- 學位論文
膝關節軟骨退化檢測用之尿液試片量產與臨床評估
Mass Production and Evaluation of Urine Strip for Osteoarthritis
摘要
臨床上可藉由X光片上關節軟骨間的空隙及是否有骨刺增生來推斷關節軟骨退化的程度。X光片診斷雖為最主要、唯一的非侵入式的診斷方式，但仍有輻射線曝露的風險，且於初期退化症狀診斷效果上卻不甚理想明顯。若能設計非侵入式且快速檢測關節軟骨的退化試片用於早期篩檢與監測軟骨退化狀況,將可大幅減少醫療診治的費用。在本論文前期部份進行免疫層析尿液試片研發與量產試製評估，探討pH 值、緩衝溶液、膠體金及多株抗體濃度對膠體金嫁接抗體的效果。研究中測試膠體金與抗體嫁接條件膠體金 OD520 = 1〜3、pH 7.5 〜9.0 、硼酸及磷酸緩衝溶液、抗體濃度 0.05〜1 mg/mL等條件。初步結果顯示膠體金 OD520 = 2 、 pH 7.5 、採用硼酸緩衝溶液及多株抗體濃度為0.8 mg/mL條件下嫁接抗體之穩定性好，再現性也較佳。使用Sartorius N95 15m的NC membrane之孔徑不會因非特異性吸附或反應時間不足而影響結果。單株抗體以濃度為1.5 mg/mL之檢測效果最佳。本論文後期則著重於將所製備的退化軟骨尿液試片進行臨床評估，並搭配X光及IKDC膝部評估表來對110位膝關節退化患者（經由X光檢查判定為II級以上之退化）進行評估，期能將退化軟骨尿液試片作為早期診斷與療效監控的工具。檢測結果顯示研究中所製備的免疫層析尿液試紙正確率可達86.0 %。IKDC問卷測試結果，正常人（54位）之問卷平均分數為85.7±14.2分，110位膝關節退化患者則降低為51.1±18.2分，且在IKDC問卷總分上二者達到顯著性的差異，顯示IKDC問卷總分能對關節軟骨進行初步的篩檢。本論文亦嘗試利用IKDC問卷總分結合尿液試片來評估關節軟骨之退化與功能，將IKDC問卷總分設定在70分，則尿液試片的敏感性為79.1 %、特異性為83.3%，正確率可達80.5%；若將IKDC問卷總分設定在80分，則尿液試片的敏感性為94.6 %、特異性則降低為68.5%，正確率可高達86.0%。由以上結果顯示：選定受測者主觀的IKDC問卷評分及結合免疫層析尿液試片檢測尿液是可提供一種有別於X光對退化性關節炎篩檢及治療之監測工具。
並列摘要
Clinically, clinicians can diagnose knee Osteoarthritis (OA) according to the degree of degeneration of articular cartilage by examining its surface and the joint space and by detecting hyperplasia or deformation of the knee on X-ray radiographs. Although X-ray radiography is the major and the only non-invasive diagnostic method, the risk of radiation exposure still exists. In addition, the outcome of the diagnosis regarding the symptoms of early degeneration is neither unambiguous nor obvious. Instead of using the abovementioned method, the study is the first trial to develop a urine strip that able to detect the presence of the marker of articular cartilage degradation, COMP (cartilage oligomeric matrix protein) in the subjects’ urine samples. This strip provides an early diagnosis of OA and effectively decreases the expense of medication on OA. There are two parts included in this thesis, the first one including the development and mass production criteria of urine strip, including pH value, buffer solution, gold colloid and multi-clonal antibody selections. The preliminary results demonstrated that the followings are the optimal production conditions for urine strip: OD520=2, pH 7.5 boric acid buffer solution, multi-clonal antibody concentration: 0.8 mg/mL, monoclonal antibody concentration: 1.5 mg/mL and using NC membrane. The second parts of this thesis are focused on, combining the prepared urine strip with x-ray examination and IKDC knee evaluation questionnaire, to evaluate the efficacy of this strip as an early diagnosis or monitoring tool for OA from the 110 OA patients (1 grade OA or worse) in E-DA hospital. The preliminary results indicated that this strip exhibited 86 % of accuracy from 110 urea tests. The IKDC scores 85.7±14.2 from 54 normal subjects, however, the IKDC scores 51.1±18.2 from 110 OA patients. These scores demonstrated statistical difference. In addition, we attempted to use this IKDC scores combing with urine strip to evaluate the degeneration and function of cartilage: 1. Setting the IKDC scores as OA threshold at 70, we can demonstrate that the sensitivity of urine strip is 79.1 %, specificity is 83.3 % and accuracy is 80.5 %. 2. Setting the IKDC scores as OA threshold at 80, we can demonstrate that the sensitivity of urine strip is 94.6 %, specificity is 68.5 % and accuracy is 86%. The results revealed that the urine strip together with the IKDC could be used to screen the OA and as an early diagnosis tool for OA, beyond the traditional X-ray examination.