利用雷射激發螢光免疫毛細管電泳法分析心肌旋光蛋白在溶液中之含量

Using laser-induce fluorescence immune-capillary electrophoresis to analyze cardiac troponin in solution.

董國華(Kuo-Hua Tung)

指導教授 ： 劉正民 博士

臺北醫學大學/醫學院/醫學檢驗生物技術學研究所/碩士(2003年)

摘要

本文利用毛細管電泳儀分析溶液中心肌旋光蛋白之含量分析方法的建立，以提供心肌梗塞在臨床診斷上的檢測。心肌旋光蛋白為目前診斷心肌梗塞最精確的指標。當心肌受損時，它在三小時即出現在血液中，持續升高在24到36小時後到達最高點，然後慢慢下降，但仍繼續較高的濃度一直到10至12天。本實驗首先以純化的抗原，心肌旋光蛋白注入兔子的皮下以產生心肌旋光蛋白多株抗體，之後將標準品之抗原及抗體結連上螢光染劑，並以雷射激發螢光方式，經由兩種免疫分析法：一、毛細管非競爭型免疫法（直接免疫分析），觀察波峰的螢光免疫復合體產生面積之改變，與加入抗原之量繪製成正比之濃度曲線圖，而為了使抗體與抗原抗體複合物在電泳圖的時間點能分開，所以使用琥珀酸化處理，使抗體的波峰移動時間變慢。經實驗結果顯示複合物形成的波峰與加入抗原量成正比，此一方法之靈敏度約為800μg/ml，未能達到實用之標準。二、毛細管競爭性免疫法，由螢光標籤之抗原與檢體抗原同時競爭一定量之抗體結合形成之波峰，觀察波峰面積之變化，而繪製成反比之濃度曲線圖。其中使用三種抗體，多株抗體、經琥珀酸化之多株抗體以及單株心肌旋光蛋白I抗體進行競爭性免疫，經實驗結果顯示，三種抗體與抗原所形成複合物的波峰，與加入抗原的量同時競爭抗體，是成反比趨勢。將兩種免疫法結果比較發現，競爭性免疫法的靈敏度較非競爭性免疫法高，可以偵測20μg/ml之含量。此一方法之建立有助於未來應用到心肌梗塞病人的診斷。

關鍵字

心肌旋光蛋白 ； 毛細管電泳 ； 雷射激發螢光法 ； 競爭性免疫測定法 ； 非競爭性免疫測定法

並列摘要

In this study, the quantitative analysis of troponin by laser-induced fluorescence capillary electrophoresis (LIF-CE) has been demonstrated. Troponin is the most important indicator for the diagnosis of myocardial infarction. Serum troponin rises about 3 hours after myocardial damage and reaching to plateau in 24 to 36 hours and keep on a relative high level for 10 to 12 days. So, the serum troponin analysis is an accurate and very useful method for the diagnosis of myocardial infarction. The homemade antibody was used in this experiment which was produced by immunization of a New Zealand rabbit from a standardized method. Both purified troponin (antigen) or anti-troponin antibody were labeled with a cyanine fluorescence (Cy5) mediated by biotin-avidin system. The LIF-CE immunoassay was performed by two modes; the first mode was non-competitive LIF-CE. In this mode, the formation of Ag-Ab complex was monitored and the correlation between complex formation and amount of Ag added could be calculated from the peak area in the resultant electropherograms. The sensitivity for the troponin detection is 800 mg which is far beyond the practical usage. The second mode was competitive LIF-CE, the fluorescence was labeled on antigen. During the assay, the labeled antigen and antigen to be assessed were incubated with a fixed amount of antibody. The antibody was either with or without succinylation were evaluated for comparing the separation between antigen and complex peaks. The resultant electropherograms showed that when succinated antibody was used, the sensitivity can reach to 20 mg/lml, which could be used for the practical usage.

並列關鍵字

Troponin ； Capillary electrophoresis ； Laser-induced fluorescence ； Competitive immunoassay ； Non-competitive immunoassay

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