另外，目前除了以醫學影像診斷外，一般的預防性的檢測都需要侵入性的抽取組織液、關節液或血液，以 ELISA或 LC/MS/MS方式分析組織液中被分解軟骨的含量來診斷軟骨受損的情形。為改善侵入式的檢體採樣方式，且期望能在軟骨退化的早期即可快速篩選或診斷出軟骨退化的狀況，若可開發非侵入性的檢驗方式，除了可早期發現早期保養，延緩軟骨退化速度，相對亦可減少醫療資源的浪費。計畫的目標便是研發及試製免疫層析分析試紙，運用抗原-抗體結合之原理發展檢驗試紙來檢測受試者尿液中生物標誌。若是能設計以試紙檢驗的方式，可利用尿液檢體，與妊娠的檢測方式相同，快速的檢測出是否患有退化性關節炎，非常適合家居或臨床門診的快速檢驗。 本論文研究包含兩部分，第一部分為尿液試片的研發，第二部分為研發出之尿液試片做實際臨床評估。 第一部分研究尿液試片的各種最佳條件，在與膠體金接合的抗體最適濃度在60~70 μg/mL；而在試片偵測區最佳的抗體濃度為1.5 g；而膠體金-抗體複合物與抗原結合最佳的時間為 15分鐘。以此實驗條件對 70 位臨床退化性關節炎病患的尿液檢體檢測，可以達到 92.8% 的正確率，但若患者有換新關節，或退化性關節炎最嚴重的末期，則因為無軟骨組織所以分泌 COMP 蛋白極少，因此也無法呈色，或被檢測出。 第二部分以產品化之尿液試片選擇臨床不同嚴重程度之病患做實際測試，結果顯示，在所有71位共有50位由試紙檢測出，檢出率為70.4%。以退化性關節炎的嚴重度來分析其檢出率，病患中在退化性關節炎第一級之病患共有10位其試紙有檢測出4位，陽性率為40%，退化性關節炎第二級的病患共有30位，其試紙有檢測出25位，陽性率為83.3%，退化性關節炎第三級的病患共有16位，其試紙有檢測出12位，陽性率為75%，退化性關節炎第四級的病患共有6位，其試紙有檢測出3位，陽性率為50%，有接受人工關節置換手術的病患共有9位，其試紙有檢測出6位，陽性率為66.7%。此臨床結果仍需更多病人做分析驗證。
並列摘要
Osteoarthritis (OA) is one of the most commonly and widely occurring degenerative disorders of joints in various parts of our body. However, according the existing non-invasive examination protocols for OA, only the very severe disorders could be readily diagnostically confirmed. In other words, by examining X-ray radiograms or MRI findings for evaluating the gaps of articular cartilage in the joints, only in severe cases of OA, where the physician might be able to find obvious differences in the voids or contacts within the gaps between the healthy and symptomatic joints. In fact, the x-ray or MRI findings could not effectively provide information for the early detection of articular cartilage degeneration. In the present study, we have developed a diagnostic test strip, based on the concept of Amperometric Immunoassay methodology, to determine the concentration of patient’s urinal fluid which containing COMP biomarker for OA. We divided the study into two parts: first part for development of the urine strips, and second part for the application of the urine strips in clinics. The first part of the study is the preliminary results demonstrated that the optimal conditions to prepare the test strip were: 60-70 μg/ml of antibody to combine the nano-gold, the optimal concentration of antibody was 1.5 μg and the optimal reaction time was 15 minutes. These preliminary prepared test strips were with 92.8 % accuracy from 70 patients’ tests (From E-DA hospital). Also, we have established a clinical degenerative index for the OA knee patient from E-DA hospital and used this index as an OA reference for this present and further study. The second part is to use the developed urine strips for the different grade of knee OA to evaluate the clinical correlation with the result of urine strips. Although, the urine samples from total 71 patients with knee OA were collected from E-DA hospital. 50 samples (70.4%) have positive results after examination by test strips. We still need more number of patients to evaluate and identify the clinical application of the developed urine strips. To date, we have met the commitment of preparation and test of test strip for OA, however, the curvet preparation and test need more periods to complete.