201132980 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種抗禽流感病毒血球凝集蛋白質之單株抗體 及其製備方法與應用，尤其係關於-種抗禽流感ΗόΝ1亞型病毒 血球凝集蛋自質之單株抗體，製倾單株錄之齡瘤細胞株， 以及應用該單株抗體之檢測套組與方法。 【先前技術】 • 家禽流行性感W(avian influenza，ΑΙ)，是由正黏液病毒科 (0▲零0咖如）之A型流行性感冒病毒感染所引起。A型流 行性感冒病毒表面有棒狀之企球凝集蛋白質（Hemagglutinin; 及簟狀之神經胺酸酶（Neuraminidase;ΝΑ)兩種醣蛋白質，依各血 球凝集蛋白質與神經胺酸酶之抗原性的不同，可分成許多免疫學 上的血清亞型（subtype)，目前共有16種血球凝集蛋白質抗原亞 型 H1 〜H16 (Fouchier et al” J Virol 79(5): 2814-22, 2005)與 9 種 φ 神經胺酸酶抗原亞型 Ν1 〜Ν9 (Webster and Laver, Virology 69(2): 511-22, 1976)。 此外’依據家禽流行性感冒病毒的病原性可分為高病原性及 低病原性’強毒型之高病原性家禽流行性感冒病毒（Highly pathogenic avian influenza virus, HPAIV)主要為 H5 及 H7 亞型，其 可引起雞隻高死亡率的全身性疾病’甚至可能感染人類導致死亡 (Lamb and Krug, Fieids virology 1487-1531, 2001; OIE，2008)。其餘 致病性較弱的稱為低病原性家禽流行性感冒病毒 (Low pathogenic 201132980 avian influenza virus，LPAIV)，此類病毒係廣泛存在於各種禽類。 ^ 依據目前田野調查，屬於低病原性家禽流行性感冒病毒（lpaiv) 之H6N1亞型禽流感病毒普遍存在於台灣本土家禽中（林仕鉦 行政院農委會家畜衛生試驗所禽流感資訊，2〇〇4;謝快樂等人」 灣省畜牧獸醫學會會報59: 45-55, 1992;鄭明珠等人，家畜衛試 所研報42: 43_5〇, 2〇〇7)。刪1過去數十年㈤，在亞洲幾乎成為 地方性的禽類流行病之-。臺灣在W2年第一次爆發h舰亞 φ 型家禽流行性感冒病毒’對於禽類有極高的致死率。卿年開始 有計畫的監控台灣的水禽和家禽疫情，在最近的 蛋雞與土雞有超過50%呈現刪丨血清陽性（揚平鱗人，X台 灣獸醫結、32:24-29,2006)，而且更有愈來越乡H6亞型病毒株被 分離出來。 腦1軸屬於低病雜，當此病毒於家禽咖形成潛伏性 感染’可能引起失重、發育不良、產蛋量下降及容易感染其他疾 #病等情況’而造成家禽養殖業者的經濟損失，更甚者，若病毒長 時間存在-群體中’將有可能會突變成高病原性家禽流行^冒 病毒（HPAIV)，或形成全新的禽流感病毒，感染無抵抗力的族群， 進而造成全球性大流行，所以建立使用便利、準禮並可以快速量 產的鑑定H6亞型家禽流行性感冒病毒檢測方式重要性不容束 視。 ’ 現今家禽流行性感胃病毒檢測方法包含以反轉錄聚合酶鍵鎖 反應及和㈣定量反轉躲合酶連鎖反應直接_病毒蛋白質或 4 201132980 核由病毒分離檢測抗原後藉由比對流感病毒樣本對HA的能 力而初步評估病毒的效價以及利用專一性抗體區分病毒血清型例 如血球凝集抑制試驗（hemagglutination inhibition test, HI teat)、神 經胺酸酶抑制試驗（neuraminidase inhibition test, NI test)與酵素 連結免疫吸附法（enzyme-linked immunosorbent assay, ELISA)等 方法。其中，又以抗體偵測禽流感病毒的方法具有簡便、快速的 優點，然而此方法之準確與靈敏度係取決於抗體是否辨認重要抗 • 原決定位（epit0Pe)及其良好的專一性，因此，多樣性（diversity) 且專一的抗體於此方法中扮演著重要的角色。 根據研究’自然界中具感染力的禽流感病毒，每一個似次 早兀大多都包含3-5個醣基化位置（Schulze，】Infcct Dis 176.S24-8，.1997)，這些具有活性的私大部份表面都覆蓋著醋類 (glycan) ’而數量、大小與翻取決於宿主的差異，位置則 由病毒基因的核酸序列決定。再者因流感病毒是驗病毒，這 ♦些釀基化位置也跟著突變而頻繁的發生改變，因而產生的多樣 性’推測是病毒對抗宿主中和抗體與各種抗病毒機制的策略。此 外，巨大的醣類會遮蔽部份HA表面，而影響其生理及免疫特 性’可能使得宿主的免疫系統無法辨識病毒，故研究指出，在流 感疫苗開發時必須要考慮到HA表面_的影響（⑽贫‘ s 1〇6(43). 18丨37_42, 2_)。除了開發疫苗外檢測用單株抗 體辨識的位置亦會受到醋類的影響，因此必須筛選避開酷類遮蔽 的位置或直接辨識特殊醋類的單株抗體。 201132980 綜上所述，由於家禽流行性感w病毒亞型眾多，故可快速且 有舰分各種不同亞到差祕為防疫制之騎，台灣家禽流 行性感胃H6N1血清普赫在於台灣本土家財且會造成 嚴重的崎減，細目前縣有對抗禽域H_亞型病毒之 專一性單株抗體或用於檢測該病毒之診斷套組，因此，為降低禽 流感所造成的禽畜病害以及驗未來新抛毒麟人類健康，抗 禽流感臓1亞型病毒之專一性單株抗體的開發係為當務之急。 φ 【發明内容】 為快速、準確檢測禽流感H6N1亞型病毒之存在，並有效區 分出H6N1亞型病毒與其他各種不同禽流感亞型病毒間的差 異，本發明之一目的為提供一種融合瘤細胞株，其可產生抗禽流 感H6N1亞型病毒之血球凝集蛋白質的單株抗體，且該單株抗體 對H6N1亞型病毒以外之至少一種其他禽流感病毒亞型（例如 H5N2亞型）之血球凝集蛋白質不產生交叉反應，進一步而言，該 • 融合瘤細胞株係命名為EB2，且於中華民國食品工業發展研 究所之寄存編號為BCRC 960409。 本發明之另一目的為提供一種具有抗原結合特性的單株 抗體，其係由寄存於中華民國食品工業發展研究所且寄存編號為 BCRC 960409之融合瘤細胞株所產生，其中該抗原結合特性 係為對禽流感H6N1亞型病毒之金球凝集蛋白質具有辨識結合 專一性，且對禽流感H5N2亞型病毒之血球凝集蛋白質不產生交 201132980 叉反應；該單株抗體係屬於IgG2b亞型，且其係可由融合瘤細胞 株EB2製備而得。 本發明之再一目的為提供一種用於_檢體中禽流感h6ni 亞型病毒存在之套組’其至少包含上述之單株抗體，且該套組係 為阻斷型ELISA (blocking ELISA)套組或西方免疫墨點轉潰 (Western immunobloting)套組。 本發明之又一目的為提供一種偵測檢體中禽流感H6Nl亞 型病毋存在之方法，其步驟包含：（1)將一檢體與上述之單株抗體 接觸；以及(2)藉免疫反應偵測該檢體中禽流感H6N1亞型病 毒之存在，其中該免疫反應係為阻斷型ELISA法（bl〇ddng ELISA)或西方免疫墨點轉潰法（WestemimmunQbbting)〇 本發明融合瘤細胞株EB2所產生之單株抗體對於禽流感 刪1亞型病毒具有高度專—性’並對其他亞型之禽流感病毒不 產生交叉反應，亦即本發明之單株抗體對禽流感H6N1亞型病毒 具良好鑑別能力，且利用該單株抗體所開發之快速、敏感性高且 特異性佳之檢驗豕禽流行型感冒的套組或方法，可達成準確偵測 並進而控制H6N1亞型病毒擴散之目的。 以下將配合圖式進一步說明本發明的實施方式，下述所列舉 的貫施例係用以闡明本發明，並非用以限定本發明之範圍，任何 熟習此技藝者，在不脫離本發明之精神和範圍内，當可做些許更 動與潤飾，因此本發明之保護範圍當視後附之申請專利範圍所界 定者為準。 201132980 【實施方式】 為達快速且準確_禽流感腦1亞型病毒，本發明係利用 纟臺灣地區分_ H6N1亞型病毒株之病毒顆粒免疫小鼠，並製 備融合瘤細胞株，且藉由病毒顆粒初步純化具完整醣類的的企球 凝集蛋白質（HA)筛選單株抗體，以增進該單株抗體與臓i亞 型禽流感病毒之結合力，並且避免獲得的單株抗體結合位受醋類 遮蔽，以期獲得對禽流感H6N1亞型病毒株具有高度專一性並且 鲁可實際運用於野外檢測之單株抗體；同時由純化後抗體之特異性 分析結果可知，本發明製備之單株抗體不會對至少一種其他亞型 的禽感病絲例如H5N2產生交又反應，故本發明之融合瘤細 胞株及單株抗體’可應肖於開發診斷試冑彳或套組，並可用於臨床 檢體十H6N1亞型病毒的檢測，有利於禽流感的有效監控。 實施例1 抗禽流感H6N1亞型病毒血球凝集蛋白質叫）之單株抗 • 製備 1.抗原之來源與製備 本發财關使狀抗縣㈣刪〗亞讀流感病毒株 A/ChiCken/TaiWan/2838V/00, GenBank 編號為 EF681878，經接種 9〜11日齡無特定病原（sPecific pathogen free，SPF)雞胚胎蛋，並 於37。(：孵蛋機中培養3〜4日，收集未死亡之雞胚胎蛋的尿囊 液’將檢測確定含有此禽域病毒者，經初步純化與濃縮，以供 201132980 後續免疫注射之用。 其中’禽流感病毒之純化與濃縮方式，係將收集之尿囊液於 4°C下以3,000rpm離心15分鐘，取上清液，並加入1%之胞， 置於37C過夜使病毒不活化。於4ΐ下以7〇，_ g超高速離心3 小時，去紅清液後’以紐積1/1G()的臟^版聰沉殿物， 將回溶液緩緩加入蔗糖梯度中(蔗糖梯度為1〇%、2〇%、3〇%、4〇% 與50%)，於4Ϊ下以50,000 g超高速離心3小時後，以針吸取可 • 目視之病毒層，並加入4 mL之NET buffer清洗，再於4°C下以 50,000 g超高速離心3小時’視沉澱物多寡以2〇〇~5〇〇祉之 NET buffer回溶，置於_2(rc保存。此外’病毒定量可以習知任 何測定蛋白質含量的方法決定，此處則是以Bi0-Rad的 microassay系統(蛋白質分析套組），利用Bradf〇rd的蛋白質 測定方法進行，並以牛血清白蛋白作為標準蛋白質。 2.融合瘤細胞株之製備 φ ⑴對小鼠進行免疫 免疫接種小鼠之抗原為已經甲醛處理不具感染活性之禽流感 H6N1亞型病毒顆粒。混合等量之合成佐劑TiterMax與前述純 化濃縮的H6N1亞型病毒製備含有抗原的乳劑，將乳化後之該 含有100 pg抗原的乳劑以腹腔注射（Intra-peritoneal，i.p.) 施打於6週齡的BALB/C雌小鼠（購自台大醫學院實驗動物 中心）’之後每2週追加注射濃度為50 pg之含有抗原的乳 劑’共注射4次。於小鼠免疫注射後之隔週，由小鼠尾部靜脈 201132980 採血收集抗血清，再以抗血清中的抗體作為一級抗體進行西方 免疫墨點轉潰法（Western immunobloting)，檢測禽流感病毒特異 性之抗體效價。直到6週後測得強烈之抗體效價反應，將不含 佐劑的5(^g之抗原以相同方式進行最後一次免疫，並於5天 後進行細胞融合。 (2)製備融合瘤細胞 融合當天取出小鼠的脾臟，分離出脾臟細胞 φ (sPlen〇cyte)，使全部之脾臟細胞與一個T80 flask (約2xl〇7 個細胞）的小鼠骨髄癌細胞（Sp2/0-Agl4, ATCC CRL-1581) 進行融合。以離心的方式收集細胞混合物，將37。(：之0.7ml 的聚乙二醇 1500 (polyethyleneglycol 1500, PEG 1500)加入細 胞混合物中，和緩地混合1分鐘使細胞進行融合。 接著，於4分鐘内慢慢加入i〇ml之37°C的DMEM培養 基（Dulbecco’s modified Eagle’s medium,購自 Biochrom AQ FG • 0435)稀釋PEG 1500，再以離心方式移除PEG 1500。將離心後 所收集的細胞和緩地重新懸浮於30ml含有次黃嘌呤-氨喋呤-胸 腺嘧啶（hypoxanthine-aminopterin-thymidine，HAT)的 DMEMX 培養基（HAT-DMEMX，添加有15%胎牛血清)中，並置於37°C 之C〇2培養箱中1〜2小時，再將融合的細胞均勻地分配到96 孔微量培養盤（microtiterplate)内。將細胞融合之日定為第0天。 融合完成後第1天，於96孔微量培養盤的每孔池加入2滴 37°C的HAT-DMEMX，之後，分別於融合完成後第6天、第11 201132980 天與第14天，將二滴37〇C的ΗΑΤ-DMEMX加入培養盤的每 一孔池中。此兩週之培養係為初步篩選，融合成功之融合瘤細胞 可繼續***生長，並可觀察到細胞聚成的群落，反之，未融合成 功之融合瘤細胞則死亡。 3.融合瘤細胞株之篩選與鑑定 初步篩選兩週後，以禽流感H6N1亞型病毒 (A/chicken/Taiwan/2838V/00)與禽流感 H5N2 亞型病毒 鲁（A/Duck/Taiwan/3233/04，此病毒係依據前列實施例丨第丨點之抗原 之來源與製備所述方法製備）同時進行西方免疫墨點轉潰法 (Western immunoblotting)以篩選融合瘤細胞。 考塁由雞胚蛋取得的H6N1亞型禽流感病毒顆粒之ha的 特疋位置具有某些巨大的醣類（glycan)，此醣類將會遮蔽部分蛋白 質表面，若是使用未含醣類的表現蛋白質或合成的短鏈胜肽進行 免疫及後續抗體篩選，雖然也可得到具效價的單株抗體，但此抗 Φ體若運贿天然的脑1亞聽域病毒齡個，其辨識蛋白 質之結合位置可能會被醣類遮蔽，而無法實際運用於檢測。為避 這個門本發明係使用由雞胚蛋取得具_類的病毒顆粒初步 純化ΗΑ (其係利用前述「抗原之來源與製備」所述方法製備得） 來筛選單株抗體’軸製備的程序較為繁複，但此方法-但_選 到具有效饧之單株抗體，則其抗體原始特性即可避開醣類的干 t自…i界中病t表面ΗΑ原本就帶有聽類的禽流感病毒進行 檢測。 201132980 於眾多產生有效價單株抗體的融合瘤細胞中，只挑選對 H6N1亞型病毒呈陽性反應’對H5N2病毒呈陰性反應之融合瘤 細胞’再以限數稀釋法（limiting diluti〇n)進行單株化，依此方法 篩選出效價最強可產生抗禽流感H6N1亞型血球凝集蛋白質之 單株抗體融合瘤細胞株，將其命名為EB2，並於中華民國99 年3月12日寄存於食品工業發展研究所（地址為新竹市食品 路331號）’其寄存編號為bCRc96〇4〇9。 • 該EB2融合瘤細胞株***快速且穩定（請參閱第一八圖），且 繼代多次（>20)後測試其效價’可稀釋達24,_倍以上(請參閱 第一B圖）。 4·早株抗體之亞型分析、產製與純化 (1) 單株抗體之亞型分析 取適量冷凍乾燥之由融合瘤細胞株EB2所產之 粉末溶於 PBS b疏中，以 Immun〇Pure m_lenai=^ • i啤ping 套組（Pierce Chemical，Rockford, IL)進行單株抗體之亞 型（isotype)種類分析。結果如第二圖所示，由該圖可知，由融合 瘤細胞株EB2所產生之單株抗體係屬於每知亞型，其輕鏈 (light chain)為 /c (kappa)鏈。 (2) 單株抗體大量製備 本發明實_係糊將融合瘤細胞株注射到㈣體内，誘導 產生腹水而量產單株抗體。 12 201132980 取八週以上之BALB/c小鼠’於每隻小鼠之腹腔注射〇 5mL 之鯊肝油烧（pristine)，以刺激小鼠腹腔。於5〜7天後，取1〇6以 上有效價之融合瘤細胞株EB2經離心去除上清液後，懸濁於〇 5 ml之37 t：的DMEM中，再以注射器打入小鼠腹腔。於一到 二週後，小鼠腹部脹大直到拖地，抽取小鼠腹腔液體，經離心後 取上清液’該上清液包含由融合瘤細胞株EB2產生之單株抗體。 (3)單株抗體之純化 將取得之小鼠腹水以PBS buffer稀釋3倍或直接收集 EB2融合瘤細胞株的上清液，進行4〇%飽和硫酸銨沉澱，將 所收集之沉澱物溶解於50 mM的碳酸氫鈉緩衝液中（pH 8.3)，並於4°C透析過夜。利用離心將透析時所產生的沉澱移 除’上清液進行DEAE_Sephacel管柱層析。 官柱先以50 mM的碳酸氫鈉緩衝液（pH 8.3)清洗，載入樣 品後，以含25至250mM氯化鈉的5〇mM的碳酸氫鈉緩衝 液（PH 8.3)線性梯度（iinear gradient)沖提，含有單株抗體的 分液於氯化鈉濃度為200mM〜300mM之處流出。 實施例2 融合瘤細胞株EB2產生之單株抗體的特異性分析 取H6N1與H5N2亞型禽流感病毒，經定量後分別取 lug/well利用15%之SDS_pAGE膠片進行蛋白質電泳，再經 由電泳轉印法將該些抗原蛋白質轉印至PVDF膜上，接著將 13 201132980 PVDF膜刀別移入不含抗體之溶液（此作為控制組）、含有祕川 -或麵2乡株抗體（該多株抗體係經2,000倍稀釋）之一次抗體 /谷液（此作為對照組）以及含有由融合瘤細胞株產生之單 株抗體（該單株抗體係經2,〇〇〇倍稀釋）❾一次抗體溶液中（此作 為實驗組）。 經抗體抗原結合反應及沖洗未結合抗體之步驟後，再使用可 辨認一次抗體的商品化二次抗體例如共軛結合過氧化酶之山羊抗 φ 小鼠抗體（horse radish peroxidase-goat-anti-mouse，HRP-GAM，此 二次抗體係經5,000倍稀釋）再進行一次專一性的辨識，由於此二 次抗體上已經連結過氧化酶（HRP)，因此可經由催化基質反應發 出冷光而進行專一性抗體之特異性分析。其結果如第三圖所示。 凊參閱第二圖，該圖結果顯示，相較於對照組之H6N1的轉 印膜上所呈現的複雜抗體免疫條帶，由融合瘤細胞株EB2產生之 單株抗體僅於H6N1的轉印膜上約55KDa大小處呈現一明顯條 • 帶，該條帶位置係為已知之HA次體HA1的相對位置，並經 LC/MS/MS定序確認其身分。至於H5N2禽流感病毒的轉印膜上 則無出現條帶，由此可知，融合瘤細胞株EB2產生之單株抗體係 可專一特異性辨識結合H6N1亞型禽流感病毒 (A/chicken/Taiwan/2838V/00)之 HA 蛋白質，而對 H5N2 禽流感 病毒不具結合能力，因此融合瘤細胞株EB2產生之單株抗體係可 應用於禽流感H6N1亞型病毒之檢測方法，例如西方免疫墨點轉 潰法或酵素連結免疫吸附法（ELISA)、生物晶片以及檢測試條 201132980 (strip)等需以抗體進行檢測之方法，亦可應用於偵測檢體中禽流 感H6N1亞型病毒存在之診斷套組，其中酵素連結免疫吸附法 (ELISA)係包含阻斷型 ELISA (blocking ELISA，bELISA)等，此 外，檢測試條部份，不需特殊儀器設備即可使用，可達到現地偵 測、即時防範之效果，對於防治禽流感病害相當有幫助。 【圖式簡單說明】 第一 A ®係本發明實施例EB2齡瘤細胞株之示意圖。 第一 B _本發日讀施例哪融合瘤細胞株經繼代多次後 檢測其效價之結果圖。 第二圖係本發明實施例單株抗體之亞型翻分析結果圖。 第-圖係本發明實施解株抗狀特異性分析結果圖。 【主要元件符號說明】201132980 VI. Description of the Invention: [Technical Field] The present invention relates to a monoclonal antibody against avian influenza virus hemagglutination protein and a preparation method and application thereof, in particular to avian avian influenza ΗόΝ1 subtype virus hemagglutination The individual antibody of the egg self-quality, the tumor cell line recorded by the single plant, and the detection kit and method for applying the monoclonal antibody. [Prior Art] • Poultry epidemic W (avian influenza, ΑΙ) is caused by a type A influenza virus infection of the Orthomyxoviridae (0▲零咖咖). Type A influenza virus has a rod-like agglutination protein (Hemagglutinin; and neuraminidase (ΝΑ) two glycoproteins, depending on the antigenicity of each blood cell agglutinating protein and neuraminidase Different, it can be divided into many immunological subtypes. There are currently 16 kinds of hemagglutination protein antigen subtypes H1 ~ H16 (Fouchier et al "J Virol 79 (5): 2814-22, 2005) and 9 species. φNeuraminidase antigen subtype Ν1 ~Ν9 (Webster and Laver, Virology 69(2): 511-22, 1976). In addition, 'based on the pathogenicity of poultry influenza virus can be divided into high pathogenicity and low pathogenicity. 'Highly pathogenic avian influenza virus (HPAIV) is mainly a subtype of H5 and H7, which can cause systemic diseases with high mortality in chickens' and may even cause human death. Lamb and Krug, Fieids virology 1487-1531, 2001; OIE, 2008). The remaining pathogenicity is called low pathogenic 201132980 avian influenza virus (LPAIV). These viruses are widely found in a variety of poultry. ^ According to the current field survey, the H6N1 subtype avian influenza virus belonging to the low pathogenic poultry influenza virus (lpaiv) is prevalent in Taiwanese native poultry (Lin Shizhen Executive Yuan Agricultural Committee livestock Information on Avian Influenza in Health Laboratory, 2〇〇4; Xie Happy, etc.” Bay Province Animal Husbandry and Veterinary Association Report 59: 45-55, 1992; Zheng Mingzhu et al., Animal Health Laboratory Research Report 42: 43_5〇, 2〇〇 7). Deleted in the past few decades (5), almost become a local poultry epidemic in Asia - Taiwan's first outbreak of the h-type φ-type poultry influenza virus in W2 'has a very high death for poultry In the year of the Qing Dynasty, there were plans to monitor the waterfowl and poultry epidemics in Taiwan. More than 50% of the recent laying hens and chickens were positive for deletion and sputum (Yang Ping Scale, X Taiwan Veterinary Junction, 32:24-29) , 2006), and more and more rural H6 subtype virus strains are isolated. Brain 1 axis belongs to low disease, when the virus forms latent infections in poultry coffee 'may cause weightlessness, dysplasia, and decreased egg production And easy to infect other diseases #病等情'The economic losses caused by poultry farmers, even worse, if the virus persists for a long time - in the population' will likely mutate into a highly pathogenic poultry epidemic virus (HPAIV), or form a new bird flu virus, infection The non-resistance group, which in turn causes a global pandemic, makes it impractical to establish a method for detecting H6 subtype poultry influenza virus that is convenient to use, can be used quickly, and can be mass-produced. ' Today's poultry epidemic gastric virus detection method consists of reverse transcription polymerase chain reaction and (4) quantitative inversion of the escape enzyme chain reaction directly _ viral protein or 4 201132980 nucleus by virus separation detection antigen by comparing influenza virus sample pairs The ability of HA to initially assess the titer of the virus and the use of specific antibodies to distinguish viral serotypes such as hemagglutination inhibition test (HI teat), neuraminidase inhibition test (NI test) and enzyme linkage Methods such as enzyme-linked immunosorbent assay (ELISA). Among them, the method of detecting the avian influenza virus by the antibody has the advantages of simplicity and rapidity. However, the accuracy and sensitivity of the method depend on whether the antibody recognizes the important anti-original epitope (epit0Pe) and its good specificity. Diversity and specific antibodies play an important role in this approach. According to the study 'infectious avian influenza viruses in nature, most of them seem to contain 3-5 glycosylation sites (Schulze, Infcct Dis 176.S24-8, .1997), which are active. The surface of the private part is covered with glycerin 'and the number, size and turnover depend on the host, and the position is determined by the nucleic acid sequence of the viral gene. Furthermore, because the influenza virus is a virus, these brewing sites are frequently changed with mutations, and the resulting diversity is presumed to be a strategy for the virus to counteract host neutralizing antibodies and various antiviral mechanisms. In addition, huge sugars will mask part of the HA surface, which affects its physiological and immunological properties. It may make the host's immune system unable to recognize the virus. Therefore, studies have pointed out that the impact of HA surface must be considered in the development of influenza vaccine ( (10) Poverty's 1〇6(43). 18丨37_42, 2_). In addition to the development of vaccines, the location of individual antibody identification for detection is also affected by vinegar. Therefore, it is necessary to screen for locations that avoid cool masking or directly identify individual antibodies to specific vinegars. 201132980 In summary, due to the large number of subtypes of poultry epidemic w virus, it can be quickly and has a variety of different sub-discriminations for epidemic prevention. Taiwanese poultry epidemic and sexy stomach H6N1 serum Puhe lies in Taiwanese local wealth and will Caused by severe downs and downs, the current county has a specific monoclonal antibody against the H_ subtype virus of the poultry domain or a diagnostic kit for detecting the virus. Therefore, in order to reduce the livestock diseases caused by avian influenza and to test the future It is imperative for the development of a specific single-antibody antibody against human avian virus, the anti-avian influenza 臓1 subtype virus. φ [Summary of the invention] In order to quickly and accurately detect the presence of avian influenza H6N1 subtype virus and effectively distinguish the difference between the H6N1 subtype virus and various other avian influenza subtype viruses, one of the objects of the present invention is to provide a fusion tumor a cell strain capable of producing a monoclonal antibody against a hemagglutination protein of avian influenza H6N1 subtype virus, and the monoclonal antibody has a blood cell of at least one other avian influenza virus subtype other than the H6N1 subtype virus (eg, H5N2 subtype) The agglutinated protein does not cross-react. Further, the fusion tumor cell line is named EB2, and the registration number of the Republic of China Food Industry Development Institute is BCRC 960409. Another object of the present invention is to provide a monoclonal antibody having antigen-binding properties, which is produced by a fusion tumor cell strain deposited with the Republic of China Food Industry Development Institute and having the accession number BCRC 960409, wherein the antigen binding property is In order to identify and bind to the agglutination protein of the avian influenza H6N1 subtype virus, the hemagglutination protein of the avian influenza H5N2 subtype virus does not produce a crossover reaction of 201132980; the monoclonal resistance system belongs to the IgG2b subtype, and It can be prepared from the fusion tumor cell line EB2. A further object of the present invention is to provide a kit for the presence of avian influenza h6ni subtype virus in a sample comprising at least the above-mentioned monoclonal antibody, and the kit is a blocking ELISA kit. Group or Western immunoblotting kits. Another object of the present invention is to provide a method for detecting the presence of avian influenza H6N1 subtype disease in a sample, the steps comprising: (1) contacting a sample with the above-mentioned monoclonal antibody; and (2) borrowing immunity The reaction detects the presence of the avian influenza H6N1 subtype virus in the sample, wherein the immune reaction is a blocking ELISA method (bl〇ddng ELISA) or a Western immune dot dot knocking method (WestemimmunQbbting). The monoclonal antibody produced by strain EB2 is highly specific for avian influenza subtype 1 virus and does not cross-react with other subtypes of avian influenza virus, that is, the monoclonal antibody of the present invention against avian influenza H6N1 subtype The virus has good discriminating ability, and the rapid, sensitive and specific test kit or method for testing the poultry epidemic cold developed by the monoclonal antibody can accurately detect and further control the spread of the H6N1 subtype virus. purpose. The embodiments of the present invention will be further described in conjunction with the drawings, which are set forth to illustrate the invention and are not intended to limit the scope of the present invention. And the scope of protection of the present invention is defined by the scope of the appended claims. 201132980 [Embodiment] In order to achieve rapid and accurate _ avian influenza brain subtype 1 virus, the present invention utilizes a virus particle of the _H6N1 subtype virus strain in Taiwan to immunize a mouse, and prepare a fusion tumor cell strain, and Viral particles are initially purified by agglutination protein (HA) with intact carbohydrates to screen for monoclonal antibodies to enhance the binding of the monoclonal antibodies to the 臓i subtype avian influenza virus, and to avoid the acquired monoclonal antibody binding sites. Vinegar masking, in order to obtain a single antibody with high specificity for the avian influenza H6N1 subtype virus strain and which can be actually used for field detection; and the specific antibody analysis results of the purified antibody, the monoclonal antibody prepared by the invention The at least one other subtype of avian susceptible filaments, such as H5N2, does not react and react, so the fusion tumor cell strain and the monoclonal antibody of the present invention can be used for clinical diagnosis or a set, and can be used for clinical purposes. The detection of the sample H6N1 subtype virus is conducive to the effective monitoring of avian influenza. Example 1 Anti-avian influenza H6N1 subtype virus hemagglutination protein called) single plant resistance • Preparation 1. Source and preparation of antigens This is the case for the anti-county (four) deletion of the sub-reading influenza virus strain A/ChiCken/TaiWan/ 2838V/00, GenBank No. EF681878, sPecific pathogen free (SPF) chicken embryo eggs were inoculated 9 to 11 days old, and 37. (: 3 to 4 days of incubation in an egg-catching machine, collecting allantoic fluid of undyed chicken embryo eggs' will be tested and confirmed to contain the poultry virus, which is initially purified and concentrated for subsequent injection of 201132980. The method of purifying and concentrating the avian influenza virus was to centrifuge the collected allantoic fluid at 3,000 rpm for 15 minutes at 4 ° C, take the supernatant, and add 1% of the cells, and place at 37 C overnight to make the virus inactivated. At 4 ΐ, 7 〇, _ g ultracentrifuged for 3 hours, after the red clear liquid 'to the 1 / 1G () of the dirty product version of the Cong Shen Temple, the solution is slowly added to the sucrose gradient (sucrose gradient 1〇%, 2〇%, 3〇%, 4〇%, and 50%), after ultracentrifugation at 50,000 g for 3 hours at 4 ,, the virus layer can be visually aspirated and 4 mL of NET is added. Buffer cleaning, then ultracentrifugation at 50,000 g for 3 hours at 4 °C. Depending on the amount of precipitate, the NET buffer is dissolved in 2〇〇~5〇〇祉, placed in _2 (rc preservation. In addition, 'virus quantification can It is customary to determine any method for determining the protein content, here using the Bi0-Rad microassay system (protein analysis kit), using Bradf〇 The protein determination method of rd is carried out, and bovine serum albumin is used as a standard protein. 2. Preparation of fusion tumor cell line φ (1) Immunization of mice The antigen of mice is avian influenza H6N1 which has been treated with formaldehyde and has no infectious activity. Type of virus particles. The emulsion containing the antigen was prepared by mixing an equal amount of the synthetic adjuvant TiterMax with the above purified and concentrated H6N1 subtype virus, and the emulsion containing 100 pg of the antigen after emulsification was intraperitoneally injected (Intra-peritoneal, ip). After 6 weeks of BALB/C female mice (purchased from the Experimental Animal Center of National Taiwan University Medical College), an additional 50 pg of antigen-containing emulsion was injected every 4 weeks for a total of 4 injections. Separately, the antiserum was collected from the tail vein of the mouse 201132980, and the antibody in the antiserum was used as the primary antibody for Western immunoblotting to detect the specific antibody titer of the avian influenza virus until 6 weeks. After the strong antibody titer reaction was measured, the 5 (^g antigen) without adjuvant was finally immunized in the same manner and was performed 5 days later. Cell fusion. (2) Preparation of fusion tumor cells The spleen of the mice was taken out on the same day, and the spleen cells φ (sPlen〇cyte) were isolated, and all the spleen cells were combined with a T80 (about 2×1〇7 cells) mouse bones. The cancer cells (Sp2/0-Agl4, ATCC CRL-1581) were fused. The cell mixture was collected by centrifugation, and 37 (: 0.7 ml of polyethylene glycol 1500 (polyethyleneglycol 1500, PEG 1500) was added to the cell mixture. The cells were gently mixed by gently mixing for 1 minute. Next, PEG 1500 was diluted by adding i〇ml of 37 ° C DMEM medium (Dulbecco's modified Eagle's medium, available from Biochrom AQ FG • 0435) over 4 minutes, and then PEG 1500 was removed by centrifugation. The cells collected after centrifugation were gently resuspended in 30 ml of DMEMX medium (HAT-DMEMX, supplemented with 15% fetal bovine serum) containing hypoxanthine-aminopterin-thymidine (HAT). The cells were placed in a C〇2 incubator at 37 ° C for 1 to 2 hours, and the fused cells were evenly distributed into a 96-well microtiter plate. The day of cell fusion was designated as day 0. On the first day after the completion of the fusion, add 2 drops of HAT-DMEMX at 37 °C in each well of a 96-well microplate, and then, on the 6th day after the completion of the fusion, on the 11th day 201111980 days and the 14th day, respectively 37 〇C of ΗΑΤ-DMEMX was added to each well of the culture dish. The two-week culture system was initially screened, and the successfully merged fusion tumor cells could continue to divide and grow, and the cells aggregated were observed. On the contrary, the fusion cells that did not fuse successfully died. 3. Screening and identification of fusion tumor cell lines Two weeks after initial screening, avian influenza H6N1 subtype virus (A/chicken/Taiwan/2838V/00) and avian influenza H5N2 subtype virus Lu (A/Duck/Taiwan/3233 /04, the virus was prepared according to the source of the antigen of the first embodiment and the preparation method described above. Simultaneous Western immunoblotting was performed to screen for fusion tumor cells. The special position of ha of H6N1 subtype avian influenza virus particles obtained from chicken embryo eggs has some huge glycan, which will mask part of the protein surface, if it is used without sugar. Protein or synthetic short-chain peptides are used for immunization and subsequent antibody screening. Although monovalent antibodies with potency can also be obtained, this anti-Φ body can be used to recognize the protein of the natural brain. The combined position may be obscured by sugar and cannot be actually used for testing. In order to avoid this, the present invention uses a preliminary purification of virions from chicken embryo eggs (which is prepared by the method described in "Source and Preparation of Antigen") to screen the preparation of monoclonal antibody 'axis preparation. More complicated, but this method - but _ selected to have a single antibody, the original characteristics of the antibody can avoid the dryness of the sugar from the surface of the disease, the surface of the disease, the original bird Influenza virus is tested. 201132980 Among the many fusion tumor cells producing antibodies with effective valency, only the hybridoma cells that responded positively to the H6N1 subtype virus, which are negative for H5N2 virus, were selected and then subjected to limiting diluti〇n. According to this method, a single antibody fusion tumor cell line with the strongest titer to produce avian influenza H6N1 subtype hemagglutination protein was selected and named as EB2, and deposited in the Republic of China on March 12, 1999. The Food Industry Development Research Institute (address No. 331, Food Road, Hsinchu City) has a registration number of bCRc96〇4〇9. • The EB2 fusion tumor cell line divides rapidly and stably (see Figure 18), and its potency can be diluted by more than 24, _ times after subculture (>20) (see first B) Figure). 4. Subtype analysis, production and purification of early strain antibodies (1) Subtype analysis of monoclonal antibodies The appropriate amount of freeze-dried powder produced by fusion tumor cell line EB2 is dissolved in PBS b, with Immun〇 Pure M_lenai = ^ • i beer ping kit (Pierce Chemical, Rockford, IL) for isotype analysis of individual antibodies. The results are shown in Fig. 2, and it is understood from the figure that the monoclonal antibody system produced by the fusion tumor cell line EB2 belongs to each subtype, and its light chain is /c (kappa) chain. (2) Large-scale preparation of monoclonal antibodies In the present invention, a fusion cell strain is injected into (IV) a body, and ascites is induced to produce a monoclonal antibody. 12 201132980 BALB/c mice with more than eight weeks were injected intraperitoneally with 5 mL of pristine in each mouse to stimulate the peritoneal cavity of the mice. After 5 to 7 days, the supernatant of the fusion cell line EB2 with an effective price of 1〇6 or more was removed by centrifugation, suspended in 5 ml of 37 mg: DMEM, and then injected into the mouse abdominal cavity with a syringe. . After one to two weeks, the mouse's abdomen swelled until the ground was pulled, and the mouse peritoneal fluid was taken, and the supernatant was taken after centrifugation. The supernatant contained the monoclonal antibody produced by the fusion tumor cell line EB2. (3) Purification of monoclonal antibody The obtained ascites of mice was diluted 3 times with PBS buffer or the supernatant of the EB2 fusion tumor cell line was directly collected, and 4% by weight of saturated ammonium sulfate was precipitated, and the collected precipitate was dissolved in 50 mM sodium bicarbonate buffer (pH 8.3) and dialyzed overnight at 4 °C. The precipitate produced during dialysis was removed by centrifugation. The supernatant was subjected to DEAE_Sephacel column chromatography. The column was first washed with 50 mM sodium bicarbonate buffer (pH 8.3), and after loading the sample, a linear gradient of 5 mM sodium bicarbonate buffer (pH 8.3) containing 25 to 250 mM sodium chloride (iinear gradient) The fractionation containing the monoclonal antibody was carried out at a concentration of 200 mM to 300 mM of sodium chloride. Example 2 Specificity analysis of monoclonal antibodies produced by fusion tumor cell line EB2 H6N1 and H5N2 subtype avian influenza viruses were obtained, and after quantification, respectively, lug/well was subjected to protein electrophoresis using 15% SDS_pAGE film, and then transferred by electrophoresis. The antigenic protein is transferred to the PVDF membrane, and then the 13 201132980 PVDF membrane knife is transferred into the antibody-free solution (this is used as a control group), and the secret drug containing the secret - or surface 2 antibody (the multi-strain resistance system) a 2,000-fold dilution of the primary antibody/trough solution (this is used as a control group) and a monoclonal antibody (the single-body anti-system diluted by 2, 〇〇〇-diluted) ❾ primary antibody solution (produced by the fusion cell strain) This is the experimental group). After the antibody antigen binding reaction and the step of washing the unbound antibody, a commercially available secondary antibody that recognizes the primary antibody, such as a goat anti-φ mouse antibody conjugated with peroxidase (horse radish peroxidase-goat-anti-mouse) is used. , HRP-GAM, this secondary anti-system is diluted 5,000 times) and then uniquely identified. Since the secondary antibody has been linked to peroxidase (HRP), it can be luminescence by catalytic substrate reaction for specificity. Specificity analysis of antibodies. The result is shown in the third figure. Referring to the second figure, the results of the figure show that the monoclonal antibody produced by the fusion tumor cell line EB2 is only transferred to the H6N1 transfer membrane compared to the complex antibody immunoslot presented on the transfer membrane of the H6N1 of the control group. An apparent strip was present at a size of about 55 KDa, which was the relative position of the known HA sub-body HA1 and its identity was confirmed by LC/MS/MS sequencing. As for the transfer film of H5N2 avian influenza virus, there is no band, and it can be seen that the monoclonal antibody system produced by the fusion cell line EB2 can specifically recognize and bind to the H6N1 subtype avian influenza virus (A/chicken/Taiwan/ 2838V/00) HA protein, but has no binding ability to H5N2 avian influenza virus. Therefore, the monoclonal antibody system produced by the fusion tumor cell line EB2 can be applied to the detection method of avian influenza H6N1 subtype virus, for example, the western immune dot collapses. Method or enzyme-linked immunosorbent assay (ELISA), biochip, and test strip 201132980 (strip), etc., which can be detected by antibodies, and can also be used to detect the presence of avian influenza H6N1 subtype virus in the sample. The enzyme-linked immunosorbent assay (ELISA) system includes a blocking ELISA (bELISA), etc. In addition, the test strip portion can be used without special equipment, and can achieve local detection and immediate prevention. The effect is quite helpful for the prevention and treatment of avian influenza diseases. BRIEF DESCRIPTION OF THE DRAWINGS The first A ® is a schematic diagram of an EB2 instar tumor cell line of the present invention. The first B _ this is the day to read the example of the fusion tumor cell line after multiple passages to test the results of the titer. The second panel is a graph showing the results of subtype analysis of individual antibodies of the present invention. The first graph is a graph showing the results of the anti-specificity analysis of the strains of the present invention. [Main component symbol description]