JP2004191073A

JP2004191073A - Immunological antigen detection method

Info

Publication number: JP2004191073A
Application number: JP2002355879A
Authority: JP
Inventors: Tatsuyuki Hachisu; 達之蜂巣; Masaaki Kojima; 正章小島
Original assignee: SHIBAYAGI KK
Current assignee: SHIBAYAGI KK
Priority date: 2002-12-06
Filing date: 2002-12-06
Publication date: 2004-07-08
Anticipated expiration: 2022-12-06
Also published as: JP3851262B2

Abstract

<P>PROBLEM TO BE SOLVED: To provide an immunological antigen detection method capable of detecting and quantitating even many allergens included in processed foods highly accurately at low cost to the utmost without requiring much time. <P>SOLUTION: A suspension wherein an insoluble antigen and a soluble antigen are intermingled is used as a sample, and the sample is allowed to pass a water-permeable carrier 2 (filter medium having hole diameters of 0.01-20.0 μm). Thus, the insoluble antigen and the soluble antigen are adsorbed on the water-permeable carrier 2. Then, a specific antigen (labeled antigen) labeled by an enzyme, a fluorescent material, a light emitting material or a radioactive material is bonded to the antigen in the state where the antigen is absorbed on the water-permeable carrier 2 (filter medium). <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【０００１】
【発明の属する技術分野】
本発明は抗原抗体反応を利用した抗原検出技術に係わり、特に加工食品中に含まれるアレルゲンをはじめとする抗原を短時間で精度良く容易に検出、定量することのできる免疫学的抗原検出法に関する。
【０００２】
【従来の技術】
近年、アトピー性皮膚炎に代表されるアレルギー症状を誘発するアレルゲンを含んだ食品の摂取による健康危害が増大する中、オボアルブミンやオボムコイドといったアレルゲンを含む卵をはじめ、乳、小麦、蕎麦、落花生を原材料として使用した食品についてはその表示が義務化され、そのほかアワビやエビなど１９品目についてはその表示が奨励された。このような食品に対する安全指向の高まりを受け、食品関連業界などでは従来に増して食品中の成分を精度良く検出、定量することが要求されている。
【０００３】
ここに、アレルゲンや血中微量成分などの抗原又は抗体を検出、定量する方法として、抗原抗体反応を利用するイムノアッセイが良く知られる。イムノアッセイには、放射性物質を標識した抗原又は抗体を用いるラジオイムノアッセイ（ＲＩＡ）をはじめ、その発展系として抗原又は抗体に酵素を結合させるエンザイムイムノアッセイ（ＥＩＡ）などがある。
【０００４】
このうち、安全性の点で放射性物質の標識を用いないＥＩＡが一般に広く利用されるが、これには非標識抗原と標識抗原を競合的に抗体と反応させる競合法ほか、ＥＬＩＳＡ（Ｅｎｚｙｍｅ−ｌｉｎｋｅｄｉｍｍｕｎｏｓｏｒｂｅｎｔａｓｓａｙ）に代表される非競合法とがある。尚、酵素標識に代えて蛍光物質を用いるなどの例もあるが、非競合法には固相担体に固定化した抗体に抗原を反応させたのち標識抗体を反応させるサンドイッチ法の原理が利用される（特許文献１、特許文献２）。
【０００５】
サンドイッチ法の原理によるＥＬＩＳＡを説明すると、図９において１１は隔壁により区画された各々が独立したウェルであり、その底部にはポリスチレンなどで成る固相担体１２が設けられる。
【０００６】
特に、固相担体１２には、特定の抗体（非標識のキャプチャー抗体）が化学的結合により予め固定化されている（図１０Ａ参照）。ここに、その抗体に対する抗原を含む一定量の試料（検体を遠心後の上澄液）を各ウェル内に分注することにより抗体に抗原Ｍを結合し（図１０Ｂ参照）、次いで抗原に対する抗体（酵素Ｅにより標識された標識抗体）を結合させ（図１０Ｃ参照）、これに基質を加えて呈色させる（図１０Ｄ参照）。そして、その吸光度を標準曲線と対比することにより、抗原量を定量する。
【０００７】
【特許文献１】
特公平２−３９７４７号公報
【特許文献１】
特公平４−２１８１８号公報
【０００８】
【発明が解決しようとする課題】
然し乍ら、上記のような従来法に用いるアッセイプレートの固相担体には、特定の抗原に対する特定の抗体が固定化されているだけなので、加工食品中の個々の原材料を特定するには、固相担体に固定された抗体の種類が異なる高価なアッセイプレートを複数枚用い、一回に数時間を要する測定を繰り返し行わねばならない。
【０００９】
しかも、加工食品は衛生上あるいは調理上の目的で加熱処理が施されるので、検出対象とする抗原（アレルゲンその他のタンパク質など）が担体上の抗体と良好に反応するような性状で存在するとは限らない。例えば、卵や牛乳を多く含む生地を高温で処理したパンケーキや小麦を主原料とする油揚げ麺といった食品では、熱変性したタンパク質がコロイド状の不溶性抗原として液相中に分散する。同様に、水溶性のタンパク質でも、Ｓ−Ｓ結合やイオン結合などにより分子構造が大型化して不溶化する。
【００１０】
よって、従来は食品中の特定抗原の有無を検出する場合、検体である加工食品ホモジナイザにより破壊するのみならず、これによって得たホモジネート（懸濁液）を遠心分離し、その上澄液を試料として測定に用いている。しかし、遠心分離を行う検体調製には時間が掛かるばかりでなく、これによって得た試料中には検出されるべき抗原の一部が可溶性成分として僅かに抽出されるだけなので、抗原抗体反応が良好に行われたとしても検出される抗原量は低い値となる。
【００１１】
本発明は以上のような事情に鑑みて成されたものであり、その目的は加工食品中に含まれる多数のアレルゲンなどでも時間を掛けずに可及的低コストで高精度に検出、定量できるようにすることにある。
【００１２】
【課題を解決するための手段】
本発明は上記目的を達成するため、不溶性抗原と可溶性抗原とが混在する懸濁液を試料とし、その試料を透水性担体に通し、これによって前記不溶性抗原と可溶性抗原とを前記透水性担体に吸着せしめた後、それら抗原を透水性担体に吸着させたまま該抗原に特定の標識抗体を結合せしめることを特徴とする。
【００１３】
又、透水性担体に吸着した複数種の抗原に対し、特定した複数種の標識抗体をそれぞれ同時に結合せしめることを特徴とし、しかも透水性担体に吸着した抗原に特定の標識抗体を接触させる前に、抗原が吸着されていない透水性担体の部位にスキンミルクなどのブロッキング液を含浸せしめて透水性担体への標識抗体の非特異的吸着を阻止することを特徴とする。
【００１４】
更に、標識抗体の標識物質として、特定の酵素、蛍光物質、発光物質、又は放射性物質を用いるほか、透水性担体として、孔径０．０１〜２０．０μｍ、好ましくは０．０２〜０．８μｍの濾材を用いることを特徴とする。
【００１５】
尚、濾材は目的とする不溶性抗原、可溶性抗原の全てを吸着させるために種々の材質及び孔径の組み合わせが可能であり、その材質としては、セルロース混合エステル、ポリテトラフルオロエチレン、ポリプロピレン、ナイロン、ポリビニリデンフロライド、ポリカーボネートフィルム、ポリエーテルスルホン、ポリビニールクロライド、セルロース、セルロースアセテート、グラスファイバー、ポリスチレン、アクリルなどがある。
【００１６】
【発明の実施の形態】
以下、本発明の適用例を詳しく説明する。図１は本発明に適用するアッセイプレートを部分的に破断して示した平面図であり、図２にはその部分拡大断面を示す。これらの図で明らかなように、このアッセイプレートは試料の注入部として縦横に配列する個々に独立した筒状のウェル１を形成し、その各ウェルの底部には試料に含まれる抗原を捕集するための透水性担体２（メンブレンフィルター）が設けられる。抗原とは抗体と結合する物質であり、これにはアレルゲン、核タンパク質（核酸）、糖タンパク質、リポタンパク質、インスリンなどのホルモン、ウィルス、細菌、グルカゴン（ペプチド）、及びハプテンなどが含まれ、そのほかダニや花粉なども係る抗原になり得るが、本発明は特にアレルギー性タンパク質、それも加工食品中におけるアレルゲンの有無を検出し、これを定量する一助となる。これには、先ず加工食品をフードカッタなどにより破砕し、これに約等量の抽出用緩衝液を加えて破砕物の組織や細胞をホモジナイザーで破壊する。そして、これにより得られたホモジネート（懸濁液）を試料とし、この試料をアッセイプレートの各ウェル１内に一定量ずつ注入する。尚、抽出用緩衝液としては、リン酸塩、ホウ酸塩、クエン酸塩、炭酸塩、尿素などの溶液を用いることができる。
【００１７】
ここに、試料中には加工食品の原材料成分が混和状態で存在するが、検出対象とするアレルゲンなどの抗原の多くは熱変性によりＳ−Ｓ結合、イオン結合、水素結合、疎水結合、又は脂質との結合などにより分子構造が大型化したコロイド状の不溶性成分（懸濁粒子）として液相中に分散し、一部は懸濁粒子より小さな可溶性成分として溶解している。
【００１８】
そして、本発明によれば、各ウェル１内に注入した試料から不溶性抗原と可溶性抗原の双方を捕集するべく、透水性担体２として不溶性抗原と可溶性抗原を共に吸着することのできる三次元連続網目構造を有する繊維質又は微細多孔質の薄膜状部材が選ばれる。これには孔径０．０１〜２０．０μｍ、好ましくは０．０２〜０．８μｍの濾材（半透膜）を用いることができる。その種の透水性担体２に試料中の不溶性抗原と可溶性抗原を吸着させるには、各ウェル１内に試料を注入せしめたアッセイプレートを例えば図３に示すような吸気装置（本例において日本ミリポア株式会社製バキュームマニホールド）の台座３上に載せ、その台座に接続する吸気源Ｖ（真空ポンプなど）により台座内の空気を吸引する。すると、その吸引力（負圧力）により、各ウェル内の試料のうち分子の小さい水などの溶媒が透水性担体を透過してウェル内から除去される一方、高分子の不溶性抗原及び可溶性抗原が透水性担体の物理的吸着作用によってこれに捕捉される。勿論、透水性担体には目的とする抗原のみならず、不要な物質も吸着されるが、それら不要物質は標識抗体の使用によるその後の抗原抗体反応操作により目的とする抗原と識別することができる。
【００１９】
上記の抗原抗体反応操作は透水性担体への抗原の吸着直後に行っても良いが、好ましくは透水性担体への抗原の吸着後、各ウェル内にブロッキング液を注入してこれを抗原が吸着されていない透水性担体の部位に含浸せしめる。このブロッキング液は吸着物の存在しない透水性担体の空き領域を埋め、抗原抗体反応に供する標識抗体が透水性担体へ非特異的に吸着するのを防止するものであり、これにはスキンミルク、ゼラチン、動物血清由来成分、又はエチレングリコールなどの合成高分子（ポリマー）を好適に用いることができる。尚、透水性担体へのブロッキング液の含浸は、図３のような吸気装置による吸引力により行われ、これによりブロッキング液中の溶質成分ｆが透水性担体２の空き領域に吸着する（図４参照）。
【００２０】
そこで、各ウェル内に特定の標識抗体を注入するのであり、これによればその標識抗体が透水性担体に非特異的に吸着せずして透水性担体に吸着した抗原にのみ接触する。尚、透水性担体に多種類の抗原が吸着している場合でも、特定の標識抗体はこれに対する特定の抗原に結合する。而して、所定の反応時間（約３０分）を置き、図３のような吸気装置を用いて透水性担体の底部から標識抗体の水分を吸引除去した後、透水性担体に吸着された抗原と非結合の標識抗体を洗浄操作によって各ウェル内から除去し、抗原との結合によって透水性担体上に残った特定の標識抗体から特定の抗原を検出、定量することができる。因に、上記の洗浄操作は所定の洗浄液（界面活性剤等の水溶液）をウェルに注入し、これを図３のような吸引装置によりウェル内から吸い出すという操作を複数回（本例において５回）繰り返すことにより達成される。
【００２１】
ここに、標識抗体とは標識物質により標識された抗体、換言すると特定の標識物質を結合した抗体であり、これには検出定量すべき抗原の種類に応じ、例えば卵アレルゲンの定量では抗オボアルブミン抗体、抗オボムコイド抗体、抗リゾチーム抗体、抗オボトランスフェリン抗体、牛乳アレルゲンの定量では抗カゼイン抗体、抗β−ラクトグロブリン抗体、抗ラクトアルブミン抗体、小麦アレルゲンの定量では抗グリアジン抗体、抗α−アミラーゼインヒビター抗体が用いられる。又、標識物質としてはペルオキシダーゼ、β−Ｄ−ガラクトシダーゼ、アルカリホスファターゼ、グルコース−６−リン酸脱水素酵素などの酵素、ルミノール、イソルミノール、アクリジニウムエステルなどの発光物質、フルオレセイン、イソチオシアネート、テトラメチルローダミンイソチオシアネートなどの蛍光物質、並びにヨード１２５、ヨード１３１などの放射性物質を用いることができる。
【００２２】
以上のような標識抗体はモノクローナル抗体でもポリクローナル抗体でも良いが、好ましくは上記したものの中から選択される複数種の混合抗体とし、これを透水性担体に吸着された複数種の抗原にそれぞれ同時に結合させるようにすると良い。例えば、卵由来のアレルゲン抗原（例えば、オボアルブミンとオボムコイド）に対する抗オボアルブミン抗体と抗オボムコイド抗体との混合抗体を用い、それらに別々の標識物質（例えば、一方の抗体に赤色の蛍光を発する蛍光物質、他方の抗体に青色の蛍光を発する蛍光物質）を結合させる。これによれば、例えば一方のオボアルブミンの全ての抗原決定基（エピトープ）が熱変性によって失活してその定量が不能な場合でも、他方のオボムコイドに抗オボムコイド抗体が結合するので、その標識とした蛍光物質の蛍光強度から抗原（オボムコイド）を検出、定量することができることは勿論、オボムコイドの検出によって試料とした加工食品に原材料として卵が使用されていると判定できる。
【００２３】
又、その他の例として、乳由来の抗原（例えば、β−ラクトグロブリン）に対する抗β−ラクトグロブリン抗体と、小麦由来の抗原（例えば、グリアジン）に対する抗グリアジン抗体との混合抗体を用い、それらに別々の標識物質（例えば、一方の抗体に赤色の蛍光を発する蛍光物質、他方の抗体に青色の蛍光を発する蛍光物質）を結合させる。これによれば、加工食品中の原材料に異なる複数種の抗原（本例においてβ−ラクトグロブリンとグリアジン）が含まれていても、一つの透水性担体、アッセイプレートによりそれらを同時に検出定量することができる。
【００２４】
尚、標識物質として、発光物質や蛍光物質を用いた場合にはその発光強度、蛍光強度などを発光光度計や蛍光光度計などを用いて測定する。一方、酵素を用いた場合には、基質として［３−（ヒドロキシフェニル）プロピオン酸、４−ｍｅｔｈｙｌｕｍｂｅｌｌｉｅｆｅｒｙｌ−β−Ｄ−ｇａｌａｃｔｏｓｉｄｅ、４−ｍｅｔｈｙｌｕｍｂｅｌｌｉｅｆｅｒｙｌ−ｐｈｏｓｐｈａｔｅ］などを加えてその蛍光強度を測定するか、基質として［ルミノール、２−ｎｉｔｒｏｐｈｅｎｙｌ−β−Ｄ−ｇａｌａｃｔｏｓｉｄｅ、グルコース−６−リン酸］などを加えてその発光強度を測定するか、又は基質として［３，３′，５，５′−ｔｅｔｒａｍｅｔｈｙｌｂｅｎｚｉｄｉｎｅ、２−ニトロフェニル−β−Ｄ−ガラクトサイド（２−ｎｉｔｒｏｐｈｅｎｙｌ−β−Ｄ−ｇａｌａｃｔｏｓｉｄｅ）、４−ｎｉｔｒｏｐｈｅｎｙｌｐｈｏｓｐｈａｔｅ］などを加えてその吸光度を測定する。
【００２５】
そして、その測定値（発光強度、蛍光強度、吸光度）を標準曲線に照らして抗原を定量することができる。標準曲線とは、既知の抗原溶液を試料とし、これに特定の標識抗体を反応させて試料濃度別に蛍光強度などを測定することにより得られるものであり、これは直交座標系に試料濃度に対応する蛍光強度などをプロットしてグラフ化されるか、又はコンピュータに蓄積されて各種抗原を定量するための尺度とされる。
【００２６】
（試験例）
鶏卵を１０ｍｇ秤量し、これに１０ｍｌの抽出用緩衝液を加えてホモジナイザーにより３０００ｒｐｍで３分間ホモジナイズし、これを２つに分け、その一方を９５℃で３分間加熱して加熱試料Ａとし、他方を非加熱試料Ｂとした。又、双方の試料Ａ，Ｂをそれぞれ希釈用緩衝液により希釈し、それぞれ濃度が異なる３つの試料Ａ１，Ａ２，Ａ３、及びＢ１，Ｂ２，Ｂ３を調製した。その一覧を下表１に示す（ここでの希釈はオボアルブミンの含有量が異なる加工食品を想定しての擬似検体を作成するための操作であり、実際の試料調製では行わない）。一方、標準溶液（既知抗原）としてオボアルブミン溶液（１００ｎｇ／ｍｌ）を用い、これを希釈用緩衝液による希釈で濃度１００％、５０％、２５％、１２．５％、６．２５％に調製した。
【００２７】
【表１】

そして、透水性担体として孔径０．４５μｍのセルロース混合エステルから成るメンブレンフィルター（日本ミリポア株式会社製ＭＡＨＡ−Ｎ４５−１０）を各ウェル（８×１２）の底部に設けたアッセイプレートを用い、その各ウェル内に濃度が異なる５つの標準溶液、並びに希釈用緩衝液でそれぞれ１０倍に希釈した試料Ａ１〜Ａ３、試料Ｂ１〜Ｂ３を０．２ｍｌずつ注入し、そのアッセイプレートを振盪機に掛けた後に３７℃下で３０分間静置した。その後、吸気装置により各ウェル内の水分が空になるまでその底部から一斉に吸液し、次いで各ウェル内にブロッキング液を０．３ｍｌずつ注入した。これを室温（２０〜２５℃下）で２０分間静置した後、吸気装置によって各ウェル内の水分が空になるまでその底部から一斉に吸液し、次いで各ウェル内を洗浄液で満たし、これを吸気装置で底部から吸い出す洗浄操作を５回繰り返して行った。
【００２８】
次に、標識抗体（フルオレセイン標識ウサギＩｇＧ抗オボアルブミン）溶液を各ウェル内に０．２ｍｌずつ注入し、これを３７℃下で３０分静置した後、吸気装置により各ウェル内の水分が空になるまでその底部から一斉に吸液し、次いで上記と同じ洗浄操作を５回繰り返し行った。
【００２９】
その後、蛍光光度計（ＴＥＣＡＮ社製ＳｐｅｃｔｒａＦｌｕｏｒＰｌｕｓ）により各ウェル毎に蛍光強度を測定し、先ず標準溶液の各濃度と蛍光強度の相関を示す標準曲線を回帰分析によって作成した（図５参照）。そして、各試料Ａ１〜Ａ３、及びＢ１〜Ｂ３の蛍光強度を図５の標準曲線又はその回帰曲線式に当てはめて対応する濃度を導き出し、これに各試料Ａ１〜Ａ３、Ｂ１〜Ｂ３の希釈倍率（１０倍）を乗じることにより、それら各試料のオボアルブミン濃度を定量した。その結果を図６に示す（３回の測定による平均値）。
【００３０】
図６から明らかなように、加熱試料と非加熱試料では同濃度の試料同士の間に大きな差異は認められない。よって、加熱、非加熱に拘わらず透水性担体に不溶性オボアルブミンと可溶性オボアルブミンの熱変性による失活以外の全てが吸着して良好な抗原抗体反応が行われたと言える。この事は、体内に経口摂取される全ての不溶性オボアルブミン量をインビトロで定量できたと言える（人が食べる加工食品を丸ごと測定するための試料となるので測定結果の信頼性が高い）。尚、本試験に用いた試薬の一覧を表２に示す。
【００３１】
【表２】

（比較試験）
上記の加熱試料Ａ１〜Ａ３を従来の検体処理法に則って遠心分離機により２０分間遠心分離し、その上澄液をぞれぞれ比較試料Ｚ１〜Ｚ３とした。そして、従来法のＥＬＩＳＡ（サンドイッチ法）に則って、各ウェル（８×１２）の底部に抗オボアルブミン抗体が固定化されたポリスチレン製の固相担体を備えたアッセイプレートを用い、その各ウェル内に希釈用緩衝液でそれぞれ１０倍に希釈した試料Ｚ１〜Ｚ３、並びに濃度調整したオボアルブミンの標準溶液を０．１ｍｌずつ注入し、そのアッセイプレートを振盪機に掛けた後に室温下で１時間静置した。その後、各ウェル内の抗体と未反応の抗原を含んだ試料Ｚ１〜Ｚ３、並び各標準溶液を捨て、次いで各ウェル内を洗浄液で満たし、これを排出する洗浄操作を６回繰り返して行った。
【００３２】
次に、標識抗体（酵素標識抗オボアルブミン抗体）溶液を各ウェル内に０．１ｍｌずつ注入し、これを室温下で３０分静置した後、未反応標識抗体溶液を捨て、次いで上記と同じ洗浄操作を６回繰り返し行った。次いで、酵素基質を各ウェル内に０．１ｍｌずつ注入し、これを室温下で１０分間静置した後、反応停止液を各ウェル内に０．１ｍｌずつ注入した。
【００３３】
その後、吸光光度計により各ウェル毎に吸光度を測定し、各試料Ｚ１〜Ｚ３の吸光度を図７の標準曲線（上記各標準溶液の吸光度に基づき作成したもの）、又はその回帰曲線式に当てはめて対応する濃度を導き出し、これに各試料Ｚ１〜Ｚ３の希釈倍率（１０倍）を乗じることにより、それら各試料のオボアルブミン濃度を定量した。その結果を試料Ａ１〜Ａ３の測定結果と対比して図８に示す（３回の測定による平均値）。
【００３４】
図８から明らかなように、従来法では低濃度のオボアルブミンが定量され、特に高濃度の試料Ａ１，Ｚ１の対比によれば、本発明に係る方法と従来法で測定結果に９倍近い差があり、本発明の優位性は歴然である。
【００３５】
【発明の効果】
以上の説明で明らかなように、本発明によれば不溶性抗原と可溶性抗原を透水性担体に吸着させるようにしていることから、従来のように目的とする抗原を固相担体に固定化した抗体に好適に結合させるべく、試料を遠心分離するという操作が不要となり、このため時間短縮を図れる上、可溶性抗原のみを測定系に供する従来法に比べて抗原の検出、定量精度を大幅に向上させることができる。
【００３６】
又、従来のように特定の抗体を固定化した担体を使用しないので、抗原の検出、定量に掛かるコストを大幅に削減することができ、しかも透水性担体に吸着した多種類の抗原を一つの透水性担体で同時に検出することができる。
【００３７】
更に、透水性担体に吸着した抗原に特定の標識抗体を結合させる前に、抗原が吸着されていない透水性担体の部位にブロッキング液を含浸せしめて透水性担体への標識抗体の非特異的吸着を阻止するようにしていることから、透水性担体への標識抗体の吸着による擬陽性を起こさない。
【図面の簡単な説明】
【図１】本発明に用いる透水性担体を備えたアッセイプレートを部分的に破断して示した平面図
【図２】同アッセイプレートの部分拡大断面図
【図３】吸気装置の使用態様を示した概略図
【図４】透水性担体にブロッキング液を含浸した状態を示す説明図
【図５】標準曲線を示すグラフ。
【図６】本発明に係る方法で検出した抗原の濃度を示す図
【図７】従来法により得た標準曲線を示すグラフ。
【図８】本発明と従来法による抗原の検出濃度を示す比較図
【図９】従来のＥＬＩＳＡで使用するアッセイプレートの部分拡大断面図
【図１０】従来のＥＬＩＳＡ原理図
【符号の説明】
１ウェル
２透水性担体[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an antigen detection technique using an antigen-antibody reaction, and more particularly to an immunological antigen detection method capable of easily and accurately detecting and quantifying allergens and other antigens contained in processed foods in a short time. .
[0002]
[Prior art]
In recent years, health hazards due to ingestion of foods containing allergens that induce allergic symptoms such as atopic dermatitis have increased, and eggs, milk, wheat, buckwheat, and peanuts including allergens such as ovalbumin and ovomucoid have been used. Labeling of foods used as raw materials was compulsory, and labeling of 19 items such as abalone and shrimp was encouraged. In response to such an increase in safety of foods, the food-related industry and the like are required to more accurately detect and quantify components in foods than ever before.
[0003]
Here, as a method for detecting and quantifying an antigen or an antibody such as an allergen or a trace component in blood, an immunoassay utilizing an antigen-antibody reaction is well known. The immunoassay includes a radioimmunoassay (RIA) using an antigen or an antibody labeled with a radioactive substance, and an enzyme immunoassay (EIA) for binding an enzyme to the antigen or the antibody as an advanced system thereof.
[0004]
Among them, EIA which does not use a radioactive substance label is generally widely used from the viewpoint of safety. Examples of the EIA include a competitive method for competitively reacting an unlabeled antigen and a labeled antigen with an antibody, and an ELISA (Enzyme-linked). There is a non-competitive method typified by immunosorbent assay). Although there is an example of using a fluorescent substance instead of enzyme labeling, the non-competitive method utilizes the principle of a sandwich method in which an antigen is reacted with an antibody immobilized on a solid support and then the labeled antibody is reacted. (Patent Documents 1 and 2).
[0005]
Explaining the ELISA based on the principle of the sandwich method. In FIG. 9, reference numeral 11 denotes independent wells defined by partition walls, and a solid support 12 made of polystyrene or the like is provided at the bottom.
[0006]
In particular, a specific antibody (unlabeled capture antibody) is immobilized on the solid phase carrier 12 in advance by chemical bonding (see FIG. 10A). Here, an antigen M is bound to the antibody by dispensing a fixed amount of a sample containing the antigen against the antibody (supernatant after centrifuging the sample) into each well (see FIG. 10B). (Labeled antibody labeled with the enzyme E) is bound (see FIG. 10C), and a substrate is added thereto to develop a color (see FIG. 10D). Then, the amount of antigen is quantified by comparing the absorbance with a standard curve.
[0007]
[Patent Document 1]
JP-B-2-39747 [Patent Document 1]
Japanese Patent Publication No. Hei 4-21818
[Problems to be solved by the invention]
However, since a specific antibody against a specific antigen is only immobilized on the solid phase carrier of the assay plate used in the conventional method as described above, it is necessary to use a solid phase to identify individual raw materials in a processed food. A plurality of expensive assay plates with different types of antibodies immobilized on a carrier must be used and the measurement requiring several hours at a time must be repeated.
[0009]
Moreover, since processed foods are subjected to heat treatment for sanitary or cooking purposes, the antigens to be detected (such as allergens and other proteins) do not exist in such a property that they react well with the antibodies on the carrier. Not exclusively. For example, in foods such as pancakes in which dough containing eggs and milk are processed at a high temperature and fried noodles mainly made of wheat, heat-denatured proteins are dispersed in the liquid phase as colloidal insoluble antigens. Similarly, even for a water-soluble protein, the molecular structure becomes large due to SS bond or ionic bond, and the protein becomes insoluble.
[0010]
Therefore, conventionally, when detecting the presence or absence of a specific antigen in a food, not only is the sample processed food homogenizer destroyed, but also the homogenate (suspension) obtained thereby is centrifuged, and the supernatant is used as a sample. Is used for measurement. However, the preparation of the sample by centrifugation is not only time-consuming, but also a part of the antigen to be detected is only slightly extracted as a soluble component in the obtained sample, so that the antigen-antibody reaction is good. The amount of the detected antigen is low even if the detection is performed at a low temperature.
[0011]
The present invention has been made in view of the circumstances as described above, and its purpose is to detect and quantify with high accuracy at a low cost as much as possible without taking much time even with a large number of allergens contained in processed foods. Is to do so.
[0012]
[Means for Solving the Problems]
In order to achieve the above object, the present invention provides a sample in which a suspension containing an insoluble antigen and a soluble antigen is mixed, and the sample is passed through a water-permeable carrier, whereby the insoluble antigen and the soluble antigen are passed through the water-permeable carrier. After the adsorption, a specific labeled antibody is bound to the antigen while the antigen is adsorbed to the water-permeable carrier.
[0013]
Further, it is characterized in that a plurality of specified labeled antibodies are simultaneously bound to a plurality of antigens adsorbed on the water-permeable carrier, respectively, and before the specific labeled antibody is brought into contact with the antigen adsorbed on the water-permeable carrier. In addition, a non-specific adsorption of a labeled antibody to the water-permeable carrier is prevented by impregnating a site of the water-permeable carrier on which the antigen is not adsorbed with a blocking solution such as skin milk.
[0014]
Further, a specific enzyme, a fluorescent substance, a luminescent substance, or a radioactive substance is used as a labeling substance of the labeled antibody, and a water-permeable carrier has a pore size of 0.01 to 20.0 μm, preferably 0.02 to 0.8 μm. It is characterized by using a filter medium.
[0015]
The filter medium can be made of various materials and a combination of pore sizes in order to adsorb all of the target insoluble antigen and soluble antigen. Examples of the material include cellulose mixed ester, polytetrafluoroethylene, polypropylene, nylon, and poly. Examples include vinylidene fluoride, polycarbonate film, polyether sulfone, polyvinyl chloride, cellulose, cellulose acetate, glass fiber, polystyrene, and acrylic.
[0016]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, application examples of the present invention will be described in detail. FIG. 1 is a plan view showing an assay plate applied to the present invention partially cut away, and FIG. 2 is a partially enlarged cross-sectional view thereof. As is clear from these figures, this assay plate forms individual cylindrical wells 1 arranged vertically and horizontally as sample injection portions, and the bottom of each well collects the antigen contained in the sample. A permeable carrier 2 (membrane filter) is provided. An antigen is a substance that binds to an antibody, including allergens, nucleoproteins (nucleic acids), glycoproteins, lipoproteins, hormones such as insulin, viruses, bacteria, glucagons (peptides), and haptens. Mite, pollen and the like can also be such antigens, but the present invention particularly helps to detect the presence or absence of allergens in allergens in processed foods and to quantify them. For this purpose, first, the processed food is crushed by a food cutter or the like, and an approximately equal amount of an extraction buffer is added thereto, and the crushed tissue or cells are destroyed by a homogenizer. The homogenate (suspension) thus obtained is used as a sample, and this sample is injected into each well 1 of the assay plate by a predetermined amount. As the extraction buffer, a solution of phosphate, borate, citrate, carbonate, urea or the like can be used.
[0017]
Here, the raw material components of the processed food are present in the sample in a mixed state, but most of the antigens, such as allergens to be detected, are subjected to thermal denaturation, such as SS bond, ionic bond, hydrogen bond, hydrophobic bond, or lipid. It is dispersed in the liquid phase as a colloidal insoluble component (suspended particles) whose molecular structure is enlarged due to bonding with the like, and a part is dissolved as a soluble component smaller than the suspended particles.
[0018]
According to the present invention, in order to collect both the insoluble antigen and the soluble antigen from the sample injected into each well 1, a three-dimensional continuous material capable of adsorbing both the insoluble antigen and the soluble antigen as the water-permeable carrier 2. A fibrous or microporous thin film member having a network structure is selected. For this, a filter medium (semi-permeable membrane) having a pore size of 0.01 to 20.0 μm, preferably 0.02 to 0.8 μm can be used. In order to adsorb the insoluble antigen and the soluble antigen in the sample to such a water-permeable carrier 2, an assay plate in which the sample is injected into each well 1 is attached to, for example, an inhaler shown in FIG. 3 (Nippon Millipore in this example). It is placed on a pedestal 3 of a vacuum manifold (manufactured by Co., Ltd.), and air in the pedestal is sucked by an intake source V (such as a vacuum pump) connected to the pedestal. Then, due to the suction force (negative pressure), a solvent such as water having a small molecule in the sample in each well passes through the water-permeable carrier and is removed from the well, while a high molecular insoluble antigen and a soluble antigen are removed. The water-permeable carrier is trapped by the physical adsorption action. Of course, not only the target antigen, but also unnecessary substances are adsorbed to the water-permeable carrier, and these unnecessary substances can be distinguished from the target antigen by a subsequent antigen-antibody reaction operation using a labeled antibody. .
[0019]
The above-described antigen-antibody reaction operation may be performed immediately after the adsorption of the antigen to the water-permeable carrier.However, preferably, after the adsorption of the antigen to the water-permeable carrier, the blocking solution is injected into each well and the antigen is adsorbed. Impregnate the part of the water-permeable carrier that has not been used. This blocking liquid fills the free area of the water-permeable carrier where there is no adsorbate, and prevents the labeled antibody to be subjected to the antigen-antibody reaction from non-specifically adsorbing to the water-permeable carrier. Gelatin, animal serum-derived components, or synthetic polymers such as ethylene glycol can be suitably used. The impregnation of the blocking liquid into the water-permeable carrier is performed by the suction force of the suction device as shown in FIG. 3, whereby the solute component f in the blocking liquid is adsorbed to the empty area of the water-permeable carrier 2 (FIG. 4). reference).
[0020]
Therefore, a specific labeled antibody is injected into each well, whereby the labeled antibody does not non-specifically adsorb to the water-permeable carrier but comes into contact only with the antigen adsorbed to the water-permeable carrier. In addition, even when various types of antigens are adsorbed on the water-permeable carrier, the specific labeled antibody binds to the specific antigen. After a predetermined reaction time (approximately 30 minutes), the water of the labeled antibody is removed by suction from the bottom of the water-permeable carrier using an air suction device as shown in FIG. 3, and then the antigen adsorbed on the water-permeable carrier is removed. The unbound labeled antibody is removed from each well by a washing operation, and the specific antigen can be detected and quantified from the specific labeled antibody remaining on the water-permeable carrier due to the binding with the antigen. In the above-mentioned washing operation, a predetermined washing solution (aqueous solution such as a surfactant) is injected into the well, and the well is sucked out of the well by the suction device shown in FIG. 3 a plurality of times (five times in this example). ) It is achieved by repeating.
[0021]
Here, the labeled antibody is an antibody labeled with a labeling substance, in other words, an antibody to which a specific labeling substance is bound, and depending on the type of antigen to be detected and quantified, for example, anti-ovalbumin in quantification of egg allergen Antibody, anti-ovomucoid antibody, anti-lysozyme antibody, anti-ovotransferrin antibody, anti-casein antibody, anti-β-lactoglobulin antibody, anti-lactalbumin antibody for quantification of milk allergen, anti-gliadin antibody, anti-α-amylase inhibitor for quantification of wheat allergen Antibodies are used. Examples of labeling substances include enzymes such as peroxidase, β-D-galactosidase, alkaline phosphatase, and glucose-6-phosphate dehydrogenase; luminescent substances such as luminol, isoluminol, and acridinium ester; fluorescein, isothiocyanate, A fluorescent substance such as methyl rhodamine isothiocyanate and a radioactive substance such as iodine 125 and iodine 131 can be used.
[0022]
The labeled antibody as described above may be a monoclonal antibody or a polyclonal antibody, but preferably is a mixed antibody of a plurality of types selected from those described above, and simultaneously binds each to a plurality of antigens adsorbed on a water-permeable carrier. It is good to make it. For example, a mixed antibody of an anti-ovalbumin antibody and an anti-ovomucoid antibody against an egg-derived allergen antigen (for example, ovalbumin and ovomucoid) is used, and a separate labeling substance (for example, fluorescence that emits red fluorescence to one antibody) is used. Substance, a fluorescent substance that emits blue fluorescence) to the other antibody. According to this, for example, even when all the antigenic determinants (epitope) of one ovalbumin are inactivated by thermal denaturation and its quantification is impossible, the anti-ovomucoid antibody binds to the other ovomucoid, so that The antigen (ovomucoid) can be detected and quantified from the fluorescence intensity of the fluorescent substance thus obtained, and of course, the egg can be determined to be used as a raw material in the processed food sampled by the detection of ovomucoid.
[0023]
As another example, a mixed antibody of an anti-β-lactoglobulin antibody against a milk-derived antigen (eg, β-lactoglobulin) and an anti-gliadin antibody against a wheat-derived antigen (eg, gliadin) is used. Separate labeling substances (for example, a fluorescent substance that emits red fluorescence to one antibody and a fluorescent substance that emits blue fluorescence to the other antibody) are bound. According to this, even if a plurality of different antigens (β-lactoglobulin and gliadin in this example) are contained in the raw material in the processed food, they can be simultaneously detected and quantified by one water-permeable carrier and assay plate. Can be.
[0024]
When a luminescent substance or a fluorescent substance is used as the labeling substance, the luminescence intensity, the fluorescence intensity, and the like are measured using a luminescence photometer or a fluorimeter. On the other hand, when an enzyme is used, the fluorescence intensity is measured by adding [3- (hydroxyphenyl) propionic acid, 4-methylbellerieferyl-β-D-galactoside, 4-methyllumbellieferyl-phosphate] as a substrate, or [Luminol, 2-nitrophenyl-β-D-galactoside, glucose-6-phosphate] or the like is added as a substrate to measure the luminescence intensity, or [3,3 ′, 5,5′-tetramethylbenzidine, 2-Nitrophenyl-β-D-galactoside (2-nitrophenyl-β-D-galactoside), 4-nitrophenylphosphate and the like are added, and the absorbance is measured.
[0025]
Then, the measured values (emission intensity, fluorescence intensity, absorbance) can be quantified by illuminating the standard curve. The standard curve is obtained by using a known antigen solution as a sample, reacting it with a specific labeled antibody, and measuring the fluorescence intensity etc. for each sample concentration, which corresponds to the sample concentration in a rectangular coordinate system. Fluorescent intensity and the like are plotted and graphed, or stored in a computer and used as a measure for quantifying various antigens.
[0026]
(Test example)
10 mg of a chicken egg is weighed, 10 ml of an extraction buffer is added thereto, and homogenized with a homogenizer at 3000 rpm for 3 minutes. The mixture is divided into two, and one of them is heated at 95 ° C. for 3 minutes to obtain a heated sample A. Was used as a non-heated sample B. Further, both samples A and B were each diluted with a diluting buffer to prepare three samples A1, A2, A3 and B1, B2, B3 having different concentrations. The list is shown in Table 1 below (the dilution here is an operation for preparing a pseudo sample assuming processed foods having different ovalbumin contents, and is not performed in actual sample preparation). On the other hand, an ovalbumin solution (100 ng / ml) was used as a standard solution (known antigen), and this was adjusted to a concentration of 100%, 50%, 25%, 12.5%, and 6.25% by dilution with a dilution buffer. did.
[0027]
[Table 1]

An assay plate provided with a membrane filter (MAHA-N45-10, manufactured by Nippon Millipore Co., Ltd.) at the bottom of each well (8 × 12) as a water-permeable carrier was used. After injecting 0.2 ml of each of five standard solutions having different concentrations, and samples A1 to A3 and samples B1 to B3 each diluted 10-fold with a dilution buffer, the assay plate was placed on a shaker. The plate was allowed to stand at 37 ° C. for 30 minutes. Thereafter, liquid was simultaneously sucked from the bottom of the wells until the water in each well was emptied by the suction device, and then 0.3 ml of the blocking solution was injected into each well. After allowing this to stand at room temperature (at 20 to 25 ° C.) for 20 minutes, the liquid is simultaneously sucked from the bottom of the well by the suction device until the water in each well becomes empty, and then the inside of each well is filled with the washing solution. The washing operation of sucking out from the bottom with the suction device was repeated 5 times.
[0028]
Next, 0.2 ml of a labeled antibody (fluorescein-labeled rabbit IgG anti-ovalbumin) solution was injected into each well, and the solution was allowed to stand at 37 ° C. for 30 minutes. , And the same washing operation as above was repeated 5 times.
[0029]
Thereafter, the fluorescence intensity was measured for each well using a fluorometer (Spectra Fluor Plus manufactured by TECAN), and a standard curve showing the correlation between each concentration of the standard solution and the fluorescence intensity was first created by regression analysis (see FIG. 5). . Then, the fluorescence intensity of each of the samples A1 to A3 and B1 to B3 is applied to the standard curve or the regression curve equation of FIG. 5 to derive the corresponding concentration, and the dilution ratio of each of the samples A1 to A3 and B1 to B3 ( 10 times), the ovalbumin concentration of each of the samples was quantified. The results are shown in FIG. 6 (average value from three measurements).
[0030]
As is clear from FIG. 6, there is no significant difference between the samples having the same concentration between the heated sample and the unheated sample. Therefore, it can be said that good antigen-antibody reaction was performed by adsorbing everything except inactivation of insoluble ovalbumin and soluble ovalbumin by thermal denaturation to the water-permeable carrier regardless of heating or non-heating. This means that the amount of all insoluble ovalbumin ingested orally into the body could be quantified in vitro (it is a sample for measuring whole processed foods eaten by humans, so the measurement results are highly reliable). Table 2 shows a list of the reagents used in this test.
[0031]
[Table 2]

(Comparative test)
The above heated samples A1 to A3 were centrifuged for 20 minutes by a centrifuge according to a conventional sample processing method, and the supernatants were used as comparative samples Z1 to Z3, respectively. Then, in accordance with a conventional ELISA (sandwich method), an assay plate provided with a polystyrene solid-phase carrier having an anti-ovalbumin antibody immobilized on the bottom of each well (8 × 12) was used. 0.1 ml of each of the samples Z1 to Z3 each diluted 10-fold with a diluting buffer, and a standard solution of ovalbumin whose concentration was adjusted were poured into the sample, and the assay plate was shaken for 1 hour at room temperature. It was left still. Thereafter, the washing operation of discarding the samples Z1 to Z3 containing the antibody and the unreacted antigen in each well and each standard solution, filling each well with a washing solution, and discharging the well, was repeated six times.
[0032]
Next, 0.1 ml of a labeled antibody (enzyme-labeled anti-ovalbumin antibody) solution was injected into each well, and the solution was allowed to stand at room temperature for 30 minutes. Then, the unreacted labeled antibody solution was discarded, and then the same as above. The washing operation was repeated six times. Next, 0.1 ml of the enzyme substrate was injected into each well, and the mixture was allowed to stand at room temperature for 10 minutes, and then 0.1 ml of the reaction stop solution was injected into each well.
[0033]
Thereafter, the absorbance is measured for each well with an absorptiometer, and the absorbance of each of the samples Z1 to Z3 is applied to the standard curve of FIG. 7 (created based on the absorbance of each of the above standard solutions) or its regression curve equation. The ovalbumin concentration of each sample was quantified by deriving the corresponding concentration and multiplying by the dilution ratio (10 times) of each sample Z1 to Z3. The results are shown in FIG. 8 in comparison with the measurement results of Samples A1 to A3 (average value by three measurements).
[0034]
As is clear from FIG. 8, the ovalbumin at a low concentration was quantified by the conventional method. Particularly, according to the comparison of the samples A1 and Z1 at the high concentration, the difference between the measurement results of the method according to the present invention and the conventional method was almost 9 times. The advantage of the present invention is obvious.
[0035]
【The invention's effect】
As is clear from the above description, since the insoluble antigen and the soluble antigen are adsorbed on the water-permeable carrier according to the present invention, the antibody in which the target antigen is immobilized on the solid-phase carrier as in the prior art The operation of centrifuging the sample is not required in order to make the binding suitable, so that the time can be reduced, and the detection and quantification accuracy of the antigen is significantly improved as compared with the conventional method in which only the soluble antigen is supplied to the measurement system. be able to.
[0036]
In addition, since a carrier on which a specific antibody is immobilized is not used as in the related art, the cost of detecting and quantifying the antigen can be significantly reduced, and moreover, various types of antigens adsorbed on the water-permeable carrier can be combined into one. It can be detected simultaneously with a permeable carrier.
[0037]
Furthermore, before binding the specific labeled antibody to the antigen adsorbed on the water-permeable carrier, the blocking liquid is impregnated into a portion of the water-permeable carrier on which the antigen is not adsorbed, and the non-specific adsorption of the labeled antibody to the water-permeable carrier is performed. , No false positives are caused by adsorption of the labeled antibody to the water-permeable carrier.
[Brief description of the drawings]
FIG. 1 is a partially cutaway plan view of an assay plate provided with a water-permeable carrier used in the present invention. FIG. 2 is a partially enlarged cross-sectional view of the assay plate. FIG. FIG. 4 is an explanatory diagram showing a state in which a blocking liquid is impregnated in a water-permeable carrier. FIG. 5 is a graph showing a standard curve.
FIG. 6 is a graph showing the concentration of an antigen detected by the method according to the present invention. FIG. 7 is a graph showing a standard curve obtained by a conventional method.
FIG. 8 is a comparison diagram showing the detected concentrations of antigens according to the present invention and the conventional method. FIG. 9 is a partially enlarged cross-sectional view of an assay plate used in a conventional ELISA. FIG.
1 well 2 permeable carrier

Claims

A suspension in which an insoluble antigen and a soluble antigen are mixed is used as a sample, and the sample is passed through a water-permeable carrier, whereby the insoluble antigen and the soluble antigen are adsorbed to the water-permeable carrier. An immunological antigen detection method, wherein a specific labeled antibody is bound to the antigen while being adsorbed on a carrier.

2. The immunological antigen detection method according to claim 1, wherein a plurality of specified labeled antibodies are simultaneously bound to a plurality of antigens adsorbed on the water-permeable carrier.

Before contacting a specific labeled antibody with the antigen adsorbed on the water-permeable carrier, the site of the water-permeable carrier on which the antigen is not adsorbed is impregnated with a blocking solution such as skin milk so that the labeled antibody is not specific to the water-permeable carrier. 2. The method for detecting an immunological antigen according to claim 1, wherein the selective adsorption is prevented.

The immunological antigen detection method according to claim 1, wherein a specific enzyme, a fluorescent substance, a luminescent substance, or a radioactive substance is used as a labeling substance of the labeled antibody.

The immunological antigen detection method according to claim 1, wherein a filter medium having a pore size of 0.01 to 20.0 µm is used as the water-permeable carrier.