以高效能分子篩層析法與酵素連結免疫分析法分析西洋參水溶性不可消化多醣之分子結構特徵

Abstract

單株抗體已普遍使用於研究植物細胞結構。本論文擬以酵素水解、陰離子樹脂層析、高效能分子篩層析法 (high performance size excluion chromatography, HPSEC) 與酵素連結免疫分析法 (enzyme-linked immunosorbent assay, ELISA) 分析西洋參水溶性不可消化多醣之分子結構特徵。將去除蛋白質的西洋參粗多醣，以酵素水解除去澱粉，得到水溶性不可消化多醣，以陰離子交換樹脂層析系統，依流洗鹽濃度分離為F1、F2、F3、及F4四個區分。將各區分以HPSEC分析，搭配ELISA assay進行分子結構快速辨識與化學分析確認，使用LM2、LM5、LM6、LM7、LM10、LM19、LM20與JIM7進行免疫親和反應。其中LM2及LM5被認為可代表arabinogalactan type II (AGII) 與 arabinogalactan type I (AGI) 結構。F1與F2多醣區分於鹽濃度為0–0.1M時出現，組成為arabinose與galactose，與LM5、LM6有親和反應，表示具有s-(1→4)-galactan及linear arabinan分支。F3、F4多醣區分於鹽濃度為0.1–018M出現，具有酸性糖galacturonic acid與glucuronic acid，此二區分除了對於LM2、LM5及LM6有親和反應外，以酸性糖為主要組成之F4對於LM19具有相當高的親和性，顯示此二區分具有partially methyl esterified HG以及RGI結構與AGI、AGII及linear arabinan等分支，此外四個區分對於LM10皆無親和反應，並不具有xylogalacturonan或xylan等結構。

This study used high performance size exclusion chromatography (HPSEC) and enzyme-linked immunosorbent assay (ELISA) to characterize the distribution of different epitopes within four fractions separated by DEAE chromatography of water soluble nondigestible polysaccharides. Water-soluble nondigestible polysaccharides were obtained from deproteinized crude polysaccharides subjected to starch hydrolyzing enzymes to remove the starch. DEAE chromatography can divide water-soluble nondigestible polysaccharides to four fractions (F1-F4) by different concentration of the eluent. Monoclonal antibodies have been widely used in studies of plant cell structures. The mAbs LM2, LM5, LM6, LM7, LM10, LM19, LM20 and JIM7 were used in this research. The LM2 and LM5 resemble the AGII and AGI structures. F1 and F2 were observed when the eluent salt concentration was 0 to 0.1M. They are composed of arabinose and galactose, and the Mw are 71 and 116 kDa, respectively. Binding of F1 and F2 to LM5 and LM6 indicated the presence of RGI material with both arabinan and the AGI side chains. On the otherhand, F3 and F4 were observed when the eluent salt concentration was 0.1 to 0.18M. In addition to the arabinose and galactose, there are acidic polysaccharides such as galacturonic acid and glucuronic acid for F3 and F4. Affinity to AGII (LM2), AGI (LM5), arabinan (LM6) and partially methyl esterified HG (LM19) were mainly observed in F3 and F4. It was also observed that F4 had a very high affinity to partially methyl esterified HG (LM19), indicating that it is mainly composed of an acid glycoprotein. Lastly, the four fractions had no observed immunoaffinity to LM10, indicating that there were no xylan or xylogalacturonan structures in the fractions.