200413715 玖、發明說明 【發明所屬之技術領域】 本發明係關於一種包含由食物過敏症病患之IgE抗體辨 識之多種天然及/或變性食物過敏原的混合物，經由利用 該混合物使動物免疫而製備得之抗體，及一種關於使用該 抗體而檢測食物過敏原及引起過敏症食物之方法。由於本 發明有用於瞭解食物過敏症開始的機制，產生及確認低過 敏原食物，及檢測食物、其原料及諸如食物製造機器及製 程之食物製造環境中之食物過敏原，而提供食物過敏症病 患安全性。 【先前技術】 食物過敏症係敏感人士的有害免疫反應。食物過敏症係 由食入食物中之引起過敏症之物質（以下稱爲食物過敏原） 所造成。食物過敏症會造成皮膚炎、氣喘、消化道障礙、 過敏反應性休克等等。過敏症視疾病開始的機制而被分類 爲第I至IV型。食物過敏症主要係由第I型過敏症所造成， 其中IgE抗體與攝入體內之食物過敏原反應。食物過敏症 病患之數目近年來逐漸增加。此一現象在醫療科學及食品 工業的領域中產生嚴重的問題。 爲防止此一危害，需經由標示而提供消費者資訊。 FAO/WHO聯合食品標準委員會（食物法典考察團（Codex A1 i m e n t a r i u s C 〇 m m i s i ο η))認知標示含有八種已知爲食物過 敏原之原料之任何一種之食物的需求，並建議各會員國實 6 326\專利說明書(補件)\92·04\92101099 200413715 施標示系統（ 1 9 99年6月）。在日本，考慮到食物過敏症的 嚴重丨生及頻率而頒佈24個項目的標示（自2QQ2年4月起實 施）。應注思此等法規既非要求標示過敏性物質亦非標示食 物過敏原的本身，而係要求標示含有食物過敏原的食物， 即引起過敏症食物。 引起過敏症食物包括蛋、奶、肉、魚、甲殼類、軟體動 物、穀物、豆類、堅果、水果、蔬菜、啤酒酵母及明膠。 同樣地’已知卵蛋白、卵類黏蛋白、溶菌酶、酪蛋白、0 _ 乳球蛋白、α -乳白蛋白、榖麩蛋白、α -澱粉酶抑制劑等 等爲引起過敏症食物。 此外，（1)不管目前的認定如何，相信仍有許多其他會導 致食物過敏症的食物及成份（食物過敏原）2)引起過敏症 食物及成份（食物過敏原）相當多樣化，且食物過敏症病患 對此等過敏原的反應各有不同；及（3)如稍後說明之實施例 所顯不’即使係於單一之引起過敏症食物中，亦存在許多 已知及未知的過敏原。然而，習知之方法皆無法容易地檢 測如此多之引起過敏症食物或食物過敏原。 食物係利用諸如加熱、冷凍、乾燥、加鹽、發酵、酵素 處理等等（以下稱爲食物製程）之程序製備得，以改良其之 消化性、儲存壽命、味道、物性等等及使其安定化。雖然 食物製程會影響蛋白質及修改其之分子結構（例如，蛋白質 之變性），但卻鮮少有人論述此種食物製程是否會導致食物 過敏原的生成。 發展本發明的硏究工作闡明以下事項：（1) 一些經加熱的 7 326\專利說明書(補件)\92-04\92101099 200413715 食物成份會表現出過敏性；（2)引起過敏症成份或個別引起 過敏症食物之表位會視所關注之食物是否經加熱而改變。 換言之，製備a)未經加熱之蛋抗原，b)經由加熱（a)中之抗 原而製備得之經加熱蛋抗原，c)經由將來自多位食物過敏 症病患之匯集血淸（稱爲匯集病患血淸）及（a)(不含未經加 熱蛋抗原之血淸）混合而製備得之血淸，及d)經由將匯集病 患血淸及（b)(不含經加熱蛋抗原之血淸）混合而製備得之血 淸。由在a)及c)或a)及d)之間及在b)及c)或b)及d)之間 的反應結果顯示不含未經加熱蛋抗原之血淸無法與未經加 熱之蛋抗原免疫反應，但可與經加熱之蛋抗原反應：同樣 地，其顯示不含經加熱蛋抗原之血淸無法與經加熱蛋抗原 免疫反應，但可與未經加熱之蛋抗原反應。總而言之，食 物過敏症病患帶有對經加工及未經加工食物兩者特異的 IgE抗體，而導致食物過敏症。然而，迄今爲止仍沒有可 檢測經加工食物或其成份中之過敏原的簡單方法。 可於商業上取得篩選雞蛋、花生、酪蛋白、/3 -乳球蛋白 及榖麩蛋白中之食物過敏原的方法（食品及發展（Food and Development)，第35冊，10-11頁）。然而，其之全部或一 些具有一或多種以下的缺點：（1)方法並非始終可檢測出食 物過敏症病患對之過敏的過敏原。換言之，其並非始終可 檢測出病患之IgE抗體所辨識出之物質；（2)方法僅可檢測 已知之過敏原，且每個方法僅可檢測單一過敏原（單一抗 原）；（3)使用單一抗原檢測抗體之方法並不可應用至具有 抑制性物質之食物；（4)使用單一抗原檢測抗體之方法無法 8 326\專利說明書(補件)\92-04\92101099 200413715 )1¾•如τρ;於稍後說明之貫施例中的引起過敏症食物準確定 量；（5)使用單一抗原檢測抗體之方法無法應用於分析不含 抗原之引起過敏症食物（例如，使用經由暴露至位於蛋白中 之卵蛋白而製備得之單一抗體之方法無法應用於分析蛋 黃、蛋黃醬等等）；（6)使用單一抗原檢測抗體之方法由於 無法檢測變性或經分子改質之過敏原，而無法應用於分析 經加工食物；及（7)對抗天然及變性々-乳球蛋白、卵蛋白 及酷蛋白之單株抗體已經發表（過敏症（Allergy)，第50 冊’ 3 09頁）。由於單株抗體僅檢測食物過敏原分子中之個 別表位，因而如表位經移除或經食物製程改質，則其之效 用會減低。 雖然已經發表使用來自對米（日本專利公告（Japanese Patent Disclosure)2000-65820)、蛋及奶（日本肉品科學 (Japanese Meat Science)，第 39 冊，166-169 頁）過敏之病患 之血淸篩選食物過敏原之方法，但其僅有用於醫院，而不 有用於檢驗機構及食品製造工廠。雖然已發表視過敏性反 應而定之食物過敏原檢測方法（FFI Ι·，Νο·180,77-82頁）， 但其由於複雜的方法及實驗室-畜牧業，而無法使用於大多 數的食品製造工廠。雖然已發展使用流動系統及酵素標示 抗體及使用微電極之過敏原感測器之方法（日本食品工業 (Japanese Food Industry)，第 42 冊，53-56 頁）’但在可實 際利用此等方法之前，仍有許多問題需要解決。 【發明內容】 本發明係要解決前述習知技術之問題。本發明之目的係 9 326\專利說明書(補件)\92-04\92101099 200413715 要提供（1)一種包含由食物過敏症病患之IgE抗體辨識之多 種已知及/或未知以及天然及/或變性食物過敏原之混合 物（爲方便起見，將其稱爲本發明之第一發明），（2) —種經 由利用混合物使動物免疫而製備得之抗體（稱爲本發明之 第二發明），及（3)—種檢測食物過敏原及引起過敏症食物 之方法（稱爲本發明之第三發明）。 【實施方式】 本發明之第一發明係由以下程序所完成： (Ι-i)將IgE抗體自食物過敏症病患之匯集血淸分離，及 (Ι-ii)經由使用前述之IgE抗體及諸如親和力層析術及免疫 沈澱作用之免疫方法自經或未經食物製程處理之食物或其 之成份（以下總稱爲食物）分離多種物質（換言之爲可被食 物過敏症病患之IgE抗體辨識之多種食物過敏原）。 本發明之第一發明亦可由以下程序所完成： (2-i)利用食物成份進行習知的SDS-PAGE，然後再將其之 膠轉移至薄膜， (2-η)利用薄膜、匯集病患血淸、接合抗人類IgE抗體及呈 色試劑進行西方墨點法（w e s t e r n b 1 〇 11 i n g)(換言之，製備由 食物過敏症病患之IgE抗體所辨識之多種食物過敏原的分 佈）， (2-in)同樣地，利用薄膜、經由以習知方式使食物成份免 疫而製備得之動物血淸、接合抗動物IgG及呈色試劑進行 西方墨點法， (2 μ v)比較兩西方墨點之分佈。測定未出現於前者，但出現 10 326\專利說明書(補件)\92-04\92101099 200413715 於後者中之帶的分子大小（換言之爲食物過敏症病患之IgE 無法辨識的多種物質），及 (2 - v )視前述結果而定，利用諸如凝膠過濾層析術之習知方 法將經食物過敏症病患之IgE抗體辨識之多種食物過敏原 自食物成份分離。 本發明之第二發明係經由利用由第一發明製備得，且可 經食物過敏症病患之I g E抗體（稱爲辨識多種抗原之抗體） 辨識之多種食物過敏原（包括經加工及/或未經加工者）使 動物免疫而製備得之對抗食物過敏原之動物抗體。 本發明之第三發明係關於使用辨識多種抗原之抗體檢 測食物過敏原及引起過敏症食物之方法。 前述之熱處理溫度範圍係相當於食品製程之範圍，以在 40及25 0 °C之間較佳，及在60及120 °C之間更佳。抗原係 經由將原始抗原混合，及將其在二至六個不同溫度下加熱 而製備得。經食物過敏症病患之I g E抗體辨識之食物過敏 原部分係利用本發明之第一發明由混合物製備得，並利用 其使動物免疫。 當檢測之目標受限於經加熱之食物時，經食物過敏症病 患之I g E抗體辨識之食物過敏原部分係利用本發明之第一 發明由經加熱之食物製備得，並利用其使動物免疫。 動物之例子爲兔子、山羊、綿羊、大鼠、小鼠、天竺鼠、 馬、豬、雞等等。在免疫作用期間中，部分收集血液及檢 查抗食物過敏原抗體之效價較佳。本發明之抗體可爲單株 或多株抗體。如稍後說明之實施例所顯示，即使係於單一 11 326\專利說明書(補件)\92-〇4\92101099 200413715 的引起過敏症食物中亦存在多種經食物過敏症病患之1 g E 抗體辨識之已知及未知的食物過敏原。此即係爲何可經由 利用此等多種食物過敏原使動物免疫而容易地製備得辨識 多種食物過敏原之多價抗體，即辨識多種抗原之抗體。 可將動物之抗血淸使用作爲對抗經食物過敏症病患之 IgE抗體辨識之食物過敏原的抗體。抗血淸可於無法經食 物過敏症病患之IgE抗體辨識之部分的吸收處理之後使 用。抗血淸可於習知之IgG純化作用之後使用。 本發明之檢測食物過敏原及引起食物過敏症之食物的 方法可應用至含過敏原之食物，而無任何限制。引起過敏 症食物之例子爲蛋、奶、肉、魚、甲殼類、軟體動物、穀 物、豆類、堅果、水果、蔬菜、啤酒酵母及明膠。更特定 言之爲蛋白、蛋黃、牛奶、乳酪、豬肉、牛肉、雞肉、羊 肉、鯖魚、竹莢魚、沙丁魚、鮪魚、鮭魚、鱈魚、比目魚、 鮭魚子醬、蟹、蝦、淡菜（moule)、魷魚、章魚、龍蝦、鮑 魚、甲殻類、軟體動物、小麥、米、薔麥、黑麥、大麥、 减麥、玉米、粟、狐尾粟（f 〇 x t a丨1 m i 11 e t)、稗、穀物、大豆、 花生、可可、豌豆、菜豆、榛實、巴西果、杏仁、椰子、 胡桃、丑類、堅果、蘋果、香蕉、橘子、桃子、奇異果、 草莓、瓜類、鱷梨、葡萄柚、芒果、梨、芝麻、芥末、水 果、番茄、紅蘿蔔、馬鈴薯、菠菜、洋蔥、大蒜、竹筍、 南瓜 '地瓜、芹菜、荷蘭芹、山芋、日本松覃、蔬菜、包 含一或多種此等項目及其成份（例如，卵蛋白、卵類黏蛋 白、溶菌酶、酪蛋白、石-乳球蛋白、α -乳白蛋白、榖魅 326\專利說明書(補件)\92·〇4\921 〇 1 〇99 200413715 蛋白、及α -澱粉酶抑制劑）之食物。此等食物可利用諸如 加熱、冷凍、乾燥、加鹽、發酵、酵素處理等等之程序製 備得。 將由機率5 0 %或以上之食物過敏症病患之I g E抗體辨識 之過敏原稱爲主要過敏原，而將低於5 0 %者稱爲次要過敏 原。食物過敏症不僅會由主要過敏原造成，並且亦會由次 要過敏原造成。一些病患不會對主要過敏原過敏，但會對 次要過敏原過敏。因此，篩選食物過敏原之方法應可檢測 主要及次要過敏原兩者。本發明之篩選方法可檢測由食物 過敏症病患之IgE抗體所辨識之天然及變性的多種過敏 原。 爲製備滿足此等需求之抗體，本發明人成功發現下列事 項。 換言之，使食物成份進行習知的SDS-PAGE分析，及將 其之膠轉移於薄膜上： (1)利用薄膜、匯集病患血淸、接合抗人類IgE抗體及呈色 試劑進行西方墨點法； (Π)同樣地，利用薄膜、經由以習知方式使食物成份對動物 免疫而製備得之血淸、接合抗動物IgG抗體及呈色試劑進 行西方墨點法， (iii)由兩西方墨點法分佈之比較，觀察到以下事實；（1)有 於兩薄膜中皆經染色的帶，（2)有未於前者中染色但於後者 中染色的帶（即爲多種非過敏性物質），及（3)分佈在較於前 者中染色之帶更高及/或更低分子量位置之僅於後者中染 13 326\專利說明書(補件)\92-04\92101099 200413715 色的帶；及 (1 V)由前述結果，利用凝膠過濾層析術將經食物過敏症病 患之IgE抗體辨識之多種食物過敏原部分自食物成份分 離，然後使動物免疫，而製備得對抗多種食物過敏原的抗 體。可使諸如兔子、山羊、綿羊、大鼠、小鼠、天竺鼠、 馬、豬、及雞的恒溫動物免疫。可採用任何熟悉技藝人士 知曉的免疫方法。 可將如前所述而製備得之抗體使用於本發明之食物過 敏原檢測方法。可將抗體固定於微量滴定板、PVDF薄膜、 硝基纖維素薄膜、層析試條（chroma to strip)、試管、珠粒、 耐綸薄膜等等上。可將抗體應用至諸如酵素免疫測定、免 疫墨點法（immunoblotting)、點墨法（dot blotting)、免疫層 析術及抗體晶片（a n t i b 〇 d y c h i p)的免疫方法。 如前所述，本發明之食物過敏原檢測方法的目標在於檢 測食物及諸如食物製造機器及程序之食物製造環境中的多 種食物過敏原。將來自食物的液體萃取物使用於分析較 佳。雖然水爲較佳的萃取劑，但可使用磷酸鹽緩衝鹽水、 Tris-HCl緩衝液、醇等等，只要可萃取過敏原即可。爲改 良自食物萃取過敏原之效率，若需要，可將蛋白質變性劑 (例如，SDS或尿素）、含SH之抗氧化劑（例如，2-硫醇乙 醇）等等加至萃取劑。可經由擦拭（塗抹）食物製造環境或經 由將其之空氣捕捉於洗滌瓶中，而測定多種過敏原。 只要本發明之食物過敏原檢測方法的原理爲免疫學，則 方法並不限定於特定的方法。二明治ELISA(sandwich 14 326\專利說明書(補件)\92-04\92101099 200413715 ELISA)、競爭法（competitive method)、直接方法（direct method)等等係酵素免疫測定之例子。當將抗體標示時，可 使用酵素（例如，過氧化酶、鹼磷酸酯酶及/3 -半乳糖苷 酶）、螢光物質（例如，螢光異硫氰酸酯）、生物發光物質（例 如，螢光素-螢光素酶）、化學發光物質（例如，胺基苯二醯 肼（lunnnol)、吖啶衍生物及金剛烷衍生物）、生物素、抗生 物素蛋白質、金膠體、放射性材料（例如，32P)等等。 關於本發明之檢測方法的例子，將三明治ELISA、競爭 及直接方法槪述於下。 爲進行三明治ELISA，製備本發明辨識多種過敏原的經 標示抗體（辨識多種抗原之經標示抗體）。經由吸附或化學 結合將本發明之辨識多種抗體的未經標示抗體固定於 ELISA板上。利用不會干擾反應系統的蛋白質，例如，明 膠及兔血淸白蛋白，將未經固定抗體之區域的板封阻。將 取自食物、食物成份或食物製造環境之萃取物（以下稱爲樣 品）或標準抗原加至板，以進行第一抗原-抗體反應。於反 應之後，洗滌板。爲進行第二抗原-抗體反應，將前述之經 標示抗體加至板’在此過敏原經固定的抗體捕捉。於將未 反應的經標示抗體洗滌及移除之後，加入檢測試劑（例如， 在過氧化酶之情況中的1，2-苯二胺及H2〇2 :在鹼磷酸酯酶 之情況中的磷酸對硝基苯酯）。經由測定在標示與檢測試劑 之間之反應物之量’而將過敏原檢測或定量。 可經由改變在ELISA板上之標準抗原或引起食物過敏症 食物（例如，蛋及奶）之加入量，而得到校準曲線。食物過 15 326\專利說明書(補件)\92-(Μ\92〗〇1〇99 200413715 敏原或引起過敏症食物之量可經由校準曲線而定量。如量 化値爲1 ppm或以上，則樣品可能爲引起過敏症食物。 使用單一抗原檢測抗體之方法由於必需將由單一抗原 檢測抗體而得之値轉變爲引起過敏症食物之含量，因而會 產生定量誤差。 舉例來說，全蛋中之卵類黏蛋白及卵蛋白之濃度分別大 約爲4%及2%。蛋白中之濃度分別大約爲8%及4%。然而， 蛋黃既不包含卵類黏蛋白，亦不包含卵蛋白。因此，（1) 僅檢測卵類黏蛋白或卵蛋白之方法無法應用於測定蛋黃蛋 白質；（2)當將由全蛋製備得之校準曲線應用於測定蛋白 時，會定量得較實際値多兩倍以上的値；及（3)當實際値超 過定量範圍時，會產生極高的定量値。 同樣地，由於全脂奶蛋白質中之酪蛋白及/3 -乳球蛋白之 濃度分別大約爲8 0 %及1 0 %，因而其有辨識單一抗原之抗 體無法精確定量經強化酪蛋白鈉、乳淸蛋白等等之經加工 奶的問題。 然而，本發明之辨識多種抗原之抗體則不具有或具有較 少的此一問題。 對於競爭法，將一定量的標準抗原直接固定於固相上， 將其以非反應性蛋白質封阻。然後將辨識過敏原之與酵素 接合之抗體及樣品同時加至板。使板靜置一定時間，並洗 滌而將未結合的物質移除。將呈色基質加至板。使在經固 定之過敏原與預先經樣品處理之與酵素接合之抗體之間的 反應停止並測定。 16 326\專利說明書(補件)\92-04\92101099 200413715 對於直接方法，使樣品直接吸附於固相上，將其以非反 應性蛋白質封阻並與辨識過敏原之與酵素接合之抗體反 應。然後應用如前所述的相同程序，以檢測樣品中之過敏 原。 對於任何前述的方法，可使用螢光、生物發光及化學發 光基質於呈色。熟悉技藝人士可視目的、樣品、測定原理 等等而修改本發明之食物過敏原檢測方法的其他條件。 本發明之食物過敏原檢測方法可檢測來自食物及其原 料之萃取物中之0.1或1.0毫微克（ng)或以上的過敏原。 (工業應用性） 包含由食物過敏症病患之IgE抗體所辨識之多種天然及 /或變性食物過敏原之混合物及本發明之抗體有用於瞭解 食物過敏症開始的機制及發展降低食物過敏原之過敏性的 技術。此外，本發明之食物過敏原檢測方法有用於確認降 低過敏性之技術的效用，及檢測食物、引起過敏症食物及 食物製造環境諸如機器及程序中之食物過敏原。因此，本 發明有用於提供食物過敏症病患安全性，及有用於解決由 近來之食物過敏症病患之數目增加所產生的醫療及工業問 題。 (實施例） 本發明由以下之實施例作更具體說明，但不應將其解釋 爲限制本發明之範圍。本說明書中所使用之縮寫係技術領 域中所常用者。 實施例1(各種食物之標準抗原的製備） 17 326\專利說明書(補件)\92-04\92101099 200413715 (1) 雞、鵪鶉及鴨蛋 將1公斤的雞蛋去殻、打散、冷凍乾燥及微細硏磨，而 製備得標準雞蛋抗原。於將1 0克之抗原懸浮於1 〇倍體積 之pH 7.0的磷酸鹽緩衝鹽水（以下簡稱爲PBS)中，然後再 分配至5個試管中之後，將其不經加熱或分別在6 0、8 0、 100及12CTC下加熱30分鐘。然後將其混合在一起並均質 化而製備得樣品。同樣地製備鵪鶉及鴨蛋之樣品。 (2) 牛奶 將1公升之牛奶攪拌以使乳脂肪固化及沈澱，將其過濾 通過吸收性棉。於將此一程序重複3次之後，將濾液冷凍 乾燥然後再微細硏磨，而製備得標準牛奶抗原。同樣地， 如（1)中之說明製備樣品。 (3) 小麥及米 將1公斤之小麥粉經由在室溫下攪拌2小時，而以5倍 量之經添加4^4尿素之〇.1“1^8-11(：1緩衝液（？118.6)萃 取，然後再離心。將上澄液透析、冷凍乾燥然後再微細硏 磨，而製備得標準小麥抗原。同樣地’如（1)中之說明製備 樣品。同樣地由米粉製備樣品。 (4) 喬麥 將1公斤之薔麥粉經由在室溫下攪拌2小時，而以5倍 量之經添加1%以(：1之〇.1“丁1七-11(：1緩衝液（1)118.4)萃 取，然後再離心。將上澄液透析、冷凍乾燥然後再微細硏 磨，而製備得標準喬麥抗原。同樣地’如（1)中之說明製備 樣品。 326\專利說明書(補件)\92-〇4\92101099 18 200413715 (5) 花生 將1公斤之花生硏磨並經由在室溫下攪拌2小時而以5 倍量之正己院脫脂。於將此一程序重複3次然後再移除正 己烷之後’經由在室溫下攪拌2小時而以5倍量之經添加 1 % N a C1之〇 · 1 Μ T r 1 s - H C1緩衝液（ρ Η 8 · 4 )萃取製劑，然後 再離心。將上澄液透析、冷凍乾燥然後再微細硏磨，而製 備得標準花生抗原。同樣地’如（1)中之說明製備樣品。 (6) 大豆 如（5)中之說明製備標準大豆抗原。同樣地，如（1)中之說 明製備樣品。 實施例2 (各種經純化之食物過敏原） (1)經純化之雞蛋過敏原 將卵蛋白（雞蛋的主要過敏原）懸浮於1 0倍體積之ρ Η 7.0 的P B S中，並分配至5個試管中，將其不經加熱或分別在 60、80、100及120 °C下加熱30分鐘。然後將其混合在一 起並均質化而製備得樣品。同樣地製備卵類黏蛋白之樣 品。卵蛋白及卵類黏蛋白係位於蛋白中之蛋白質。 (2 )經純化之牛奶過敏原 如實施例2(1)中之說明製備酪蛋白、/3 -乳球蛋白及α -乳白蛋白之樣品。/3 -乳球蛋白及α -乳白蛋白係位於乳漿 中之蛋白質。 實施例3(抗體之製備） (1)對抗各種標準食物抗原之兔子抗體的製備 利用佛恩得（Freund)完全佐劑（使用於第一免疫作用）及 19 326\專利說明書(補件)\92-04\92101099 200413715 不完全佐劑（使用於第二及其後的免疫作用）將於實施例1 中製備得之各樣品乳化，並使日本白兔免疫4至6次。在 此同時，部分收集血液，以確認對抗各抗原之抗體的產生。 收集所有的血液並製備抗體。 (2)對抗各種經純化食物抗原之抗體的製備 同樣地，如前所述製備對抗各種經純化食物過敏原之抗 體。 實施例4(利用免疫墨點方法檢測各種食物過敏原） (1) 匯集病患血淸及兔子抗體 經由將來自20位具二或多種（特異IgE抗體>0.7UA /毫 升）對抗蛋、奶或小麥之RAST(放射過敏吸收試驗）分數之 病患之等量的血淸混合，而製備得匯集血淸。對抗蛋、奶 及小麥之兔子抗體係於實施例3 (1)中所製備得者。 (2) 十二基硫酸鈉·聚丙烯醯胺膠電泳（SDS-PAGE) 將前述之標準蛋、奶及小麥抗原與2-硫醇乙醇加熱3分 鐘，並於具10%濃度之迷你厚板膠中電泳。然後將膠電轉 移於PVDF(聚偏二氟乙烯）薄膜上。利用金膠體染色套組 (BioRad)使一部分的薄膜進行蛋白質染色。 (3) 免疫染色 利用1%人類血淸白蛋白（HSA)將前述的PVDF薄膜封 阻。將薄膜以含0.05% Tween 20之PBS(PBST)洗滌，與匯 集病患血淸（1 0 0倍稀釋）或兔子抗體（1 0 0 0倍稀釋）在室溫 下反應2小時，洗滌，然後再與經接合鹼磷酸酯酶之抗人 類IgE- ε鏈山羊抗體之次級抗體（25 00倍稀釋）或經接合鹼 20 326\專利說明書(補件)\92-04\92101099 200413715 磷酸酯酶之抗兔子IgG (4000倍稀釋）在室溫下反應1小 時。將薄膜以PBST洗滌，以4-甲基- 4(3-磷酸酯苯基）螺[1,2-二環氧丙烷-3,2 -金剛烷]二鈉鹽-化學發光試劑 (Lumi-Phos 530，Wako Pure Chemical)在室溫下處理 30 分 鐘，並暴露於感光性薄膜上，以檢測由鹼磷酸酯酶之去磷 作用所產生的光子。將此等結果示於圖1。 由圖1，觀察到匯集病患血淸與多個帶反應（圖1之1、4、 及7行），及於各食物中存在由病患IgE所辨識的多個帶。 同時，兔子抗體檢測由食物過敏症病患之IgE抗體所辨 識的食物過敏原（圖1之2、5、及8行）。 由兩血淸辨識的物質如下：蛋之卵蛋白、卵類黏蛋白、 溶菌酶及卵輸鐵蛋白；奶之酪蛋白、Θ -乳球蛋白及α -乳 白蛋白；小麥之麥膠蛋白及α ·澱粉酶抑制劑；薔麥之 132-、84-、27-及 ΙΙ-kDa 物質；及花生之 107-、72-、35-及28-kDa物質。 此外，兔子抗體並未檢測病患IgE無法辨識的非食物過 敏原。 (4)過敏原之濃縮及分級分離 如下製備無法辨識除過敏原外之任何物質的抗體： 比較經匯集病患血淸染色及經動物抗體染色之帶的分 子量，注意到後者的一些帶分佈在較前者高及/或低的分 子量位置上（圖1)。利用凝膠過濾層析術自標準抗原收集相 當於經食物過敏症病患之IgE抗體辨識之食物過敏原之分 子量的部分（以下稱爲過敏原部分）。如實施例丨中之說明 21 326\專利說明書(補件)\92-04\92101〇99 200413715 製備樣品，並使動物免疫，而製備得抗過敏原部分抗體。 由如以上關於抗體之說明而進行的西方墨點法，發現抗體 辨識到由匯集病患血淸所辨識的食物過敏原（圖1之3、6、 及9行）。 此種過敏原的濃縮及分級分離可利用免疫沈澱及親和 力層析術利用病患IgE抗體及離子交換層析術而完成。 實施例5 (主要過敏原及其之經加熱製劑的點墨法檢測） 於將經浸泡PBS之PVDF薄膜裝設於點墨裝置中之後， 使經純化之主要過敏原（卵蛋白、卵類黏蛋白、卵輸鐵蛋 白、溶菌酶、酪蛋白、乳球蛋白、α -乳白蛋白、α -澱 粉酶抑制劑及麥膠蛋白）及其之經加熱製劑吸收至薄膜，將 其以經加入3 % R S Α之T B S封阻，並以T B S Τ洗滌。將對 抗蛋過敏原部分、奶過敏原部分及小麥過敏原部分之抗體 (經稀釋2000倍）加至薄膜，並使其在室溫下靜置1小時。 接著將經接合生物素之抗兔子IgG綿羊抗體及HRP-接合抗 生物素蛋白質（4000倍）加至薄膜，將其洗滌並進行經由與 化學發光試劑反應所產生之光子的測定及於感光性薄膜上 檢測。 前述之抗體檢測卵蛋白、卵類黏蛋白、卵輸鐵蛋白、溶 菌酶、酪蛋白、Θ -乳球蛋白、α -乳白蛋白、α ·殿粉酶抑 制劑及麥膠蛋白之主要過敏原。此外，抗體檢測前述經純 化過敏原之經加熱製劑。 實施例6(利用三明治ELISA檢測蛋、奶及小麥過敏原） U)抗體 22 32ό\專利說明書(補件)\92-〇4\92101099 200413715 將於實施例4(4)中製備得之對抗蛋、奶及小麥之過敏原 部分的抗體及根據習知之方式製備得之與生物素接合的抗 體使用於下。 (2) 將抗體塗布於微量滴定板上並封阻 將100微升份量的前述抗體（10微克/毫升）分配於 ELISA板（Nunc)的槽中，使其在4 °C下靜置隔夜，洗滌（經 加入 150 mM-NaCl 及 0.05% Tween 20 之 2〇 mM Tris-HCl 緩 衝液，p Η 7.4)，並以0 · 1 % R S A ( S i g m a)在2 5 °C下封阻1小 時。 (3) 蛋、奶及小麥過敏原之檢測 於將槽中之封阻溶液移除後，將95微升份量之稀釋劑 (經加入 0.1%-RSA、150 mM-NaCl 及 0.05% Tween 20 之 20 m Μ Τη s-Η Cl緩衝液，pH 7.4)及5微升份量之來自各種食 物的PBS萃取物加至槽，並使其在25 °C下靜置2小時。同 樣地，將5微升份量之於實施例1中提及之蛋、奶及小麥 標準抗原的懸浮液加至槽，並使其在25 °C下靜置2小時。 於以300微升洗滌液體洗滌5次後，將100微升份量之經 接合生物素的抗體（ 1 0000倍稀釋）加至槽，並使其在25 °C 下靜置1小時，然後再將1 00微升份量之經接合過氧化酶 的抗生物素蛋白質（25 00倍稀釋）加至槽，並使其在25 t下 靜置30分鐘。於洗滌後，將100微升份量之3,3’，5,5’-四 聯苯胺溶液加至槽，並使其在25 °C在遮光下反應30分鐘。 然後加入1 00微升份量之1 N硫酸，以停止反應。利用微 量滴定板讀數計讀取槽之光學密度（分別在主及次波長之 23 326\專利說明書(補件)\92-04\92101099 200413715 45 0及6 3 0奈米下）。 結果示於表1。如表1所顯示，可檢測各種食物萃取物 中之蛋、奶及小麥過敏原。 表1.各種食物中之蛋、奶及小麥的檢測 食物 對抗蛋過敏原 部分之抗體· 對抗奶過敏原 部分之抗體 對抗小麥過敏原 部分之抗體 煮熟的蛋 〇 X X 奶 X 〇 X 麵包(於實驗室中 X X 〇 製備） 布丁(於實驗室中 〇 〇 X 製備：含蛋及奶） 法國吐司（於實驗 〇 〇 〇 室中製備：含蛋、 奶及小麥） 〇；檢測到。X ;未檢測到。（以下表中相同) 實施例7(利用三明治ELISA方法之食物過敏原檢測套組之 基礎性質的評估） (1) 標準抗原之稀釋試驗 根據實施例6，將於實施例1中製備得之蛋、奶及小麥 標準抗原定量。將此等結果繪於繪圖紙上，其證實可得到 幾乎延伸通過原點的校準曲線。 (2) 同時測定之間之再現性的評估 24 326\專利說明書(補件)\92_〇4\92101099 200413715 根據前述之方法，製備在可檢測範圍內之五個不同濃度 之蛋標準抗原的樣品A至E，並進行五個同時測定。如表 2所示，得到低於5 %之C V値。因此，證實優異的同時再 現性。 表2.在利用三明治ELISA方法之蛋標準抗原檢測之同時測定之間之再現性 的評估 1 2 3 4 5 平均 SD CV(%) A 1.165 1.079 1.068 1.075 1.064 1.090 0.042 3.87 B 0.756 0.711 0.699 0.689 0.693 0.710 0.027 3.84 C 0.544 0.505 0.500 0.497 0.507 0.511 0.019 3.74 D 0.233 0.222 0.236 0.222 0.227 0.228 0.006 2.79 E 0.175 0.165 0.179 0.173 0.170 0.172 0.005 3.06 (3)在每日測定之間之再現性的評估 根據前述之方法，製備在可檢測範圍內之五個不同濃度 之奶標準抗原的樣品A至E，並進行連續5天的測定。如 表3所示，得到低於5 %之CV値。因此，證實在每日測定 之間之優異的再現性。 25 326\專利說明書(補件)\92-〇4\92101099 200413715 表3.在利用三明治ELISA方法之奶標準抗原檢測之每日測定之間之再現性 的評估 1 2 3 4 5 平均 SD CV(%) A 2.356 2.387 2.346 2.321 2.241 2.330 0.055 2.37 B 1.353 1.306 1.274 1.246 1.254 1.287 0.044 3.40 C 0.956 0.949 0.928 0.921 0.899 0.931 0.023 2.45 D 0.295 0.292 0.275 0.281 0.278 0.284 0.009 3.10 E 0.195 0.183 0.186 0.184 0.182 0.186 0.005 2.82 此等結果顯示本方法可快速及穩定地檢測分佈於食物及 其成份中之多種天然及變性食物過敏原。經證實此方法可 檢測0 · 5毫微克/毫升或以上之食物過敏原或含食物過敏 原之食物。 實施例8 (食物過敏症病患帶有對抗天然及經加熱食物過敏 原兩者之IgE抗體） 舉蛋過敏症爲例，經證實食物過敏症病患不僅帶有對抗 天然食物過敏原之IgE抗體，並且亦帶有對抗經加熱食物 過敏原之IgE抗體。換言之，將全蛋溶解於PBS(1.0%，w/v) 中。一半的溶液不經加熱（未經加熱之蛋抗原），及將另一 半的溶液在120°C下加熱30分鐘（經加熱的蛋抗原）。將100 微升份量之各經稀釋10倍的溶液分配於ELISA板之槽 中’將其洗滌，以經加入1 % H A S之P B S封阻並洗滌。因 此’製備得經塗布天然蛋抗原之板及經塗布經加熱蛋抗原 之板。 接下來’將天然蛋抗原或經加熱之蛋抗原（1、5、5〇、1〇〇、 26 326\專利說明書(補件)\92-〇4\92101099 200413715 5 0 0及1 0 0 0毫微克/毫升）加至說明於實施例4 (1)中之匯集 病患血淸（1 0 0 0倍稀釋），在3 7 °C下反應2小時，並離心。 如此製備得兩種血淸。分別將前者及後者稱爲不含天然蛋 抗原之血淸及不含經加熱蛋抗原之血淸。 然後將各100微升份量之不含天然蛋抗原之血淸及不含 經加熱蛋抗原之血淸分配於各經塗布天然蛋抗原之板及經 塗布經加熱蛋抗原之板上。使其在3 7 °C下靜置2小時，並 以PBS丁洗滌。將100微升份量之經接合生物素之抗人類 IgE- ε山羊抗體（25 00倍稀釋）加至板，使其在37t下靜置 1小時，並以P B S T洗滌。於加入經接合鹼磷酸酯酶之抗生 物素蛋白質及發光基質並在37 °C下靜置30分鐘後，讀取 發光度（照度計CT-9000D，Diatron)。結果示於圖2。 如圖2所示，經證實不含經加熱蛋抗原之血淸（於圖2中 示爲〇）可特異地與天然蛋抗原反應（圖2之左圖），但血淸 則無法與經加熱抗原反應（圖2之右圖）。同時，經證實不 含天然蛋抗原之血淸（於圖2中示爲_)可特異地與經加熱 蛋抗原反應（圖2之右圖），但血淸則無法與天然抗原反應 (圖2之左圖）。 由此等結果，證實食物過敏症病患帶有明確辨識天然及 經加熱蛋兩者的IgE抗體。 因此，檢測食物過敏原之方法應可檢測經加熱及天然過 敏原兩者。 實施例9(利用由經加熱抗體之免疫作用製備得之抗體增強 ELISA強度） 27 326\專利說明書(補件)\92-04\92101099 200413715 即使使如實施例4中所說明之抗過敏原部分抗體進行測 定，由在抗體與經加熱樣品之間之反應製得之ELISA強度 (光學密度，OD値）亦可能較由在抗體與天然樣品之間之反 應所得者低。此可能係因（1)自經加熱樣品萃取得之蛋白質 濃度較自天然樣品萃取得者低；及（2)在經加熱樣品與抗體 之間的反應性較在天然樣品與抗體之間的反應性低，即使 樣品之蛋白質濃度經均勻調整。因此，硏究具增進反應性 之抗體的製備。 將如實施例4( 1)中說明之蛋過敏原部分於熱壓釜中在 120°C下處理30分鐘，冷卻，與經加入4M尿素之PBS均 質化，並離心。將上澄液冷凍乾燥及微細硏磨而製備得抗 原，使其進行如實施例3 (1)中所說明之兔子抗體之製備（以 下稱爲抗經熱壓釜處理之蛋抗原的抗體）。根據如實施例6 所說明之方法，使抗體與如實施例8所說明之經塗布天然 蛋抗原之板及經塗布經加熱蛋抗原之板進行反應。 結果示於表4。表4顯示天然及經加熱蛋抗原係以與經 由使來自經熱壓釜處理之食物過敏原之萃取物免疫而製備 得之抗體幾乎相同的ELISA強度檢測得。因此，前述的問 題經解決。 28 326\專利說明書(補件)\92·〇4\92101099 200413715 表4.利用經由使經加熱抗體免疫而製備得之抗體增強ELISA強度 抗體 ELISA強度 經塗布天然蛋抗原 之板 經塗布經加熱蛋 抗原之板 抗蛋過敏原部分抗體 100 36 抗經熱壓釜處理之蛋 100 118 抗原抗體 將經由在抗蛋過敏原部分抗體與標準蛋抗原板之間之反應 而得之ELISA強度（OD値）示爲100。 實施例10(食物過敏原及引起食物過敏症食物之測定-1) 檢驗本發明之辨識多種抗原之抗體（如說明於實施例3 中者）及如說明於實施例7中之方法是否可檢測含食物過 敏原之食物。使用如實施例7中之說明製備得的校準曲線 進行定量及計算定量指數（測量値/實際値X 1 00)。表5顯 示蛋黃、蛋白、蛋黃醬及全蛋蛋黃醬的結果。 當使由位於蛋白中之單一抗原製備得之抗體進行測定 時，（1)無法檢測得蛋黃，而僅可檢測蛋白，及（2)利用校準 曲線測得之標準蛋抗原的量較實際値多二至60倍。 當使本發明之辨識多種抗原之抗標準蛋抗原之抗體進 行測定時，（3)可檢測得蛋黃及蛋白兩者，及（4)標準蛋抗原 之測定量與實際値幾乎相同。由此等結果，注意到本發明 之辨識多種抗原之抗標準蛋抗原抗體可檢測及定量經加工 蛋食品中之蛋或其成份之引起過敏症食物。 29 326\專利說明書(補件)\92-04\92101099 200413715 表5.利用各種抗體檢測及/或定量蛋及蛋產品之可能性 樣品 測定 辨識多種抗 原之抗體(抗 標準蛋抗原 抗體） 辨識單一抗 原之抗體(抗 卵類黏蛋白 抗體） 辨識單一抗 原之抗體(抗 卵蛋白抗體） 蛋黃 檢測之可能性 〇 X X 定量指數 74-106 蛋白 檢測之可能性 〇 〇 〇 定量指數 105-170 240-310 6,330-13,150 蛋黃醬 檢測之可能性 〇 X X 全蛋蛋 檢測之可能性 〇 〇 〇 黃醬 實施例1 1(食物過敏原及引起過敏症食物之測定-2) 如實施例1 0中之說明檢測乳漿、酪蛋白、乳鐵蛋白、及 水解酪蛋白。結果示於表6。 表6顯示（1)本發明之辨識多種抗原之抗標準奶抗原抗體 可檢測及定量引起食物過敏症之奶及其之成份；但（2)經由 使位於乳漿中之物質免疫而製備得之辨識單一抗原之抗體 則無法將其定量。表6中之△之符號顯示可完成定量，但 定量指數低於5。 30 326\專利說明書(補件)\92-04\92101099 200413715 表6.利用各種抗體檢測及/或定量標準奶抗原 樣品 測定 辨識多種抗 原之抗體(抗 標準奶抗原 抗體） 辨識單一抗 原之抗體(抗 酪蛋白抗體） 辨識單一抗 原之抗體(抗 /5-乳球蛋白 抗體） 乳漿 檢測之可能性 〇 X 〇 定量指數 160-230 320-1,080 酪蛋白 檢測之可能性 〇 〇 X 定量指數 38-47 170-180 乳鐵蛋 檢測之可能性 〇 △ X 白 水解酪 檢測之可能性 〇 X △ 蛋白 【圖式簡單說明】 圖1顯示在標準抗原與病患IgE抗體之間的反應，及在 標準抗原與對抗本發明之標準抗原抗體之抗體之間的反 應。 圖2顯示病患血淸帶有對天然及變性蛋之蛋白質兩者特 異之IgE抗體。 31 326\專利說明書(補件)\92-04\92101099200413715 发明 Description of the invention [Technical field to which the invention belongs] The present invention relates to a mixture containing a variety of natural and / or denatured food allergens recognized by IgE antibodies of food allergic patients, and is prepared by using the mixture to immunize animals The obtained antibody, and a method for using the antibody to detect food allergens and foods that cause allergies. Since the present invention has a mechanism for understanding the onset of food allergies, generating and confirming hypoallergenic foods, and detecting food allergens in foods, their raw materials, and food manufacturing environments such as food manufacturing machines and processes, to provide food allergies Suffering from safety. [Prior art] Food allergies are harmful immune responses in sensitive people. Food allergies are caused by allergic substances (hereinafter referred to as food allergens) ingested into food. Food allergies can cause dermatitis, asthma, digestive disorders, anaphylactic shock, and more. Allergies are classified as types I to IV depending on the mechanism by which the disease begins. Food allergies are mainly caused by type I allergies, where IgE antibodies react with food allergens that are ingested into the body. The number of food allergies has gradually increased in recent years. This phenomenon creates serious problems in the fields of medical science and the food industry. To prevent this hazard, consumer information is required through labeling. The Joint FAO / WHO Food Standards Committee (Codex A1 imentarius C ommisi ο η) recognizes the demand for foods containing any one of eight raw materials known to be food allergens, and recommends that Member States implement 6 326 \ Patent Specification (Supplement) \ 92 · 04 \ 92101099 200413715 Marking System (June 1999). In Japan, in consideration of the severity and frequency of food allergies, 24 items have been issued (implemented since April 2QQ2). It should be noted that these regulations neither require the labelling of allergens nor the labelling of food allergens, but require the labelling of foods containing food allergens, ie foods that cause allergies. Foods that cause allergies include eggs, milk, meat, fish, crustaceans, molluscs, cereals, beans, nuts, fruits, vegetables, beer yeast, and gelatin. Similarly, it is known that egg proteins, ovomucoids, lysozyme, casein, 0_lactoglobulin, α-lactalbumin, bran protein, α-amylase inhibitors, etc. are foods that cause allergies. In addition, (1) regardless of the current identification, it is believed that there are still many other foods and ingredients (food allergens) that can cause food allergies 2) foods and ingredients (food allergens) that cause allergies are quite diverse and food allergies Patients have different reactions to these allergens; and (3) as shown in the examples described later, 'even in a single allergenic food, there are many known and unknown allergens . However, none of the known methods can easily detect so many allergic foods or food allergens. Food is prepared by using procedures such as heating, freezing, drying, salting, fermentation, enzyme treatment, etc. (hereinafter referred to as food process) to improve its digestibility, storage life, taste, physical properties, etc. and to stabilize it Into. Although food processes affect proteins and modify their molecular structure (for example, protein denaturation), few people have discussed whether such food processes can cause the production of food allergens. The research work to develop the present invention clarifies the following: (1) some heated 7 326 \ patent specifications (supplements) \ 92-04 \ 92101099 200413715 food ingredients will show allergies; (2) ingredients that cause allergies or The epitope of individual foods that cause allergies will change depending on whether the food of interest is heated. In other words, preparing a) unheated egg antigen, b) heating egg antigen prepared by heating the antigen in (a), and c) collecting blood maggots from multiple food allergy patients (called Pooled blood sacrifice of patients) and (a) (free blood sacrifice of unheated egg antigen) prepared by mixing, and d) pooled sera of patients and (b) (excluding heated eggs) Antigen blood pupae) are prepared by mixing blood pupae. The results of reactions between a) and c) or a) and d) and between b) and c) or b) and d) show that blood cells that do not contain unheated egg antigen cannot The egg antigen is immunoreactive, but can react with the heated egg antigen: Similarly, it is shown that the blood pupae that do not contain the heated egg antigen cannot immunoreact with the heated egg antigen, but can react with the unheated egg antigen. In summary, patients with food allergies carry IgE antibodies specific to both processed and unprocessed foods, leading to food allergies. However, to date there is no easy way to detect allergens in processed foods or their ingredients. Methods for screening food allergens in eggs, peanuts, casein, / 3-lactoglobulin, and bran protein are commercially available (Food and Development, Vol. 35, pp. 10-11). However, all or some of them have one or more of the following disadvantages: (1) The method does not always detect allergens to which food allergy patients are allergic. In other words, it is not always detectable by the patient's IgE antibodies; (2) methods can only detect known allergens, and each method can only detect a single allergen (single antigen); (3) use The single antigen detection antibody method cannot be applied to foods with inhibitory substances; (4) the method using single antigen detection antibody cannot be 8 326 \ Patent Specification (Supplement) \ 92-04 \ 92101099 200413715) 1¾ • such as τρ; Accurate quantification of allergenic foods in the examples described later; (5) The method using single antigen detection antibodies cannot be applied to the analysis of allergenic foods that do not contain antigen (for example, by using The method of single antibody prepared from egg protein cannot be applied to the analysis of egg yolk, mayonnaise, etc.); (6) The method of using a single antigen detection antibody cannot be used for the analysis of denatured or molecularly modified allergens. Processed foods; and (7) monoclonal antibodies against natural and denatured peptone-lactoglobulin, egg protein, and keratin have been published (Allergy, No. 50 volumes ’3 09 pages). Monoclonal antibodies only detect individual epitopes in food allergen molecules, so if the epitope is removed or modified by the food process, its effectiveness will be reduced. Although it has been published to use blood from patients allergic to rice (Japanese Patent Disclosure 2000-65820), eggs and milk (Japanese Meat Science, Vol. 39, pp. 166-169)方法 The method for screening food allergens, but it is only used in hospitals, not for inspection agencies and food manufacturing plants. Although food allergen detection methods based on allergic reactions have been published (FFI I, No. 180, 77-82), they cannot be used in most foods due to complex methods and laboratory-animal husbandry. Manufacturing plant. Although methods have been developed that use mobile systems, enzyme-labeled antibodies, and allergen sensors using microelectrodes (Japanese Food Industry, Vol. 42, pp. 53-56), these methods can be put to practical use Before that, there were still many issues to be resolved. SUMMARY OF THE INVENTION The present invention is to solve the problems of the aforementioned conventional technologies. The purpose of the present invention is 9 326 \ Patent Specification (Supplement) \ 92-04 \ 92101099 200413715 To provide (1) a variety of known and / or unknown and natural and / Or a mixture of denatured food allergens (referred to as the first invention of the present invention for convenience), (2) an antibody prepared by immunizing animals with the mixture (referred to as the second invention of the present invention) ), And (3) —a method for detecting food allergens and foods that cause allergies (referred to as the third invention of the present invention). [Embodiment] The first invention of the present invention is completed by the following procedures: (I-i) separating IgE antibodies from pooled blood cells of food allergic patients, and (I-ii) by using the aforementioned IgE antibodies and Immune methods such as affinity chromatography and immunoprecipitation isolate a variety of substances from foods or their components (hereinafter collectively referred to as foods) that have not been processed by a food process (in other words, they are recognized by IgE antibodies in food allergic patients Multiple food allergens). The first invention of the present invention can also be completed by the following procedures: (2-i) Perform conventional SDS-PAGE using food ingredients, and then transfer the gel to the film, (2-η) Use the film to collect patients Blood 淸, anti-human IgE antibodies, and coloring reagents were subjected to western blotting (in other words, the distribution of multiple food allergens identified by IgE antibodies in food allergic patients), (2 -in) Similarly, the western blot method was performed using a thin film, animal blood pupa prepared by immunizing food ingredients in a conventional manner, joining anti-animal IgG, and a coloring reagent, (2 μv) comparing the two western blots Its distribution. The measurement does not appear in the former, but appears in 10 326 \ Patent Specification (Supplements) \ 92-04 \ 92101099 200413715 The molecular size of the band in the latter (in other words, multiple substances that are not recognized by IgE in food allergy patients), and (2-v) Depending on the foregoing results, a variety of food allergens recognized by IgE antibodies of food allergic patients are separated from food ingredients using conventional methods such as gel filtration chromatography. The second invention of the present invention is based on the use of a variety of food allergens (including processed and / (Or unprocessed) animal antibodies against food allergens prepared by immunizing animals. The third invention of the present invention relates to a method for detecting food allergens and foods that cause allergies using antibodies that recognize multiple antigens. The aforementioned heat treatment temperature range is equivalent to the range of food processing, preferably between 40 and 25 0 ° C, and more preferably between 60 and 120 ° C. Antigen lines are prepared by mixing the original antigens and heating them at two to six different temperatures. The food allergen portion identified by the I g E antibody of a food allergy patient was prepared from a mixture using the first invention of the present invention, and used it to immunize the animal. When the detection target is limited to heated food, the food allergen portion identified by the IgE antibody of a food allergy patient is prepared from the heated food using the first invention of the present invention, and uses it to make Animal immunity. Examples of animals are rabbits, goats, sheep, rats, mice, guinea pigs, horses, pigs, chickens, and the like. During the period of immune action, partial collection of blood and detection of anti-food allergen antibodies have better titers. The antibodies of the present invention may be single or multiple antibodies. As shown in the example described later, even in allergic foods that are tied to a single 11 326 \ Patent Specification (Supplements) \ 92-〇4 \ 92101099 200413715, there are 1 g of multiple food allergic patients in E Known and unknown food allergens recognized by antibodies. This is why polyvalent antibodies that recognize multiple food allergens, that is, antibodies that recognize multiple antigens can be easily prepared by immunizing animals with these multiple food allergens. Animal anti-blood can be used as an antibody against food allergens recognized by IgE antibodies in food allergic patients. The antiserum can be used after the absorption treatment of the part which is not recognized by the IgE antibody of the food allergy patient. Antiserum can be used after the conventional IgG purification. The method for detecting food allergens and foods that cause food allergies of the present invention can be applied to foods containing allergens without any restrictions. Examples of foods that cause allergies are eggs, milk, meat, fish, crustaceans, molluscs, cereals, beans, nuts, fruits, vegetables, beer yeast and gelatin. More specifically, protein, egg yolk, milk, cheese, pork, beef, chicken, lamb, mackerel, horse mackerel, sardines, anchovies, salmon, cod, flounder, salmon caviar, crab, shrimp, mussels ( moule), squid, octopus, lobster, abalone, crustaceans, molluscs, wheat, rice, rose rye, rye, barley, reduced wheat, corn, millet, foxtail millet (f 0xta 丨 1 mi 11 et), Coriander, cereal, soybean, peanut, cocoa, pea, kidney bean, hazelnut, brazilian fruit, almond, coconut, walnut, ugly, nut, apple, banana, orange, peach, kiwi, strawberry, melon, avocado, Grapefruit, mango, pear, sesame, mustard, fruit, tomato, carrot, potato, spinach, onion, garlic, bamboo shoot, pumpkin 'sweet potato, celery, parsley, potato, Japanese pine quince, vegetable, contains one or more of this And other items and their ingredients (eg, egg protein, ovomucoid, lysozyme, casein, stone-lactoglobulin, α-lactalbumin, fascination 326 \ Patent Specification (Supplement) \ 92 · 〇4 \ 921 〇1 〇99 200413715 Protein, and alpha-amylase inhibitors). Such foods can be prepared using procedures such as heating, freezing, drying, salting, fermentation, enzyme treatment, and the like. Allergens identified by Ig E antibodies in food allergy patients with a 50% or higher probability are referred to as primary allergens, and those with less than 50% are referred to as secondary allergens. Food allergies are caused not only by major allergens but also by minor allergens. Some patients will not be allergic to major allergens but will be allergic to minor allergens. Therefore, methods for screening food allergens should detect both major and minor allergens. The screening method of the present invention can detect a variety of natural and denatured allergens recognized by IgE antibodies of food allergic patients. To prepare antibodies that meet these needs, the inventors have successfully discovered the following. In other words, the conventional SDS-PAGE analysis of the food ingredients and the transfer of the gel to the film: (1) Western blotting method using the film, pooling the patient's blood pupae, joining anti-human IgE antibodies, and coloring reagent (Π) Similarly, Western blotting was performed using a thin film, blood sacrifice prepared by immunizing food ingredients to animals in a conventional manner, conjugating anti-animal IgG antibodies, and coloring reagents, and (iii) using two western inks. The comparison of the point method distributions observed the following facts: (1) there are bands dyed in both films, (2) there are bands that are not dyed in the former but dyed in the latter (that is, a variety of non-allergic substances) , And (3) bands distributed in higher and / or lower molecular weight positions than those dyed in the former and dyed in the latter only 13 326 \ Patent Specification (Supplement) \ 92-04 \ 92101099 200413715; and (1 V) From the foregoing results, gel filtration chromatography was used to separate multiple food allergens identified by IgE antibodies from food allergic patients from food ingredients and then immunize animals to prepare multiple food allergens Antibodies. Immune to isothermal animals such as rabbits, goats, sheep, rats, mice, guinea pigs, horses, pigs, and chickens. Any immunization method known to those skilled in the art can be used. The antibody prepared as described above can be used in the food allergen detection method of the present invention. Antibodies can be fixed on microtiter plates, PVDF films, nitrocellulose films, chroma to strips, test tubes, beads, nylon films, etc. Antibodies can be applied to immunological methods such as enzyme immunoassay, immunoblotting, dot blotting, immunoassay, and antibody wafers (an t i b 0 d y c h i p). As mentioned before, the object of the food allergen detection method of the present invention is to detect a variety of food allergens in food and food manufacturing environments such as food manufacturing machines and procedures. Liquid extracts from food are better for analysis. Although water is the preferred extractant, phosphate buffered saline, Tris-HCl buffer, alcohol, etc. can be used as long as the allergen can be extracted. To improve the efficiency of extracting allergens from food, if necessary, protein denaturing agents (for example, SDS or urea), SH-containing antioxidants (for example, 2-thiol ethanol), etc. can be added to the extracting agent. Various allergens can be measured by wiping (smearing) the food manufacturing environment or by trapping its air in a wash bottle. As long as the principle of the food allergen detection method of the present invention is immunology, the method is not limited to a specific method. The Meiji ELISA (sandwich 14 326 \ Patent Specification (Supplement) \ 92-04 \ 92101099 200413715 ELISA), competitive method, direct method, etc. are examples of enzyme immunoassays. When labeling antibodies, enzymes (for example, peroxidase, alkaline phosphatase, and / 3-galactosidase), fluorescent substances (for example, fluorescent isothiocyanate), bioluminescent substances (for example, , Luciferin-luciferase), chemiluminescent substances (for example, lunnnol, acridine derivatives and adamantane derivatives), biotin, avidin, gold colloid, radioactivity Materials (for example, 32P) and so on. As examples of the detection method of the present invention, sandwich ELISA, competition and direct methods are described below. To perform a sandwich ELISA, labeled antibodies (labeled antibodies that recognize multiple antigens) that recognize multiple allergens of the present invention are prepared. The unlabeled antibodies that recognize a variety of antibodies of the present invention are immobilized on an ELISA plate by adsorption or chemical binding. Use proteins that do not interfere with the reaction system, such as gelatin and rabbit blood albumin, to block the plate in areas where antibodies have not been immobilized. An extract (hereinafter referred to as a sample) or standard antigen taken from food, food ingredients, or food manufacturing environment is added to the plate to perform a first antigen-antibody reaction. After the reaction, the plate was washed. To perform the second antigen-antibody reaction, the aforementioned labeled antibody is added to the plate 'where the allergen is captured by the immobilized antibody. After washing and removing unreacted labeled antibodies, detection reagents (eg, 1,2-phenylenediamine and H2O2 in the case of peroxidase: phosphoric acid in the case of alkaline phosphatase) are added. P-nitrophenyl ester). The allergen is detected or quantified by measuring the amount of reactants' between the label and the detection reagent. The calibration curve can be obtained by changing the standard antigen on the ELISA plate or the amount of food (such as eggs and milk) that causes food allergies. Food over 15 326 \ Patent Specification (Supplement) \ 92- (Μ \ 92) 〇10〇99 200413715 The amount of allergens or foods that cause allergies can be quantified through a calibration curve. If the quantification is 値 1 ppm or more, The sample may be a food that causes allergies. The method of using a single antigen detection antibody will cause a quantitative error because it is necessary to convert the maggot obtained from a single antigen detection antibody to the content of a food that causes allergies. For example, eggs in whole eggs The concentrations of mucin-like protein and egg protein are approximately 4% and 2%, respectively. The concentrations in the protein are approximately 8% and 4%, respectively. However, egg yolk contains neither oval mucin nor egg protein. Therefore, ( 1) The method of detecting only ovomucoid or egg protein cannot be used to determine egg yolk protein; (2) When the calibration curve prepared from whole eggs is used to determine protein, it will be quantified more than twice as much ; And (3) When the actual radon exceeds the quantitative range, an extremely high quantitative radon will be produced. Similarly, since the concentrations of casein and / 3-lactoglobulin in whole milk protein are about 80% and 1 respectively. 0% because It has the problem that antibodies that recognize a single antigen cannot accurately quantify processed milk with enhanced casein sodium, lactoprotein, etc. However, the antibodies that recognize multiple antigens of the present invention do not have or have fewer such problems. For the competition method, a certain amount of standard antigen is directly immobilized on the solid phase, and it is blocked with non-reactive protein. Then the antibody and sample that identify the allergen and the enzyme are conjugated to the plate at the same time. The plate is allowed to stand for a certain period of time. Time, and washing to remove unbound material. A colored substrate is added to the plate. The reaction between the immobilized allergen and the enzyme-conjugated antibody pretreated with the sample is stopped and measured. 16 326 \ Patent Specification (Supplement) \ 92-04 \ 92101099 200413715 For the direct method, the sample is directly adsorbed on the solid phase, blocked with non-reactive protein, and reacted with the enzyme-conjugated antibody that recognizes the allergen. Then apply The same procedure as previously described to detect allergens in a sample. For any of the foregoing methods, fluorescent, bioluminescent, and chemiluminescent substrates can be used for color development. Those skilled in the art can modify other conditions of the food allergen detection method of the present invention according to the purpose, sample, measurement principle, etc. The food allergen detection method of the present invention can detect 0. 0 from extracts of food and its raw materials. 1 or 1. Allergens of 0 nanograms (ng) or above. (Industrial applicability) A mixture containing a variety of natural and / or denatured food allergens recognized by IgE antibodies of food allergic patients and the antibodies of the present invention are useful for understanding the mechanism of the onset of food allergies and the development of food allergen reduction Allergic technology. In addition, the food allergen detection method of the present invention is useful for confirming the effectiveness of a technique for reducing allergies, and detecting foods, foods that cause allergies, and food allergens in food manufacturing environments such as machines and procedures. Therefore, the present invention is useful for providing the safety of food allergy patients and for solving the medical and industrial problems caused by the recent increase in the number of food allergy patients. (Examples) The present invention will be described in more detail by the following examples, but it should not be construed as limiting the scope of the present invention. The abbreviations used in this specification are commonly used in the technical field. Example 1 (Preparation of standard antigens for various foods) 17 326 \ Patent Specification (Supplement) \ 92-04 \ 92101099 200413715 (1) Chicken, quail and duck eggs 1 kg of eggs are shelled, broken, freeze-dried and dried. Finely pulverized to prepare a standard egg antigen. Suspend 10 grams of the antigen in 10 volumes of pH 7. Phosphate buffered saline (hereinafter abbreviated as PBS) in 0, and then dispensed into 5 test tubes, and heated for 30 minutes without heating or at 60, 80, 100, and 12 CTC. The samples were then mixed together and homogenized. Samples of quail and duck eggs were prepared in the same way. (2) Milk Stir 1 liter of milk to solidify the milk fat and precipitate, and filter it through absorbent cotton. After repeating this procedure three times, the filtrate was freeze-dried and then finely pulverized to prepare a standard milk antigen. Similarly, prepare samples as described in (1). (3) Wheat and rice 1 kg of wheat flour was stirred at room temperature for 2 hours, and 4 ^ 4 of urea was added in a 5 times amount. 1 "1 ^ 8-11 (: 1 buffer (? 118. 6) Extract and centrifuge again. The supernatant was dialyzed, freeze-dried and then finely pulverized to prepare a standard wheat antigen. Similarly, a sample was prepared as described in (1). Samples were similarly prepared from rice flour. (4) Qiao Mai 1 kg of rose wheat flour is stirred at room temperature for 2 hours, and 5% of the amount is added by 1% to (: 1 to 0. 1 "Ding 1 Qi -11 (: 1 buffer (1) 118. 4) Extract and centrifuge again. The supernatant was dialyzed, freeze-dried, and then finely pulverized to prepare a standard Qiaomai antigen. Similarly, a sample was prepared as described in (1). 326 \ Patent Specification (Supplements) \ 92-〇4 \ 92101099 18 200413715 (5) Peanuts 1 kg of peanuts was honed and degreased in 5 times the amount of Zhengjiyuan by stirring at room temperature for 2 hours. After repeating this procedure 3 times and then removing the n-hexane ', by stirring at room temperature for 2 hours, 5% of the amount of 1% NaCl was added to 0.1 M T r 1 s-H C1 buffer. Liquid (ρ Η 8 · 4) extract the preparation, and then centrifuge. The supernatant was dialyzed, freeze-dried, and then finely honed to prepare a standard peanut antigen. Similarly, a sample was prepared as described in (1). (6) Soybean Prepare standard soybean antigen as described in (5). Similarly, prepare samples as described in (1). Example 2 (Various purified food allergens) (1) Purified egg allergens Egg protein (the main allergen for eggs) was suspended in a volume of 10 times ρ Η 7. 0 P B S and distribute to 5 test tubes and heat them without heating or at 60, 80, 100 and 120 ° C for 30 minutes. The samples were then mixed together and homogenized. A sample of ovomucoid was prepared in the same manner. Ovalin and ovomucoid are proteins located in proteins. (2) Purified milk allergen Samples of casein, / 3-lactoglobulin, and α-lactalbumin were prepared as described in Example 2 (1). / 3-lactoglobulin and α-lactalbumin are proteins in the milk. Example 3 (Preparation of antibodies) (1) Preparation of rabbit antibodies against various standard food antigens Complete Freund adjuvant (used for the first immune effect) and 19 326 \ Patent Specification (Supplement) \ 92-04 \ 92101099 200413715 Incomplete adjuvant (used for second and subsequent immunization) will emulsify each sample prepared in Example 1 and immunize Japanese white rabbits 4 to 6 times. At the same time, blood was partially collected to confirm the production of antibodies against each antigen. Collect all blood and prepare antibodies. (2) Preparation of antibodies against various purified food antigens Similarly, antibodies against various purified food allergens were prepared as described above. Example 4 (Detection of various food allergens by immunodot method) (1) Pooling of blood pupa and rabbit antibodies from patients By passing 20 or more (specific IgE antibodies> 0. 7UA / mL) The same amount of blood pupa was mixed against patients with RAST (Radiation Allergy Absorption Test) scores of eggs, milk or wheat to prepare pooled pupa. A rabbit resistance system against eggs, milk and wheat was prepared in Example 3 (1). (2) Sodium lauryl sulfate · polyacrylamide gel electrophoresis (SDS-PAGE) The aforementioned standard egg, milk, and wheat antigens were heated with 2-thiol ethanol for 3 minutes, and then placed on a mini-thick plate with a concentration of 10% Electrophoresis in gel. The gel was then transferred to a PVDF (polyvinylidene fluoride) film. A portion of the film was stained for protein using a gold colloid staining kit (BioRad). (3) Immunostaining The aforementioned PVDF film was blocked with 1% human serum albumin (HSA). Place the film with 0. Wash with 05% Tween 20 in PBS (PBST), react with pooled patients' blood pupa (100-fold dilution) or rabbit antibody (100-fold dilution) at room temperature for 2 hours, wash, and then conjugate with Alkaline phosphatase anti-human IgE-ε chain goat antibody secondary antibody (25 00 times dilution) or conjugate base 20 326 \ Patent Specification (Supplement) \ 92-04 \ 92101099 200413715 Phosphatase anti-rabbit IgG (4000-fold dilution) for 1 hour at room temperature. The film was washed with PBST, and 4-methyl-4 (3-phosphate phenyl) spiro [1,2-dioxirane-3,2-adamantane] disodium salt-chemiluminescent reagent (Lumi-Phos 530, Wako Pure Chemical) was treated at room temperature for 30 minutes and exposed to a photosensitive film to detect photons generated by dephosphorization of alkaline phosphatase. These results are shown in FIG. 1. From FIG. 1, it was observed that the patient's blood salamander reacted with multiple bands (lines 1, 4, and 7 of FIG. 1), and the presence of multiple bands identified by the patient's IgE in each food. At the same time, rabbit antibodies detect food allergens identified by IgE antibodies in patients with food allergies (lines 1, 5, and 8 of Figure 1). The substances identified by the two blood crickets are as follows: egg egg protein, ovomucoid, lysozyme, and egg transferrin; casein milk, Θ-lactoglobulin and α-lactalbumin; wheat gluten and α -Amylase inhibitors; 132-, 84-, 27-, and 11-kDa substances of rosewood; and 107-, 72-, 35-, and 28-kDa substances of peanut. In addition, rabbit antibodies did not detect non-food allergens that were not recognized by the patient's IgE. (4) Concentration and fractionation of allergens: Prepare antibodies that cannot identify any substance except allergens: Compare the molecular weights of the bands stained by blood sacral staining of animal patients and those stained by animal antibodies. Higher and / or lower molecular weight positions than the former (Figure 1). A fraction corresponding to the molecular weight of a food allergen recognized by an IgE antibody of a food allergy patient (hereinafter referred to as an allergen fraction) was collected from a standard antigen using gel filtration chromatography. As described in Example 丨 21 326 \ Patent Specification (Supplement) \ 92-04 \ 92101〇99 200413715 Prepare samples and immunize animals to prepare anti-allergen partial antibodies. From the western blotting method performed as described above for antibodies, it was found that the antibodies identified food allergens that were identified by pooling the patients' blood pupae (lines 3, 6, and 9 of Figure 1). The concentration and fractionation of such allergens can be accomplished by immunoprecipitation and affinity chromatography using patient IgE antibodies and ion exchange chromatography. Example 5 (Ink method detection of major allergens and heated preparations thereof) After the PVDF film soaked in PBS was installed in an inking device, the purified main allergens (egg protein, egg viscous) were purified. Protein, egg transferrin, lysozyme, casein, lactoglobulin, α-lactalbumin, α-amylase inhibitor, and gluten) and their heated preparations are absorbed into the film, which is added by 3% The TBS of RS A was blocked and washed with TBS T. Antibodies against the egg allergen portion, milk allergen portion, and wheat allergen portion (2,000-fold diluted) were added to the film, and allowed to stand at room temperature for 1 hour. Next, the biotin-conjugated anti-rabbit IgG sheep antibody and HRP-conjugated avidin protein (4000 times) were added to the film, washed, and the photons generated by the reaction with the chemiluminescence reagent were measured and applied to the photosensitive film. On detection. The aforementioned antibodies detect major allergens of egg protein, ovomucoid, ovulin transferrin, lysozyme, casein, Θ-lactoglobulin, α-lactalbumin, α · diaminase inhibitor and glutenin. In addition, the antibody detects the aforementioned heated preparation of purified allergens. Example 6 (Detection of egg, milk and wheat allergens by sandwich ELISA) U) Antibody 22 32ό \ Patent Specification (Supplement) \ 92-〇4 \ 92101099 200413715 The anti-egg prepared in Example 4 (4) Antibodies to the allergens of milk, wheat, and wheat, and biotin-conjugated antibodies prepared in a conventional manner are used below. (2) Coating the antibody on a microtiter plate and blocking the distribution of 100 microliters of the aforementioned antibody (10 micrograms / ml) in a slot of an ELISA plate (Nunc), and let it stand at 4 ° C overnight. Wash (after adding 150 mM-NaCl and 0. 05% Tween 20 in 20 mM Tris-HCl buffer, p Η 7. 4), and blocked with 0 · 1% R S A (S i g m a) at 25 ° C for 1 hour. (3) Detection of egg, milk and wheat allergens After removing the blocking solution from the tank, add 95 microliters of diluent (after adding 0. 1% -RSA, 150 mM-NaCl and 0. 05% Tween 20 of 20 m Μηη s-ΗCl buffer, pH 7. 4) and 5 microliter portions of PBS extracts from various foods were added to the tank, and allowed to stand at 25 ° C for 2 hours. Similarly, a 5 microliter portion of the suspension of the egg, milk and wheat standard antigens mentioned in Example 1 was added to the tank and allowed to stand at 25 ° C for 2 hours. After washing 5 times with 300 microliters of washing liquid, 100 microliters of the biotin-conjugated antibody (10,000-fold dilution) was added to the tank and allowed to stand at 25 ° C for 1 hour, and then A 100 microliter portion of the avidin-conjugated peroxidase protein (2,500-fold dilution) was added to the tank and allowed to stand at 25 t for 30 minutes. After washing, a 100 microliter portion of the 3,3 ', 5,5'-tetrabenzidine solution was added to the tank and allowed to react at 25 ° C for 30 minutes under light-shielding. Then, 100 microliters of 1 N sulfuric acid was added to stop the reaction. Use a microtiter plate reader to read the optical density of the slot (at 23 326 \ Patent Specification (Supplement) \ 92-04 \ 92101099 200413715 45 0 and 6 3 0 nm for the primary and secondary wavelengths, respectively). The results are shown in Table 1. As shown in Table 1, egg, milk and wheat allergens can be detected in various food extracts. Table 1. Detection of eggs, milk and wheat in various foods. Antibodies against egg allergens. Antibodies against milk allergens. Antibodies against wheat allergens. Cooked eggs 〇XX milk X 〇X bread (in laboratory) XX 〇 Preparation) Pudding (prepared in laboratory OX: containing eggs and milk) French toast (prepared in experiment OO: containing eggs, milk and wheat) 〇; detected. X; not detected. (Same in the following table) Example 7 (Evaluation of the basic properties of a food allergen detection kit using a sandwich ELISA method) (1) Dilution test of standard antigens According to Example 6, eggs prepared in Example 1 , Milk and wheat standard antigen quantification. These results were plotted on a drawing paper and it was confirmed that a calibration curve extending almost through the origin can be obtained. (2) Evaluation of reproducibility between simultaneous determinations 24 326 \ Patent Specification (Supplement) \ 92_〇4 \ 92101099 200413715 According to the method described above, five different concentrations of egg standard antigen in a detectable range were prepared. Samples A to E and five simultaneous determinations were performed. As shown in Table 2, a C V 値 of less than 5% was obtained. Therefore, excellent simultaneous reproducibility was confirmed. Table 2. Evaluation of reproducibility between assays with egg standard antigen detection using sandwich ELISA method 1 2 3 4 5 Mean SD CV (%) A 1. 165 1. 079 1. 068 1. 075 1. 064 1. 090 0. 042 3. 87 B 0. 756 0. 711 0. 699 0. 689 0. 693 0. 710 0. 027 3. 84 C 0. 544 0. 505 0. 500 0. 497 0. 507 0. 511 0. 019 3. 74 D 0. 233 0. 222 0. 236 0. 222 0. 227 0. 228 0. 006 2. 79 E 0. 175 0. 165 0. 179 0. 173 0. 170 0. 172 0. 005 3. 06 (3) Evaluation of reproducibility between daily measurements According to the method described above, samples A to E of five milk standard antigens at different concentrations within a detectable range were prepared and measured for 5 consecutive days. As shown in Table 3, a CV 値 of less than 5% was obtained. Therefore, excellent reproducibility between daily measurements was confirmed. 25 326 \ Patent Specification (Supplement) \ 92-〇4 \ 92101099 200413715 Table 3. Evaluation of reproducibility between daily measurements of milk standard antigen detection using sandwich ELISA method 1 2 3 4 5 Mean SD CV (%) A 2. 356 2. 387 2. 346 2. 321 2. 241 2. 330 0. 055 2. 37 B 1. 353 1. 306 1. 274 1. 246 1. 254 1. 287 0. 044 3. 40 C 0. 956 0. 949 0. 928 0. 921 0. 899 0. 931 0. 023 2. 45 D 0. 295 0. 292 0. 275 0. 281 0. 278 0. 284 0. 009 3. 10 E 0. 195 0. 183 0. 186 0. 184 0. 182 0. 186 0. 005 2. These results show that this method can quickly and stably detect a variety of natural and denatured food allergens distributed in food and its ingredients. This method has been proven to detect food allergens or food allergens containing 0.5 ng / ml or more. Example 8 (IgE antibodies against natural and heated food allergens in food allergic patients) Taking egg allergy as an example, it has been confirmed that food allergic patients not only have IgE antibodies against natural food allergens It also carries IgE antibodies against heated food allergens. In other words, whole eggs were dissolved in PBS (1. 0%, w / v). Half of the solution is unheated (unheated egg antigen), and the other half of the solution is heated at 120 ° C for 30 minutes (heated egg antigen). A 100 microliter portion of each diluted 10-fold solution was dispensed into a tank of an ELISA plate ', washed, blocked and washed by adding P B S with 1% H A S. Thus, a plate coated with natural egg antigen and a plate coated with heated egg antigen were prepared. Next, the natural egg antigen or the heated egg antigen (1, 5, 50, 100, 26, 326 \ Patent Specification (Supplement) \ 92-〇4 \ 92101099 200413715 5 0 0 and 1 0 0 0 Nanograms / ml) was added to the pooled patient's blood salamander (100-fold dilution) described in Example 4 (1), reacted at 37 ° C for 2 hours, and centrifuged. Thus, two kinds of blood pupa were prepared. The former and the latter are referred to as blood pupa without natural egg antigen and blood pupa without heated egg antigen, respectively. Then, each 100 microliter portions of the blood pupa without natural egg antigen and the blood pupa without heated egg antigen were dispensed on each plate coated with natural egg antigen and the plate coated with heated egg antigen. It was allowed to stand at 37 ° C for 2 hours and washed with PBS. A 100 microliter portion of the conjugated biotin-conjugated anti-human IgE-ε goat antibody (2,500-fold dilution) was added to the plate, allowed to stand at 37t for 1 hour, and washed with P B S T. After adding the antibiotic protein and the luminescent matrix conjugated alkaline phosphatase and let stand at 37 ° C for 30 minutes, read the luminescence (illumination meter CT-9000D, Diatron). The results are shown in Fig. 2. As shown in Fig. 2, blood pupa (shown as 〇 in Fig. 2) that does not contain heated egg antigen can specifically react with natural egg antigen (left picture in Fig. 2), but blood pupa cannot react with heated egg pudding. Antigen response (Figure 2 right). At the same time, the blood pupa (shown as _ in Figure 2) that has been confirmed to contain no natural egg antigen can specifically react with the heated egg antigen (Figure 2 on the right), but the blood pupa cannot react with the natural antigen (Figure 2 (Left). From these results, it was confirmed that patients with food allergy have IgE antibodies that clearly recognize both natural and heated eggs. Therefore, methods for detecting food allergens should be able to detect both heated and natural allergens. Example 9 (Enhanced ELISA strength using an antibody prepared by the immune effect of heated antibodies) 27 326 \ Patent Specification (Supplement) \ 92-04 \ 92101099 200413715 Even if the anti-allergen portion as described in Example 4 is used For the determination of antibodies, the ELISA intensity (optical density, OD 値) obtained by the reaction between the antibody and the heated sample may also be lower than that obtained by the reaction between the antibody and the natural sample. This may be due to (1) the protein concentration extracted from the heated sample is lower than that extracted from the natural sample; and (2) the reactivity between the heated sample and the antibody is higher than the reaction between the natural sample and the antibody Low performance, even if the protein concentration of the sample is evenly adjusted. Therefore, the preparation of antibodies with enhanced reactivity was investigated. The egg allergen portion as described in Example 4 (1) was processed in an autoclave at 120 ° C for 30 minutes, cooled, homogenized with PBS added with 4M urea, and centrifuged. The supernatant was lyophilized and finely pulverized to prepare an antigen, and the rabbit antibody was prepared as described in Example 3 (1) (hereinafter referred to as an antibody against an autoclaved egg antigen). According to the method described in Example 6, the antibody was reacted with the plate coated with natural egg antigen and the plate coated with heated egg antigen as described in Example 8. The results are shown in Table 4. Table 4 shows that natural and heated egg antigens were detected with almost the same ELISA strength as antibodies prepared by immunizing extracts from autoclaved food allergens. Therefore, the aforementioned problems are resolved. 28 326 \ Patent Specification (Supplement) \ 92 · 〇4 \ 92101099 200413715 Table 4. Uses antibodies prepared by immunizing heated antibodies to increase the ELISA strength. The ELISA strength is coated with a plate of natural egg antigen. The plate is coated with a heated egg antigen. Anti-egg allergen antibody 100 36 Anti-autoclaved egg 100 118 Antigen antibody The ELISA strength (OD 値) obtained by the reaction between the anti-egg allergen partial antibody and the standard egg antigen plate is shown as 100. Example 10 (Determination of food allergens and foods that cause food allergies -1) Check whether the antibodies that recognize multiple antigens of the present invention (as described in Example 3) and the method as described in Example 7 are detectable Food containing food allergens. The calibration curve prepared as described in Example 7 was used for quantification and calculation of the quantification index (measurement 値 / actual 値 X 1 00). Table 5 shows the results of egg yolk, egg white, mayonnaise and whole egg mayonnaise. When an antibody prepared from a single antigen located in a protein is measured, (1) egg yolk cannot be detected but only the protein can be detected, and (2) the amount of standard egg antigen measured using a calibration curve is greater than the actual amount Two to 60 times. When the antibodies against standard egg antigens that recognize multiple antigens of the present invention are measured, (3) both egg yolk and protein can be detected, and (4) the measured amount of standard egg antigens is almost the same as the actual radon. From these results, it is noted that the anti-standard egg antigen antibody that recognizes multiple antigens of the present invention can detect and quantify allergic foods of eggs or their ingredients in processed egg foods. 29 326 \ Patent Specification (Supplement) \ 92-04 \ 92101099 200413715 Table 5. Use various antibodies to detect and / or quantify the possibility of eggs and egg products. Sample determination. Antibodies that recognize multiple antigens (anti-standard egg antigen antibodies). Antibodies that recognize a single antigen (anti-oval mucin antibodies). Protein antibody) Possibility of egg yolk detection XX Quantitative index 74-106 Possibility of protein detection 〇 Quantitative index 105-170 240-310 6,330-13,150 Possibility of mayonnaise detection XX Possibility of whole egg detection 〇〇 黄酱 例 11 (Determination of food allergens and foods that cause allergies-2) The milk, casein, lactoferrin, and hydrolyzed casein were tested as described in Example 10. The results are shown in Table 6. Table 6 shows that (1) the anti-standard milk antigen antibody that recognizes multiple antigens of the present invention can detect and quantify milk and its components that cause food allergies; but (2) prepared by immunizing substances located in the milk Antibodies that recognize a single antigen cannot be quantified. The symbol of △ in Table 6 shows that the quantification can be completed, but the quantification index is lower than 5. 30 326 \ Patent Specification (Supplement) \ 92-04 \ 92101099 200413715 Table 6. Use various antibody detection and / or quantification of standard milk antigen samples to determine antibodies that recognize multiple antigens (anti-standard milk antigen antibodies). Antibodies that recognize single antigens (anti-casein antibodies). Antibodies that recognize single antigens (anti- / 5-lactoglobulin antibodies) ) Possibility of milk test 〇 〇 Quantitative index 160-230 320-1,080 Casein test possibility 〇X Quantitative index 38-47 170-180 Possibility of lactoferrin egg test △ X Test of white hydrolyzed casein Possibility OX △ protein [Schematic description] Figure 1 shows the reaction between the standard antigen and the patient's IgE antibody, and the reaction between the standard antigen and the antibody against the standard antigen antibody of the present invention. Figure 2 shows that the patient's blood sacrum carries IgE antibodies specific for both natural and denatured egg proteins. 31 326 \ Patent Specification (Supplement) \ 92-04 \ 92101099