篇名:
應用酵素結合免疫吸附分析(ELISA)方法檢測牛白血病Diagnosis of Bovine Leucosis by Enzyme-Linked Immunosorbent Assay
作者:
林敬覆;張惟茗;吳義興;蘇杰夫;蕭終融
中文摘要:
分別以一般含胎牛血清之組織培養生長液 (Hank’s MEM solution 90 % 及gentamycine 0.04 mg/mL, A) 及不含胎牛血清之代用添加完全組織培養生長液 (basal medium supplement solution及 gentamycine 0.04 mg/mL, B:係Biochrom KG 廠商業成品) 大量增殖持續感染牛白血病病毒 (bovine leukemia virus; BLV) 之 FLK 細胞,再將所收集之培養液,經離心,不活化處理及濃縮處理後,分別製得粗抗原 A-Ag 及 B-Ag。再應用上述粗抗原分別以瓊膠免疫擴散法 (agar gel immunodiffusion test; AGID) 測定牛隻白血病 (bovine leucosis, BL) 抗體,結果以 B-Ag 為抗原者較佳,二者分別為38.74 % (289/746)及39.41 % (294/746),接著將所製得之 B-Ag 粗抗原,以Sephacryl S-300層析所收集之各階收集液,進行免疫轉置 (immunoblot) 分析後,取其第一峰之收集液,以硫酸銨濃縮透析後,經光電比色計分析後,再將濃度調到 5 μg/mL,製成牛白血病 (BL) 抗體測定用之 BL antibody test ELISA-plate (BL-AbT- ELISA-plate),以其與 AGID 進行牛隻 BL 陽性牛抗體檢測結果,分別為 38.84% (294/757) 及 36.20% (274/757)。
英文摘要:
SUMMARY The FLK cells with bovine leucosis virus (BLV) persisting infection were cultured in Hank’s base growth solution (containing 10% fetal calf serum and 0.04 mg/mL gentamycine, used for preparing the A antigen) and in basal medium supplement growth solution (Biochrom KG, used for preparing the B antigen). The supernatants were harvested, inactivated, concentrated, and prepared for antigen production. Both the A (A-Ag) and B (B-Ag) antigens were used for the agar gel immunodiffusion (AGID) test and Enzyme-linked immunosorbent assay (ELISA). A total of 746 bovine sera collected from 21 prefectures were tested with the A-Ag and B-Ag in the AGID test. Results indicated that the positive rates were 38.74% (289/746) for the A antigen and 39.41% (294/746) for the B antigen. In ELISA, the coating antigen was prepared from B antigen fractionated with Sephacryl S-300. The ELISA-plates were coated with the purified B antigen for overnight at 4℃. Serum samples from 7 prefectures were assayed for anti-BLV antibodies with ELISA and AGID test. Results showed that positive rates were 38.84% (394/757) for ELISA and 36.2%274/757) for the AGID test.
備註:
年份:西元 2001 年
期別:第 37 期
詳細內容:
本病係為一種慢性病毒性傳染病,歷年來對養牛業者所造成之經濟損失實在無從估計, 早在 1987 年楊等調查台灣地區 31,518 頭乳牛血清之牛白血病 ( BL) 抗體,結果發現以瓊脂免疫擴散法 (AGID) 測定顯示其陽性反應率達17.31% (5,466/31,518) ﹝3﹞,可見本病已嚴重為害台灣地區乳牛,由於以 AGID 測定本病,其操作繁複且費時,為簡化操作程序,縮短其測定時間及增加其敏感度,以提升診斷品質及時效,同時藉以所測得之結果加以分析比較,以瞭解本病之流行病學現況,而進行本計畫,今就所使用之方法、材料及結果等,分述如下:
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