人類載脂蛋白，Apolipoprotein A-I，之單株抗體的製備及其特性研究

Abstract

冠狀動脈性心臟病是一種高致死率的心臟性疾病，動脈粥狀化(atherosclerosis)及由於血管硬化、管腔變窄而形成血栓 (thrombosis)，是造成冠狀動脈性心臟病最主要的原因。肝臟製造一類特別的脂蛋白（Lipoproteins），負責運送膽固醇。在眾多脂蛋白中，高密度脂蛋白（High Density Lipoproteins或HDLs）的功能是將細胞及血液中，多餘的脂肪及膽固醇送返肝臟代謝，避免膽固醇在血管壁堆積，引發心血管疾病產生。從高密度脂蛋白的功能，可推知心血管疾病生成與高密度脂蛋白濃度具有負相關性。載脂蛋白apoA-I是高密度脂蛋白的主要成分，約佔其中的70%。前人文獻報導中亦指出，位於高密度脂蛋白的載脂蛋白apoA-I為負責膽固醇運送之主要蛋白質，故測量載脂蛋白apoA-I含量，即可作為心血管疾病的指標。本研究成功製備三株載脂蛋白apoA-I的單株抗體，分別為2C7、2E10與3F8。利用酵素連結免疫分析法及西方免疫墨點法，可得知均對於載脂蛋白apoA-I具有很高之專一性。利用不同的化學方法（Acetylation 及Trypsin digestion）處理載脂蛋白apoA-I，發現載脂蛋白apoA-I上正電胺基酸是抗原與抗體連接的主要部位。由於載脂蛋白apoA-I結構中有被脂肪遮蔽的部分，故本研究欲利用單株抗體與抗原具有強親合力的特性，找出載脂蛋白apoA-I不被脂肪包裹的部位。且利用競爭型酵素連結免疫分析法發現單株抗體3F8，可以成功偵測載脂蛋白apoA-I的裸露部位，此3F8單株抗體具有與載脂蛋白apoA-I不被脂肪包裹的部位結合特性，可以應用於未來臨床分析。

Coronary artery disease (CAD) is a high mortality heart disorder causing by formation of atherosclerosis and thrombosis. High-density lipoproteins (HDL) in plasma are functioning as to remove the arterial cholesterol from the atheroscleroic lesions via reverse transport. An inverse relationship between the concentrations of HDL and the development of CAD is well established. Since apoA-I is a major protein moiety of HDL (~70%), we have previously demonstrated that apoA-I is a superior marker in patients with CAD. In the present study, monoclonal antibodies 2C7, 2E10 and 3F8 specific to apoA-I were prepared and characterized. Using immunoassay and Western blot analyses, we show that these antibodies recognized different antigenic domains of apoA-I. By chemical modification and limited trypsinization, we further demonstrated that the positively charged groups of apoA-I were involved in maintaining the epitope structure. Since lipid moiety in HDL substantially masks the apoA-I immunoreactivity, we attempted to identify a monoclonal antibody that may react with apoA-I without the interference by HDL lipids. Using the competitive enzyme linked immunosorbent assay (ELISA) we demonstrated that monoclonal antibody 3F8 could determine apoA-I in human plasma without the lipid interference. This 3F8 antibody will be therefore novel for the future clinical determinant of apoA-I.