利用單株抗體於酵素連接免疫吸附分析、免疫細胞分佈和免疫親合性管柱以檢測及分離天然物活性成分   全文下載

ELISA, Western Blotting, Immunocytolocalization and Immunoaffinity Column for Naturally Occuring Bioactive Compounds Using Monoclonal Antibodies

抗番瀉?甲（sennoside A ；SA）及抗甘草甜素（glycyrrhizin ；GC）之單株抗體已經過選篩（screening）及選殖（cloning），並且應用於酵素連接免疫吸附分析（enzyme link immuno sorbent assay；ELISA）。檢測抗甘草甜素及人參皂?（ginsenoside）之新西方墨點法（new western blotting method）已建立。首先將甘草甜素和人參皂?以矽膠薄層層析板分離，再轉移到聚偏二氟乙烯（polyvinylidene difluoride ；PVDF）膜，隨後以碘酸鈉（NaIO4）溶液處理，再以牛血清蛋白（bovine serum albumin ；BSA）浸潤，結果PVDF膜就附上人參皂?-血清蛋白複合物之抗原，接下來的染色步驟與傳統西方墨點法相同。檢驗甘草屬（Glycyrrhizaspecies）植物新鮮根部中甘草甜素在細胞內之分配情形，可先將甘草甜素以上述方法轉移到聚偏二氟乙烯膜，再以抗甘草甜素之單株抗體偵測。應用免疫親合性管柱（immunoaffinity column）接上人參皂?Rbl（GRb1）之單株抗體來分離人參皂?Rbl ，遠優於先前所發表由人參粗萃取物分離Rb1的方法。另一方面，solasodine glycosides的總含量測定，可藉由免疫親合性管柱接上anti-solamargine（SM）之單株抗體，經廣範性交叉反應來分離。

Anti-sennoside A (SA) and anti-glycyrrhizin (GC) MAbs were used for screening and cloning. And they were also used for ELISA. New western blotting method of determination for GC and ginsenoside was established that compounds separated by silica gel TLC were transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was treated with NaIO4 solution followed by BSA, resulting in a ginsenoside-BSA conjugate on the PVDF membrane which was stained by the general western blotting. Immunocytolocalization of GC in the fresh root of Glycyrrhiza species was conducted using anti-GC MAb after blotting to PVDF membrane. Immunoaffinity column chromatography using anti-ginsenoside Rb1 (GRb1)MAb performs better than previously published separation methods resulting in pure GRb1 from the crude extracts of ginseng. On the other hand, total solasodine glycosides were separated by an immunoaffinity column using a wide-cross reactive anti-solamargine (SM)MAb. 抗番瀉苷甲（sennoside A ；SA）及抗甘草甜素（glycyrrhizin ；GC）之單株抗體已經過選篩（screening）及選殖（cloning），並且應用於酵素連接免疫吸附分析（enzyme link immuno sorbent assay；ELISA）。檢測抗甘草甜素及人參皂苷（ginsenoside）之新西方墨點法（new western blotting method）已建立。首先將甘草甜素和人參皂苷以矽膠薄層層析板分離，再轉移到聚偏二氟乙烯（polyvinylidene difluoride ；PVDF）膜，隨後以碘酸鈉（NaIO4）溶液處理，再以牛血清蛋白（bovine serum albumin ；BSA）浸潤，結果PVDF膜就附上人參皂苷-血清蛋白複合物之抗原，接下來的染色步驟與傳統西方墨點法相同。檢驗甘草屬（Glycyrrhizaspecies）植物新鮮根部中甘草甜素在細胞內之分配情形，可先將甘草甜素以上述方法轉移到聚偏二氟乙烯膜，再以抗甘草甜素之單株抗體偵測。應用免疫親合性管柱（immunoaffinity column）接上人參皂苷Rbl（GRb1）之單株抗體來分離人參皂苷Rbl ，遠優於先前所發表由人參粗萃取物分離Rb1的方法。另一方面，solasodine glycosides的總含量測定，可藉由免疫親合性管柱接上anti-solamargine（SM）之單株抗體，經廣範性交叉反應來分離。