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標題: 應用競爭性酵素接合免疫吸附法檢測雞隻家禽里奧病毒抗體
Application on competitive enzyme-linked immunosorbent assay for detecting antibodies to avian reovirus
作者: 林雅玲
Lin, Ya-Ling
關鍵字: avian reovirus;家禽里奧病毒;competitive ELISA;antibody detection;競爭性酵素接合免疫吸附法;抗體檢測
出版社: 獸醫學系
為了探討在不同家禽里奧病毒之免疫計畫及感染狀況下雞隻體內抗體出現情形,本實驗配合家禽里奧病毒病毒蛋白(結構蛋白σA,非結構蛋白μNS及σNS)之單源抗體建立一競爭性酵素接合免疫吸附法來偵測之。以雞纖維胚芽母細胞攻毒後之細胞萃取液當作塗鍍抗原,利用棋盤格稀釋法找出抗原、五株病毒蛋白的單源抗體(MAb(23)、MAb(81)、MAb(7)、MAb(H1E1)及MAb(4B9))及待測血清之最佳稀釋濃度後,分別檢測雞隻在不同免疫狀況及不同路徑感染病毒之抗體產生情形,同時也檢測這些血清之中和抗體。酵素接合免疫吸附法吸光值(O.D.)換算成抑制百分比(percentage inhibition;PI),顯示在給予雞隻死毒疫苗後約2週,抗體的抑制百分比可上升40-60%,反之活毒疫苗或強毒感染則上升不明顯,通常小於10%。某些雞隻無法維持長時間的高抗體力價,顯示個體間有差異存在。而就不同病毒蛋白抗體而論,血清之結構性蛋白抗體抑制百分比可上升40-60%,非結構蛋白則為10-50%。至於不同蛋白抗體的出現似乎沒有時間上的差異。中和抗體的情形則是先給予活毒疫苗再補強死毒疫苗者上升情況較佳,而若與競爭性酵素接合免疫吸附法比較,則於第3週才開始測得中和抗體上升。另外以競爭性酵素接合免疫吸附法檢測野外雞隻血清抗體,所得結果與商品化酵素接合免疫吸附法檢測所得之力價有所差異,以後者檢測較易有高力價的結果。因此,此次建立之競爭性酵素接合免疫吸附法確實可偵測雞隻血清中家禽里奧病毒抗體的存在,且敏感性較病毒中和抗體試驗佳,是研究家禽里奧病毒在免疫學上的一項工具;而與商品化酵素接合免疫吸附法套組的不同,在於可檢測出單一種病毒蛋白的抗體。

A competitive enzyme-linked immunosorbent assay (c-ELISA) was developed for detection of avian reovirus (ARV) antibodies. Cell lysates of chicken embryo fibroblast (CEF) infected with ARV (S1133 strain) was used as the coating antigen, and ARV protein-specific monoclonal antibodies (MAb(23), MAb(81), MAb(7), MAb(H1E1) and MAb(4B9)) as competitive factors in the c-ELISA. Sera of several groups of SPF chickens with different vaccination programs or infected with S1133 in different routes were collected and antibody against ARV proteins was analyzed by c-ELISA and virus neutralization test. The OD value of c-ELISA was transformed to percentage inhibition (PI), and the results showed that the PI of antibodies raised up to 40-60 % in 2 weeks postinoculation with inactivated vaccine. Instead, when chickens were inoculated with attenuated live vaccine or infected once with S1133, the PI of antibodies had no significant raise (< 10%). The PI of antibodies against structural proteins raised to 40-60%, and that against nonstructural proteins raised to 10-50%. Besides, antibodies against different viral proteins seemed to appear at the same time. In general, the antibody titers were variable, and some obvious positive responses were of short duration. In virus neutralization test, chickens primed with attenuated and boosted with inactivated vaccine induced more neutralizing antibody production. Compared with c-ELISA titers, the neutralizing antibody titers started to raise in 3 weeks postinoculation and seemed to have lower sensitivity. Using c-ELISA to analyse antibodies of field samples, the results were different from those tested by commercial ELISA kit. However, this c-ELISA was sensitive in detecting ARV-specific antibodies in chicken sera, and was more advantageous than virus neutralization test. The c-ELISA would be an efficient test for ARV serological analysis.
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