JP4178632B2 - Methods and reagents for avoiding interference effects - Google Patents
Methods and reagents for avoiding interference effects Download PDFInfo
- Publication number
- JP4178632B2 JP4178632B2 JP35247498A JP35247498A JP4178632B2 JP 4178632 B2 JP4178632 B2 JP 4178632B2 JP 35247498 A JP35247498 A JP 35247498A JP 35247498 A JP35247498 A JP 35247498A JP 4178632 B2 JP4178632 B2 JP 4178632B2
- Authority
- JP
- Japan
- Prior art keywords
- complement
- antibody
- fragment
- activate
- human immunoglobulin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Description
【０００１】
【発明の属する技術分野】
本発明は、試料中の補体価を定量分析するための測定方法及び試薬に関する。
【０００２】
【従来の技術】
補体系はヒトなどの動物の血清中に存在する約20種の蛋白質の総称であり、主として免疫複合体により活性化される古典的経路と、多糖類などにより活性化される第二経路の二つの活性化経路を持つ。古典的経路とは、主に細菌等の細胞膜上の抗原に、これに対する抗体が結合して形成される免疫複合体により補体成分が秩序をもって次々と活性化され、最後は細胞膜を破壊し、細菌等は死滅又は溶解するという作用を示す。また、溶血素で感作された赤血球が補体により溶血に到る反応や、ハプテンで感作されたリポソームに抗ハプテン抗体を反応させると補体によりリポソーム膜が破壊される反応は古典的経路の作用による。一方、第二経路では抗体の関与は必要なく、例えば細菌の細胞壁の構成成分である多糖やウイルスとの接触のみで補体が活性化される。
【０００３】
近年、補体系の活性、即ち補体価の測定は、急性糸球体腎炎、自己免疫疾患等の診断や、治療の指標として注目されている。
【０００４】
現在、一般的に補体価の測定は、溶血素で感作したヒツジ赤血球を用いて、補体の溶血活性を測定するMayerの50％溶血法及びその変法が広く用いられている ［「臨床検査法提要」,1233〜1234頁,第29版第5刷,昭和60年,金原出版(株)；J.Clin.Chem.,12,143(1983)等］。しかし、この方法では同一検体につき何種類かの希釈度に希釈した試料を用意しなければならず、それが測定に誤差を与える原因となっており、また、50％溶血が起こる量をグラフより求めるという繁雑な方法でもある。更に、ヒツジ赤血球を用いるが、生体由来の赤血球は不安定であり、動物の個体差により赤血球の補体に対する感受性が異なる（ロット間差がある）等の問題がある。
【０００５】
一方、より簡便に補体価を測定するために反応時間を短くし（５〜10分）、自動分析装置への適応を可能とした方法として、感作血球により活性化された補体によって生じる感作血球懸濁液の濁度の変化に基づいて補体活性値を求める、赤血球を用いる補体価測定方法（臨床検査、32(12)、1537-1540、1998）や、赤血球の代わりに、より安定でロット間差が少なく、補体活性により膜損傷を受けるリポソームを用いた補体価測定方法（YAMAMOTO.S Clin.Chem.41/4,586-590,1994、特開平7-110331号公報、特開平7-140147号公報等）が近時提案されている。
【０００６】
しかし、Mayer法では補体価を測定する際の反応時における血清試料の希釈倍率が、約160〜480倍であるのに対し、自動分析装置への適応を可能とした補体価測定用試薬に於けるその希釈倍率は、赤血球を使う試薬で約100〜110倍、リポソームを使う試薬では35〜45倍程度である。そのため、血清試料の試薬に対する比率が相対的に高くなるため、自動分析装置による補体価測定の際には、血清試料による測定系への干渉作用が懸念されており、実際に、赤血球及びリポソームを用いた補体価測定において、リウマチ因子陽性血清試料を用いると、試料により干渉作用を受けている可能性が示唆されている（「第４４回日本臨床衛生検査学会要旨集」,演題365,1995、「医学と薬学」35(5),1163-1167,1996）。
【０００７】
【本発明が解決しようとする課題】
本発明は、上記した如き状況に鑑みなされたもので、自動分析装置による補体価測定における血清試料等の測定試料による測定系への干渉作用を軽減させた測定方法及び測定用試薬を提供することを目的とする。
【０００８】
【問題を解決するための手段】
本発明は、上記課題を解決するためになされたものであり、
「ヒト免疫グロブリンに特異的に結合する抗体の存在下で補体価を測定することを特徴とする、補体価測定方法。」，
「ヒト免疫グロブリンに特異的に結合する抗体を含有する、補体価測定用試薬。」，
「ヒト免疫グロブリンに特異的に結合する抗体を含有する試薬と、膜上に抗原が固定化された標識物質内包リポソームを含有する試薬と、膜上に固定化された抗原に対する抗体を含有する試薬とを含んでなる補体価測定用試薬キット。」及び
「ヒト免疫グロブリンに特異的に結合する抗体を含有する試薬と、溶血素感作血球を含有する試薬とを含んでなる補体価測定用試薬キット。」に関する。
【０００９】
即ち本発明者らは、赤血球及びリポソームを用いて補体価を測定するに際し、測定試料中の例えばリウマチ因子等から受ける干渉作用を抑制する方法を見出すべく鋭意研究の結果、測定時にヒト免疫グロブリンに特異的に結合する抗体（以下、本発明に係る抗体と略記する場合がある。）を共存させることにより、測定試料中の共存物質から受ける干渉作用を軽減し、より正確な補体価測定が可能となることを見出し、本発明を完成するに至った。
【００１０】
本発明に係る抗体としては、ヒト免疫グロブリンに特異的に結合する抗体であればいずれでもよいが、それ自身が古典的経路の補体活性化能を有しない抗体が望ましく、例えば、マウスIgG1、マウスIgA、ラットIgG1、ラットIgA等のそれ自体が古典的経路の補体価活性化能を有しない抗体や、古典的経路の補体活性化能を有する抗体を、常法、例えば「免疫生化学研究法、第１版第１刷、（株）東京化学同人,1986」等に記載の方法に準じて、ペプシン消化やパパイン消化等を行い、補体の結合部位であるFc部分を除いた、F(ab')2フラグメントやFab'フラグメント等が挙げられる。中でもマウスIgG1が好ましい。
【００１１】
これらの抗体は、血清中或は腹水中に含有された状態で使用してもよいが、例えば「免疫生化学研究法，第１版第１刷，（株）東京化学同人,1986」等に記載の方法に準じて、硫安分画、イオン交換クロマトグラフィー、ゲル濾過クロマトグラフィー、アフィニティークロマトグラフィー等の方法により、精製して用いることが望ましい。また、Fc部分の除去操作を行う場合は、当該操作後、精製して用いることが望ましい。
【００１２】
また、本発明に係る抗体は、例えば「免疫実験学入門、第２刷、松橋直ら、（株）学会出版センター、1981」等に記載の方法に準じて、馬、牛、羊、兎、山羊、ラット、マウス等の動物にヒト免疫グロブリンあるいはそのフラグメントを免疫して作製されるポリクローナル抗体でも、或は、ケラーとミルスタイン（Nature,256巻,495頁,1975）により確立された細胞融合法に従い、マウスの腫瘍ラインからの細胞と、ヒト免疫グロブリンあるいはそのフラグメントで予め免疫されたマウスの脾細胞とを融合させて得られるハイブリドーマが産生するモノクローナル抗体の何れでもよいが、モノクローナル抗体が好ましい。これらは単独で用いてもよく、或は適宜組み合わせて用いてもよい。
【００１３】
本発明に係る抗体が特異的に結合するヒト免疫グロブリンとしては、特に限定されないが、ヒトIgA、ヒトIgM、ヒトIgG等が挙げられる。
【００１４】
本発明の補体価測定方法は、上記した如き本発明に係る抗体を測定時に存在させる以外は、リポソームや赤血球を用いる自体公知の測定法、例えば、YAMAMOTO.S Clin.Chem.41/4,586-590,1994、特開平7-110331号公報、特開平7-140147号公報や、臨床検査，32(12)，1537-1540，1998に準じて実施すればよく、使用されるその他の試薬類もこれら自体公知の測定法に準じて適宜選択すればよい。
【００１５】
即ち、測定試料を、本発明に係る抗体の存在下で上記した如きリポソームや赤血球を用いる自体公知の測定法に準じて測定することにより、試料中の補体価を簡便に且つ精度よく測定することができる。
【００１６】
また、本発明に係る抗体は、測定時に、例えば、血清、血漿等の測定試料１μｌに対する抗体蛋白量が、通常１×10-6〜10ｍｇ、好ましくは５×10-5〜１×10-2ｍｇとなるように使用される。
【００１７】
本発明の測定方法に於いては、リポソーム又は赤血球と、試料とを反応させる際に、最終的に本発明に係る抗体を上記した如き濃度範囲で共存させればよく、その方法については特に限定されない。
【００１８】
具体的には、例えば、上記した如き自体公知の補体価測定用試薬中に、本発明に係る抗体を含有させ、これと試料とを混合する方法、例えば本発明に係る抗体を含有する緩衝液等の溶液で試料を希釈し、該希釈試料と上記した如き補体価測定用試薬とを混合する方法等が挙げられる。
【００１９】
本発明に係る抗体を測定系に添加する時期は補体価の測定を開始する前であればいつでもよいが、補体が活性化される以前に添加することが望ましい。即ち、本発明に係る抗体を測定系に添加する時期としては、リポソームを用いた方法に於いては、リポソーム表面に形成された抗原抗体複合物によって試料中の補体が活性化される以前が、また、赤血球を用いた方法に於いては、赤血球膜上の免疫複合体によって試料中の補体が活性化される以前が、夫々望ましい。
また、上記した如き自体公知の補体価測定用試薬に用いられるリポソーム、当該リポソーム内に内包される標識物質、当該リポソームに固定される抗原及び当該抗原に対する抗体或いは溶血素で感作させた赤血球は、通常用いられるものであれば何れにてもよく、特に限定されない。
【００２０】
即ち、本発明に用いられるリポソームとしては、通常この分野で使用されているものは全て使用可能であるが、例えば、ジステアロイルホスファチジルコリン，ジミリストイルホスファチジルグリセロール（ＤＭＰＧ），卵黄ホスファチジルグリセロール等を原料として、自体公知の調製方法、例えば、J.liposome Res.,1(3),339〜377(1989-90)、「ライフサイエンスにおけるリポソーム実験マニュアル」,寺田弘,吉村哲郎編著,シュプリンガー・フェアラーク東京(株),1992年やClin.Chem.41/4.586〜590(1995)等に記載された方法等を用いて調製されたもの等が望ましい。中でも、Clin.Chem.41/4.586〜590(1995)の方法により調製されたものが好ましい。
【００２１】
また、リポソーム内に内包される標識物質としては、通常この分野で使用されているものは全て使用可能であるが、例えば、酵素、補酵素、色素、水溶性の蛍光物質、糖類、イオン性化合物、キレート指示薬、色素、スピンラベル化合物等、リポソームの膜傷害によって膜外に放出されて検出され得るもの等が好ましく、中でも、酵素を標識物質に用いることが望ましい。また、その内包方法も、従来公知の調製方法、例えば、「ライフサイエンスにおけるリポソーム実験マニュアル」,寺田弘,吉村哲郎編著，60〜89頁,シュプリンガー・フェアラーク東京(株),1992年、特開平7-110331号公報、特開平7-140147号公報等に記載されている方法に準じて行えばよい。
【００２２】
リポソーム膜表面に固定させる抗原としては、リポソーム膜に組み込むことができる抗原であれば何れにてもよいが、例えば、フォルスマン抗原、ＧＭ1等の糖脂質抗原、ジニトロベンゼン、トリニトロベンゼン等の芳香族有機化合物、サイロキシン等のホルモン、テオフィリン、フェニトイン等の薬物等が挙げられる。また、これらをリポソーム膜に組み込む方法としては、フォルスマン抗原については、例えばBiochemistry,8,4149(1969)に記載の方法、糖脂質抗原については、例えばJ.Immunol.Methods,85,53〜63(1985)に記載の方法、ジニトロベンゼンやトリニトロベンゼンについては、例えばBiochemistry,Vol.11,No.22,4085(1972)やJ.Immunol.Methods,123,19〜24(1989)に記載の方法、サイロキシンについては、例えばBio.Technology,April,349(1984)に記載の方法、テオフィリンやフェニトインについては、例えばJ.Chem.Pharm.Bull.,36,1086(1988)やClin.Chem.,38,808(1992)に記載の方法等が挙げられる。
【００２３】
また、リポソーム膜表面に抗原を固定させる方法としては、通常この分野で使用される方法である、例えば、架橋法［「続生化学実験講座５」,免疫生化学実験法,第1版第1刷,(社)日本生化学会編,(株)東京化学同人,144〜148頁,1986年3月14日；Biochemistry,20,4229〜4238(1981)；J.Biol.Biochem.,257,286〜288(1982)］も挙げられる。
【００２４】
本発明に於て使用されるリポソーム膜表面上の抗原に対する抗体としては、リポソームに感作している抗原に対する抗体であれば何れでもよく、その由来に特に制限はない。例えばヤギ、ウサギ、ウマ、ヒツジ、マウス由来のもの等が挙げられる。
【００２５】
一方、赤血球としては、ヒツジ、ウサギ、ウマ、ウシ、ヤギ、ラット、マウス等の動物から得られる赤血球が使用可能であり、また、感作させる溶血素（抗体）としては、用いられる赤血球に対する抗体が挙げられ、これらは常法により得られるモノクローナル抗体でも、例えば、ウサギ、ヒツジ、ウマ、ウシ、ヤギ、ラット、マウス等の動物から得られるポリクローナル抗体でも良いが、ヒツジの赤血球と抗ヒツジ赤血球ウサギ抗体から調製された感作ヒツジ赤血球が望ましい。
【００２６】
リポソームを用いた本発明の補体価測定方法は、例えば下記のごとく行えばよい。
【００２７】
即ち、先ず、標識物質を内包し、その膜上に抗原が固定化されたリポソームと、試料（例えば補体を含むヒト血清等）とリポソーム膜上に固定された抗原に対する抗体及び標識物質の検出に必要な物質ならびに抗ヒト免疫グロブリン抗体とを適当な緩衝液中で混合し、37℃で５〜10分間反応させる。次いで、リポソーム膜上に形成された免疫複合体で活性化された補体によりリポソーム膜が損傷を受けた結果リポソーム外に流出した標識物質量をその性質に基づいて測定し、得られた値を、例えば、あらかじめ補体価既知の血清を用い同様の操作をおこなって得た、補体価と標識物質量との関係を表す検量線に当てはめることにより、試料中の補体価を求めることができる。
【００２８】
また、感作ヒツジ赤血球を用いた本発明の補体価測定方法は例えば下記のごとく行えばよい。
【００２９】
即ち、試料（例えば補体を含むヒト血清等）と、感作血球適当量と、抗ヒト免疫グロブリン抗体とを適当な緩衝液中で混合し、37℃で５〜10分間反応させると、赤血球膜上の免疫複合体で活性化された補体により、感作赤血球膜が溶血するので、感作血球懸濁液の濁度が減少する。この濁度の減少量を測定し、この値を、例えば、あらかじめ補体価既知の血清を用い同様の操作をおこなって得た、補体価と濁度の減少量との関係を表す検量線に当てはめることにより試料中の補体価を求めることができる。また、用手法で行う場合は、反応液を冷却後遠心し、その上清のヘモグロビン量を測定し、この値を用いることよっても、補体価を測定することができる。
【００３０】
なお、ヒト補体価の単位は、例えば、前述のMayerの方法ではCH50値として表現されているが、CH50に限定されるものではない。
【００３１】
本発明の補体価測定用試薬は、リポソームや赤血球を用いる補体価測定用試薬に本発明に係る抗体を含有させた点に特徴を有するものであり、当該抗体の好ましい実施態様や使用濃度等は上記で述べたとおりである。
【００３２】
本発明の補体価測定用試薬は、緩衝剤を含んでいてもよく、これに用いられる緩衝剤としては、例えばリン酸及びその塩、ホウ酸及びその塩、トリス(ヒドロキシメチル)アミノメタン（Tris），グッド緩衝剤(Good's Buffer)，ベロナール緩衝剤等が挙げられるが、これらに限定されるものではなく、通常用いられる緩衝剤であれば何れも使用可能である。
【００３３】
また、本発明の測定用試薬には、牛血清アルブミン、ゼラチン等の蛋白質、糖、キレート剤、還元剤、防腐剤等の、通常この分野に於いて使用される添加物等や、リポソームを用いる場合であって内包された標識物質が酵素である場合は、更にその基質等を、必要に応じて適宜添加してもよい。
【００３４】
本発明に於いて用いられる各種試薬類や、標識物質が酵素である場合に用いられる基質等は、自体公知の酵素測定法に於いて通常用いられる濃度範囲から適宜選択して用いればよい。
【００３５】
本発明の測定法に用いられる試薬キットは、自体公知の補体価測定のための試薬キットに本発明に係る抗体を加えたものである。即ち、例えば、リポソームを用いる補体価測定用試薬キットとしては、標識物質を内包し且つこの膜表面に抗原が固定化されたリポソーム含有試薬、当該抗原に対する抗体含有試薬及び本発明に係る抗体等を含有させた試薬等を含んでなるものが挙げられ、又、赤血球を用いる補体価測定用試薬キットとしては、赤血球と抗赤血球抗体とから調製された感作血球を含有する試薬と本発明に係る抗体等を含有させた試薬等を含有してなるものが挙げられる。
【００３６】
本発明のキットには、補体標準液を添付してもよく、補体標準液としては、ヒト、ラット、ヤギ又はヒツジ由来の血清が挙げられる。これら動物種の系統は特に限定されない。また、これらの動物種由来の補体標準液としては、各種新鮮血清の凍結乾燥品を水又は適当な溶解液で溶解させたものでもよく、また、これを更に適当な溶液で希釈したもの、限外濾過法等で濃縮したもの、補体反応に関与しない成分を除いたもの等も使用可能である。また、これに必要に応じて通常この分野で用いられる糖、蛋白質、防腐剤、安定化剤、緩衝剤等を添加してもよい。
【００３７】
以下に実施例をあげて本発明を更に具体的に説明するが、これらの実施例は本発明を何ら制限するものではない。
【実施例】
実施例１．
（１）補体価測定用リポソームの調製
グルコース-６-リン酸脱水素酵素(G6PDH)を内包し、ジニトロベンゼンが膜上に固定化されたリポソームを、ライフサイエンスにおけるリポソーム実験マニュアル（寺田弘、吉村哲郎編著:シュプリンガー・フェアラーク東京株式会社,60-89 1992年）に記載されたボルテックスイング法によるリポソーム調製法に準じて以下のように調製した。
【００３８】
即ち、ジミリストイルホスファチジルコリン(DMPC)71μmol、ジミリストイルホスファチジルグリセロール(DMPG)8μmol、コレステロール82μmol、及びジニトロベンゼンのホスファチジルエタノールアミン誘導体（AVANTI社製）0.8μmolとをナスフラスコに計りこみ５mlのクロロホルムを加えて溶解した後、ロータリーエバポレーターを用いて減圧乾燥させた。これに G6PDH 水溶液 7.5ml[ 2500U/ml、in 10mM トリス（ヒドロキシメチル）アミノメタン(Tris/HCl)緩衝液(pH7.8)]を加えボルテックスミキサーで混和した。このようにして得られた脂質水和液をポアサイズ0.2μmのフィルターを通して整粒した。得られたリポソーム懸濁液を遠心チューブに移し、４℃、36000rpmで遠心してリポソームに内包されなかった酵素を除き、最後に 100mM Tris/HCl緩衝液(pH7.8)に懸濁して、補体価測定用リポソームを得た。
【００３９】
（２）補体価測定用リポソーム試薬の調製
上記（１）で調製した補体価測定用リポソームを脂質濃度が５nmol/mlとなるように、145mmol/L NaCl含有60mM Tris/HCl緩衝液(pH8.0）で希釈し、補体価測定用試液１を調製した。また、十分量のヤギ抗ＤＮＰ抗体、酵素基質［24mM グルコース-6-リン酸（Ｇ６Ｐ）、９mM ニコチンアミド アデニンジヌクレオチド（ＮＡＤ）］、145mmol/L NaCl、1.5mmol/L MgCl2及び0.45mmol/L CaCl2を含む、10mMマレイン酸／NaOH緩衝液（pH5.5）を調製し、補体価測定用試液２とした。
【００４０】
（３）抗ヒトIgMモノクローナル抗体の作製
精製ヒトIgM（オリエンタル酵母社製）をフロイント完全アジュバントとともにBALB/cマウス（♀）に免疫（２回）後、摘出した脾臓細胞とミエローマ細胞（F0）とをポリエチレングリコールを用いる常法（例えば特開平５−244983号に記載された方法等）により融合させた。その後、常法により抗ヒトIgＭモノクローナル抗体産生ハイブリドーマを選別し、これを培養して抗ヒトIgＭモノクローナル抗体IgG1（クローンNo.101及び102）を得た。
【００４１】
（４）F(ab')2化抗体の作製
ヤギの抗ヒトIgM抗体（ＩＩＣ社製）をイオン交換クロマトグラフィーを用いる常法により精製して得た、ヤギの抗ヒトIgM抗体IgG分画を、ペプシン消化後、ゲルろ過を行う常法（例えば「免疫生化学研究法、第１版第１刷、（株）東京化学同人,1986」に記載された方法）により精製してそのF(ab')2を得た。
【００４２】
（５）干渉作用回避効果の比較
上記（２）で調製した補体価測定用試液２に種々の抗体を添加し、補体価への干渉作用回避効果の比較を行った。なお、補体価測定用試液２への添加量は抗体蛋白量として各々０.１mg/mlとした。
【００４３】
尚、干渉作用回避効果は、以下に示す方法で調べた。
【００４４】
補体価への干渉作用があることが確認されている血清試料10μlと補体価測定用試薬１ 250μlとを混合し、37℃で５分間インキュベーションした後、 所定の抗体を添加した補体価測定用試薬２を125μlを加え、更に37℃で５分間反応させ４〜５分間放置後、G6PDH の活性値を、１分間あたりの340nmの吸光度変化（△Ａ１）として測定した。さらに、先に用いた干渉作用がある血清試料と同等の補体価を有し、干渉作用が無いことが確認されている血清についても、各々の試薬を用いて同様の操作を行い△Ａ２を得た。これらの値を下記式に当てはめ、干渉作用回避率を求めた。
【００４５】
得られた値を表１に示す。また、抗体無添加の補体価測定用試薬２を用い、干渉作用のない血清試料を測定して求めた△Ａ２を100とし、各種抗体を添加した補体価測定用試薬２を用いて求めた△Ａ２のこれに対する比率を、補体活性維持率として表２に示す。
【００４７】
【表１】
【００４８】
表１
表１の結果から、抗ヒトIgMモノクローナル抗体・クローンNo.101、抗ヒトIgMモノクローナル抗体・クローンNo.102及びＦ(ab')2化抗ヒトIgMヤギ抗体等のヒト免疫グロブリンを認識する抗体に、試料の補体価への干渉作用を回避する効果があることがわかった。特に、抗ヒトIgMモノクローナル抗体・クローンNo.101でその効果が最も大きかった。
【００５０】
【表２】
【００５１】
表２
表２の結果から、抗ヒトIgMモノクローナル抗体・クローンNo.101、抗ヒトIgMモノクローナル抗体・クローンNo.102及びＦ(ab')2化抗ヒトIgMヤギ抗体の、補体活性維持率はほぼ100％であり、これらの抗体は補体活性を阻害しないことがわかった。
【００５３】
実施例２．
（１）補体価測定用赤血球試薬の調製
1mmol/L MgCl2、0.15mmol/L CaCl2及び0.1%ゼラチンを含む、イオン強度0.147のベロナール緩衝液（pH7.4）を調製し、補体価測定用試液３とした。また、この一部に実施例１で用いた抗ヒトIgMモノクローナル抗体（クローンNo.101）を0.014mg/mlとなるように添加し、補体価測定用試液４とした。
【００５４】
ヒツジ赤血球に溶血素［抗ヒツジ赤血球抗体（ウサギ）］を常法（例えば「補体学」稲井ら著、122-126頁,第2刷,昭和58年,医歯薬出版株式会社、）により感作し、感作ヒツジ赤血球を得た。これを3.3×108個／ｍｌとなるように補体価測定用試液３で希釈し、補体価測定用試液５を調製した。
【００５５】
（２）干渉作用回避効果の比較
上記（１）で調製した補体価測定用試薬を用い、以下に示す方法で測定を行って干渉作用回避効果を調べた。
【００５６】
補体価への干渉作用があることが確認されている血清試料3μlと、補体価測定用試薬３又は４ 260μlとを混合し、37℃で５分間インキュベーションした後、 補体価測定用試薬５を60μlを加え、更に37℃で５分間反応させ、その５分間に減少した濁度を660nmで測定した（△Ａ３）。さらに、先に用いた干渉作用があることが確認されている血清試料と同等の補体価を有し、干渉作用が無いことが確認されている血清についても、それぞれの試薬で同様の操作を行い△Ａ４を得た。これらの値を下記式に当てはめ、干渉作用回避率を求めた。
【００５７】
得られた値を表３に示す。また、補体価測定用試薬３を用い、干渉作用のない血清試料を測定して求めた△Ａ４を100とし、補体価測定用試薬４で求めた△Ａ４の比率を、補体活性維持率として表４に示す。
【００５９】
【表３】
【００６０】
表３
【表４】
【００６２】
表４
表３及び４の結果から、抗ヒトIgMモノクローナル抗体・クローンNo.101は血清試料の補体活性を阻害せず、しかも測定試料による補体価への干渉作用を回避できることがわかった。
【００６４】
【発明の効果】
本発明は、測定用試薬にヒト免疫グロブリンを認識する抗体を添加することを特徴とする補体価測定方法及びその試薬を提供するものであり、本発明を使用することによって、測定試料中のリウマチ因子等から受ける補体価への干渉作用を軽減し、良好な再現性で高精度に補体価が測定できるという顕著な効果を奏する。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a measurement method and a reagent for quantitative analysis of complement value in a sample.
[0002]
[Prior art]
The complement system is a collective term for about 20 proteins present in the sera of animals such as humans, and is divided into a classical pathway activated mainly by immune complexes and a second pathway activated by polysaccharides. Has one activation pathway. The classical pathway is that the complement components are activated one after another in order by the immune complex formed by binding antibodies against the antigens mainly on cell membranes such as bacteria, and finally destroying the cell membrane, Bacteria and the like have the effect of being killed or dissolved. The classical pathway is the reaction in which red blood cells sensitized with hemolysin reach hemolysis with complement, or when the anti-hapten antibody is reacted with liposomes sensitized with hapten, the liposome membrane is destroyed by complement. By the action of. On the other hand, the antibody does not need to be involved in the second pathway. For example, complement is activated only by contact with polysaccharides or viruses that are constituents of bacterial cell walls.
[0003]
In recent years, the activity of the complement system, that is, the measurement of complement value, has attracted attention as an indicator for the diagnosis and treatment of acute glomerulonephritis, autoimmune diseases and the like.
[0004]
Currently, the Mayer's 50% hemolysis method, which measures the hemolytic activity of complement using sheep erythrocytes sensitized with hemolysin, and its modified method are widely used for the measurement of complement value. "Procedure for clinical laboratory test", pp. 1233-1234, 29th edition, 5th edition, 1985, Kanbara Publishing Co., Ltd .; J. Clin. Chem., 12 , 143 (1983), etc.]. However, this method requires preparation of samples diluted to several dilutions for the same sample, which causes measurement errors, and shows the amount of hemolysis that occurs at 50% from the graph. It is also a complicated way of seeking. Furthermore, sheep erythrocytes are used, but erythrocytes derived from living organisms are unstable, and there are problems such as differences in sensitivity of erythrocytes to complements due to individual differences among animals (difference between lots).
[0005]
On the other hand, the reaction time is shortened (5 to 10 minutes) in order to more easily measure the complement value, and it is produced by complement activated by sensitized blood cells as a method enabling adaptation to an automatic analyzer. Complement value measurement method using erythrocytes (clinical test, 32 (12) , 1537-1540, 1998) to obtain complement activity value based on turbidity change of sensitized blood cell suspension, instead of erythrocytes Complement value measurement method using liposomes that are more stable and have less lot-to-lot differences, and membrane damage due to complement activity (YAMAMOTO.S Clin.Chem. 41/4 , 586-590,1994, JP 7-110331 A Recently, Japanese Laid-Open Patent Publication No. 7-140147 and the like have been proposed.
[0006]
However, in the Mayer method, the dilution factor of the serum sample during the reaction for measuring the complement value is about 160 to 480 times, whereas the reagent for measuring the complement value that can be applied to an automatic analyzer The dilution ratio is about 100 to 110 times for a reagent using erythrocytes and about 35 to 45 times for a reagent using liposomes. Therefore, since the ratio of the serum sample to the reagent is relatively high, there is a concern about the interference effect of the serum sample on the measurement system when measuring the complement value by the automatic analyzer. It has been suggested that when rheumatoid factor-positive serum samples are used in the measurement of complement titer using the Rheumatoid factor, there is a possibility that they may be interfered with by the samples (“The 44th Annual Meeting of the Japanese Society for Clinical Hygiene Testing”, Title 365, 1995, “Medicine and Pharmacy” 35 (5) , 1163-1167, 1996).
[0007]
[Problems to be solved by the present invention]
The present invention has been made in view of the situation as described above, and provides a measurement method and a reagent for measurement in which the interference effect on a measurement system by a measurement sample such as a serum sample in complement value measurement by an automatic analyzer is reduced. For the purpose.
[0008]
[Means for solving problems]
The present invention has been made to solve the above problems,
“A method for measuring complement titer, comprising measuring complement titer in the presence of an antibody that specifically binds to human immunoglobulin.”,
"Complement value measuring reagent containing an antibody that specifically binds to human immunoglobulin.",
“A reagent containing an antibody that specifically binds to human immunoglobulin, a reagent containing a labeling substance-encapsulating liposome with an antigen immobilized on the membrane, and a reagent containing an antibody against the antigen immobilized on the membrane Complement value measurement reagent kit comprising: a reagent containing an antibody that specifically binds to human immunoglobulin, and a reagent containing hemolysin-sensitized blood cells. Reagent kit. "
[0009]
That is, the present inventors, as a result of intensive research to find out a method for suppressing interference effect received from, for example, rheumatoid factor in a measurement sample when measuring complement values using erythrocytes and liposomes, By coexisting with an antibody that specifically binds to the antibody (hereinafter sometimes abbreviated as the antibody according to the present invention), the interference effect received from the coexisting substance in the measurement sample is reduced, and more accurate complement value measurement As a result, the present invention has been completed.
[0010]
The antibody according to the present invention may be any antibody that specifically binds to human immunoglobulin, but is preferably an antibody that does not itself have the ability to activate the complement of the classical pathway, such as mouse IgG1, Antibodies such as mouse IgA, rat IgG1, and rat IgA that do not themselves have the ability to activate the complement of the classical pathway, or antibodies that have the ability to activate the complement of the classical pathway, can be obtained by conventional methods such as “immunogenic In accordance with the method described in “Chemical Research Method, First Edition, First Print, Tokyo Chemical Co., Ltd., 1986”, etc., pepsin digestion, papain digestion, etc. were performed, and the Fc portion that is the binding site of complement was removed , F (ab ') 2 fragments or Fab' fragments and the like. Of these, mouse IgG1 is preferred.
[0011]
These antibodies may be used in a state where they are contained in serum or ascites. For example, “Immunobiochemistry Research Method, First Edition, First Print, Tokyo Chemical Dojin, 1986” etc. According to the described method, it is desirable to purify and use by methods such as ammonium sulfate fractionation, ion exchange chromatography, gel filtration chromatography, affinity chromatography and the like. Moreover, when performing the removal operation of Fc part, it is desirable to refine | purify and use after the said operation.
[0012]
In addition, the antibody according to the present invention can be used in accordance with a method described in, for example, “Introduction to Immunological Experiments, Second Printing, Nao Matsuhashi, Society Publishing Center, 1981”, etc. Polyclonal antibodies produced by immunizing animals such as rats and mice with human immunoglobulins or fragments thereof, or the cell fusion method established by Keller and Milstein (Nature, 256, 495, 1975) According to the present invention, any monoclonal antibody produced by a hybridoma obtained by fusing cells from a mouse tumor line with spleen cells of a mouse previously immunized with human immunoglobulin or a fragment thereof is preferable, but a monoclonal antibody is preferred. These may be used alone or in appropriate combination.
[0013]
The human immunoglobulin to which the antibody according to the present invention specifically binds is not particularly limited, and examples thereof include human IgA, human IgM, and human IgG.
[0014]
The complement titer measuring method of the present invention is a per se known measuring method using liposomes or erythrocytes other than the presence of the antibody according to the present invention as described above, for example, YAMAMOTO.S Clin.Chem. 41/4 , 586-590, 1994, Japanese Patent Laid-Open No. 7-110331, Japanese Patent Laid-Open No. 7-140147, and clinical tests, 32 (12) , 1537-1540, 1998 The kind may be appropriately selected according to a measurement method known per se.
[0015]
That is, by measuring a measurement sample according to a known measurement method using liposomes or erythrocytes as described above in the presence of the antibody according to the present invention, the complement value in the sample is easily and accurately measured. be able to.
[0016]
In addition, the antibody according to the present invention usually has an antibody protein amount of 1 × 10 −6 to 10 mg, preferably 5 × 10 −5 to 1 × 10 −2 for 1 μl of a measurement sample such as serum or plasma at the time of measurement. Used to be mg.
[0017]
In the measurement method of the present invention, when the liposome or erythrocyte and the sample are reacted, the antibody according to the present invention may be finally allowed to coexist in the concentration range as described above, and the method is particularly limited. Not.
[0018]
Specifically, for example, a method for containing the antibody according to the present invention in a reagent for measuring complement value as described above and mixing the sample with the sample, for example, a buffer containing the antibody according to the present invention Examples include a method of diluting a sample with a solution such as a liquid and mixing the diluted sample with a reagent for measuring complement value as described above.
[0019]
The antibody according to the present invention may be added to the measurement system at any time before the start of the measurement of complement value, but it is desirable to add it before complement is activated. That is, when the antibody according to the present invention is added to the measurement system, in the method using liposomes, before the complement in the sample is activated by the antigen-antibody complex formed on the liposome surface. In addition, in the method using erythrocytes, it is preferable that complements in the sample are activated by immune complexes on the erythrocyte membrane.
In addition, liposomes used in the above-mentioned reagents for measuring complement valency as described above, labeling substances encapsulated in the liposomes, antigens immobilized on the liposomes, and antibodies against the antigens or erythrocytes sensitized with hemolysin Is not particularly limited as long as it is usually used.
[0020]
That is, as the liposomes used in the present invention, all those usually used in this field can be used. For example, distearoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol (DMPG), egg yolk phosphatidylglycerol, etc. Preparation methods known per se, for example, J. liposome Res., 1 (3) , 339-377 (1989-90), “Liposome Experiment Manual in Life Science”, edited by Hiroshi Terada, edited by Tetsuro Yoshimura, Springer Fairlake Tokyo ( Co., Ltd., 1992, Clin. Chem. 41 / 4.586-590 (1995) and the like are preferable. Among them, those prepared by the method of Clin. Chem. 41 / 4.586 to 590 (1995) are preferable.
[0021]
In addition, as the labeling substance encapsulated in the liposome, any substance usually used in this field can be used. For example, enzymes, coenzymes, dyes, water-soluble fluorescent substances, saccharides, ionic compounds In addition, a chelate indicator, a dye, a spin label compound, and the like that can be detected by being released from the membrane due to membrane damage of the liposome are preferable. Among them, it is preferable to use an enzyme as a labeling substance. The encapsulation method is also a conventionally known preparation method, for example, “Liposome Experiment Manual in Life Science”, edited by Hiroshi Terada, edited by Tetsuro Yoshimura, pages 60-89, Springer Fairlark Tokyo, 1992, The methods described in JP-A-7-110331 and JP-A-7-140147 can be performed.
[0022]
Antigens to be fixed on the liposome membrane surface, may be at any as long as antigens that can be incorporated into the liposome membrane, for example, Forssman antigen, glycolipid antigens such as GM 1, dinitrobenzene, aromatic birds nitrobenzene Group organic compounds, hormones such as thyroxine, drugs such as theophylline and phenytoin, and the like. Further, as a method for incorporating these into the liposome membrane, Forsman antigen, for example, the method described in Biochemistry, 8 , 4149 (1969), for glycolipid antigen, for example, J. Immunol. Methods, 85 , 53-63. The method described in (1985), for dinitrobenzene and trinitrobenzene, for example, the method described in Biochemistry, Vol. 11, No. 22, 4085 (1972) and J. Immunol. Methods, 123 , 19-24 (1989) , for thyroxine, for example Bio.Technology, the method described in April, 349 (1984), the theophylline and phenytoin, for example J.Chem.Pharm.Bull., 36, 1086 (1988 ) and Clin. Chem., 38 808 (1992).
[0023]
In addition, as a method of immobilizing an antigen on the surface of a liposome membrane, a method usually used in this field, for example, a cross-linking method [“Second Life Chemistry Laboratory Lecture 5”, Immunobiochemical Experiment Method, First Edition, First Edition] Pp., Japan Biochemical Society, Tokyo Chemical Co., Ltd., pp. 144-148, March 14, 1986; Biochemistry, 20 , 4229-4238 (1981); J. Biol. Biochem., 257 , 286 -288 (1982)].
[0024]
The antibody against the antigen on the surface of the liposome membrane used in the present invention may be any antibody as long as it is an antibody against the antigen sensitized to the liposome, and its origin is not particularly limited. Examples include those derived from goats, rabbits, horses, sheep and mice.
[0025]
On the other hand, as red blood cells, red blood cells obtained from animals such as sheep, rabbits, horses, cows, goats, rats and mice can be used, and as a hemolysin (antibody) to be sensitized, antibodies against the red blood cells used are used. These may be monoclonal antibodies obtained by conventional methods, for example, polyclonal antibodies obtained from animals such as rabbits, sheep, horses, cows, goats, rats, mice, etc., but sheep red blood cells and anti-sheep red blood cells rabbits Sensitized sheep erythrocytes prepared from antibodies are preferred.
[0026]
The complement value measuring method of the present invention using liposomes may be performed, for example, as follows.
[0027]
That is, first, a liposome in which a labeling substance is encapsulated and an antigen is immobilized on the membrane, a sample (eg, human serum containing complement), an antibody against the antigen immobilized on the liposome membrane, and a labeling substance are detected. Substances necessary for the preparation and anti-human immunoglobulin antibody are mixed in an appropriate buffer and reacted at 37 ° C. for 5 to 10 minutes. Next, the amount of the labeled substance that flows out of the liposome as a result of damage to the liposome membrane caused by the complement activated by the immune complex formed on the liposome membrane is measured based on its property, and the obtained value is calculated. For example, the complement value in the sample can be determined by applying a calibration curve representing the relationship between the complement value and the amount of the labeled substance obtained by performing the same operation using serum having a known complement value in advance. it can.
[0028]
Further, the complement value measuring method of the present invention using sensitized sheep erythrocytes may be carried out as follows, for example.
[0029]
That is, when a sample (such as human serum containing complement), an appropriate amount of sensitized blood cells, and an anti-human immunoglobulin antibody are mixed in an appropriate buffer and reacted at 37 ° C. for 5 to 10 minutes, Complement activated by immune complexes on the membrane results in hemolysis of the sensitized erythrocyte membrane, thus reducing the turbidity of the sensitized blood cell suspension. The amount of decrease in turbidity was measured, and this value was obtained by, for example, performing a similar operation using a serum having a known complement value in advance, and a calibration curve representing the relationship between the complement value and the decrease in turbidity. The complement value in the sample can be determined by applying to the above. Moreover, when carrying out by a method, a reaction liquid is centrifuged after cooling, Complement value can be measured also by measuring the hemoglobin amount of the supernatant liquid, and using this value.
[0030]
The unit of human complement value is expressed as, for example, the CH50 value in the aforementioned Mayer method, but is not limited to CH50.
[0031]
The reagent for measuring complement value of the present invention is characterized in that the antibody according to the present invention is contained in a reagent for measuring complement value using liposomes or erythrocytes, and preferred embodiments and concentrations of the antibody are used. Etc. are as described above.
[0032]
The reagent for measuring complement value of the present invention may contain a buffer, and examples of the buffer used therefor include phosphoric acid and salts thereof, boric acid and salts thereof, tris (hydroxymethyl) aminomethane ( Tris), Good's Buffer, Veronal Buffer, and the like, but are not limited to these, and any buffer that is usually used can be used.
[0033]
The measuring reagent of the present invention uses proteins such as bovine serum albumin and gelatin, sugars, chelating agents, reducing agents, preservatives and the like, additives and the like normally used in this field, and liposomes. In the case where the encapsulated labeling substance is an enzyme, its substrate or the like may be added as necessary.
[0034]
The various reagents used in the present invention, the substrate used when the labeling substance is an enzyme, etc. may be appropriately selected from a concentration range usually used in a known enzyme measurement method.
[0035]
The reagent kit used in the measurement method of the present invention is a reagent kit for measuring complement value known per se, to which the antibody according to the present invention is added. That is, for example, as a reagent kit for measuring complement value using liposome, a liposome-containing reagent containing a labeling substance and having an antigen immobilized on the membrane surface, an antibody-containing reagent against the antigen, the antibody according to the present invention, etc. And a reagent kit for measuring the complement using erythrocytes is a reagent containing sensitized blood cells prepared from erythrocytes and anti-erythrocyte antibodies and the present invention. And those containing a reagent containing the antibody according to the above.
[0036]
A complement standard solution may be attached to the kit of the present invention, and examples of the complement standard solution include serum derived from human, rat, goat or sheep. The strain of these animal species is not particularly limited. Further, as a complement standard solution derived from these animal species, lyophilized products of various fresh sera may be dissolved in water or an appropriate lysis solution, and further diluted with an appropriate solution, What concentrated by the ultrafiltration method etc., what remove | excluded the component which does not participate in a complement reaction, etc. can be used. In addition, sugars, proteins, preservatives, stabilizers, buffers and the like that are usually used in this field may be added as necessary.
[0037]
The present invention will be described more specifically with reference to the following examples. However, these examples are not intended to limit the present invention.
【Example】
Example 1.
(1) Preparation of Liposome for Complement Value Measurement Liposomes containing glucose-6-phosphate dehydrogenase (G6PDH) and dinitrobenzene immobilized on the membrane were prepared as a liposome experiment manual in life science (Hiroshi Terada, It was prepared as follows according to the liposome preparation method by the vortex swing method described in Tetsuro Yoshimura edited by Springer Fairlark Tokyo Co., Ltd., 60-89 (1992).
[0038]
Specifically, 71 μmol of dimyristoyl phosphatidylcholine (DMPC), 8 μmol of dimyristoyl phosphatidylglycerol (DMPG), 82 μmol of cholesterol, and 0.8 μmol of phosphatidylethanolamine derivative of dinitrobenzene (manufactured by AVANTI) were weighed into an eggplant flask and 5 ml of chloroform was added. After dissolution, it was dried under reduced pressure using a rotary evaporator. To this, 7.5 ml of G6PDH aqueous solution [2500 U / ml, in 10 mM tris (hydroxymethyl) aminomethane (Tris / HCl) buffer (pH 7.8)] was added and mixed with a vortex mixer. The lipid hydration solution thus obtained was sized through a filter having a pore size of 0.2 μm. Transfer the resulting liposome suspension to a centrifuge tube, centrifuge at 4 ° C., 36000 rpm to remove the enzyme that was not encapsulated in the liposomes, and finally suspend in 100 mM Tris / HCl buffer (pH 7.8). A liposome for titration measurement was obtained.
[0039]
(2) Preparation of Liposome Reagent for Complement Value Measurement The liposome for complement value measurement prepared in (1) above was added to a 60 mM Tris / HCl buffer solution (pH 8) containing 145 mmol / L NaCl so that the lipid concentration was 5 nmol / ml. 0.0) to prepare Complement Value Measuring Solution 1. In addition, a sufficient amount of goat anti-DNP antibody, enzyme substrate [24 mM glucose-6-phosphate (G6P), 9 mM nicotinamide adenine dinucleotide (NAD)], 145 mmol / L NaCl, 1.5 mmol / L MgCl 2 and 0.45 mmol / A 10 mM maleic acid / NaOH buffer solution (pH 5.5) containing L CaCl 2 was prepared and used as Complement Value Measuring Solution 2.
[0040]
(3) Preparation of anti-human IgM monoclonal antibody Purified human IgM (manufactured by Oriental Yeast) was immunized (double) to BALB / c mice (c) together with Freund's complete adjuvant, and then extracted spleen cells and myeloma cells (F0) Were fused by a conventional method using polyethylene glycol (for example, the method described in JP-A-5-244983). Thereafter, anti-human IgM monoclonal antibody-producing hybridomas were selected by a conventional method and cultured to obtain anti-human IgM monoclonal antibody IgG1 (clone Nos. 101 and 102).
[0041]
(4) Preparation of F (ab ′) 2 antibody A goat anti-human IgM antibody IgG fraction obtained by purifying goat anti-human IgM antibody (manufactured by IIC) by a conventional method using ion exchange chromatography was obtained. After purification with pepsin, purification by the conventional method of gel filtration (for example, the method described in “Immunobiochemical Research, First Edition, First Printing, Tokyo Chemical Dojin, 1986”) and its F (ab ') Got two .
[0042]
(5) Comparison of interference action avoidance effect Various antibodies were added to the complement value measuring test solution 2 prepared in the above (2), and the interference action avoidance effect on complement value was compared. The amount added to Complement Value Measuring Solution 2 was 0.1 mg / ml as the amount of antibody protein.
[0043]
The interference avoidance effect was examined by the following method.
[0044]
10 μl of a serum sample that has been confirmed to interfere with complement titer and 250 μl of complement titration reagent 1 are mixed, incubated at 37 ° C. for 5 minutes, and then complemented with a specific antibody. 125 μl of measurement reagent 2 was added, and the mixture was further reacted at 37 ° C. for 5 minutes. After standing for 4 to 5 minutes, the activity value of G6PDH was measured as an absorbance change (ΔA1) at 340 nm per minute. In addition, for the serum having a complement value equivalent to that of the serum sample having the interference action previously used and confirmed to have no interference action, the same operation was performed using each reagent, and ΔA2 was calculated. Obtained. These values were applied to the following equation to determine the interference avoidance rate.
[0045]
The obtained values are shown in Table 1. In addition, ΔA2 obtained by measuring a serum sample having no interference using the antibody-free complement 2 measuring reagent 2 is set to 100, and the complement-value measuring reagent 2 added with various antibodies is used. The ratio of ΔA2 to this is shown in Table 2 as the complement activity maintenance rate.
[0047]
[Table 1]
[0048]
Table 1
From the results of Table 1, anti-human IgM monoclonal antibody / clone No. 101, anti-human IgM monoclonal antibody / clone No. 102 and antibodies that recognize human immunoglobulin such as F (ab ′) 2 anti-human IgM goat antibody It was found that there is an effect of avoiding interference with the complement value of the sample. In particular, the anti-human IgM monoclonal antibody / clone No. 101 was most effective.
[0050]
[Table 2]
[0051]
Table 2
From the results shown in Table 2, the complement activity maintenance rate of anti-human IgM monoclonal antibody / clone No. 101, anti-human IgM monoclonal antibody / clone No. 102 and F (ab ′) 2 anti-human IgM goat antibody is almost 100%. % And these antibodies were found not to inhibit complement activity.
[0053]
Example 2
(1) Preparation of red blood cell reagent for complement value measurement
A Veronal buffer solution (pH 7.4) containing 1 mmol / L MgCl 2 , 0.15 mmol / L CaCl 2 and 0.1% gelatin and having an ionic strength of 0.147 was prepared and used as Complement Value Measuring Solution 3. In addition, to this part, the anti-human IgM monoclonal antibody (clone No. 101) used in Example 1 was added to a concentration of 0.014 mg / ml to obtain Complement Value Determination Test Solution 4.
[0054]
Hemolysin [anti-sheep erythrocyte antibody (rabbit)] is applied to sheep erythrocytes by conventional methods (for example, "Complementology" written by Inai et al., Pages 122-126, 2nd edition, 1983, Ishiyaku Shuppan Publishing Co., Ltd.) Sensitized to obtain sensitized sheep erythrocytes. This was diluted with Complement Value Measuring Reagent 3 so as to be 3.3 × 10 8 pieces / ml, and Complement Value Measuring Reagent 5 was prepared.
[0055]
(2) Comparison of interference action avoidance effect Using the reagent for measuring complement value prepared in the above (1), measurement was performed by the following method to investigate the interference action avoidance effect.
[0056]
3 μl of a serum sample that has been confirmed to interfere with complement value and 3 or 4 260 μl of complement titration reagent are mixed and incubated at 37 ° C. for 5 minutes. 60 μl of 5 was added, and the mixture was further reacted at 37 ° C. for 5 minutes. The turbidity decreased in 5 minutes was measured at 660 nm (ΔA3). In addition, for the serum that has a complement value equivalent to that of the serum sample that has been confirmed to have the interference action previously used and that has been confirmed to have no interference action, the same operation is performed with each reagent. To obtain ΔA4. These values were applied to the following equation to determine the interference avoidance rate.
[0057]
The obtained values are shown in Table 3. In addition, ΔA4 obtained by measuring a serum sample having no interference action using the complement value measuring reagent 3 is set to 100, and the ratio of ΔA4 obtained by the complement value measuring reagent 4 is set to maintain complement activity. It shows in Table 4 as a rate.
[0059]
[Table 3]
[0060]
Table 3
[Table 4]
[0062]
Table 4
From the results of Tables 3 and 4, it was found that the anti-human IgM monoclonal antibody / clone No. 101 does not inhibit the complement activity of the serum sample and can avoid the interference effect on the complement value by the measurement sample.
[0064]
【The invention's effect】
The present invention provides a complement value measuring method characterized in that an antibody recognizing human immunoglobulin is added to a measuring reagent and the reagent, and by using the present invention, The effect of interfering with the complement value received from rheumatoid factors is reduced, and the complement value can be measured with high reproducibility and high accuracy.
Claims (11)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP35247498A JP4178632B2 (en) | 1998-12-11 | 1998-12-11 | Methods and reagents for avoiding interference effects |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP35247498A JP4178632B2 (en) | 1998-12-11 | 1998-12-11 | Methods and reagents for avoiding interference effects |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2000180446A JP2000180446A (en) | 2000-06-30 |
| JP4178632B2 true JP4178632B2 (en) | 2008-11-12 |
Family
ID=18424324
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP35247498A Expired - Fee Related JP4178632B2 (en) | 1998-12-11 | 1998-12-11 | Methods and reagents for avoiding interference effects |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP4178632B2 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2609280T3 (en) * | 2007-08-23 | 2017-04-19 | Lsi Medience Corporation | Non-specific reaction inhibitor |
| US20180088110A1 (en) * | 2015-08-17 | 2018-03-29 | Sekisui Medical Co., Ltd. | Immunological detection method and test strip used therefor |
| CN110650729B (en) | 2017-03-29 | 2024-08-20 | 住友制药株式会社 | Vaccine adjuvant formulations |
| CN110780082B (en) * | 2019-11-04 | 2023-05-12 | 上海昆涞实业发展有限公司 | Total complement activity quality control product, reagent, preparation method and application |
| KR20240150777A (en) * | 2022-02-18 | 2024-10-16 | 피에이치씨 주식회사 | Nonspecific reaction inhibitor |
-
1998
- 1998-12-11 JP JP35247498A patent/JP4178632B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JP2000180446A (en) | 2000-06-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA1149277A (en) | Process for determining immuno-complexes | |
| AU659797B2 (en) | Analyte determination in biological fluids in the presence of interfering substances | |
| CN113671193A (en) | Immunoassay method for L-FABP and assay reagent used for the same | |
| JP4178632B2 (en) | Methods and reagents for avoiding interference effects | |
| US5302512A (en) | Agglutinant complex as blood typing reagent | |
| EP1347298A2 (en) | Immunoassay method, test strip for use therein, and immunoassay apparatus | |
| WO2022091923A1 (en) | Sensitizer for immunochromatographic assays, and assay | |
| JPS58211662A (en) | Uniform system combination analysis method for measuring sample in test sample and reagent system used for said method | |
| NZ231727A (en) | Specific binding type assay (e.g. immunoassay) involving binding compound coupled or adsorbed on inner surface of reaction vessel and lyophilised conjugate | |
| JP2005505755A (en) | Cat blood type determination method and determination kit | |
| WO1988009933A1 (en) | Immunoassay method | |
| JP7144039B2 (en) | Severe Fever Thrombocytopenia Syndrome Virus Antibody Detection Kit and Severe Fever Thrombocytopenia Syndrome Virus Antibody Detection Method | |
| JPS628055A (en) | Enzymatic immunoassay | |
| JP4491572B2 (en) | Reagents and methods for measuring substances to be measured in samples by enzyme immunoassay | |
| RU2149406C1 (en) | Method for determining functional activity of human c4 complement component | |
| JP4422291B2 (en) | Immunological assay for human medalacin | |
| RU2144676C1 (en) | Method of assay of functional activity of human c1q subcomponent of complement | |
| JPS61228353A (en) | Immunological concentration determination method of amine, monochronal antibody and reagent set for executing said method | |
| JP3512545B2 (en) | Method for measuring hemoglobin A1C | |
| JPS6175262A (en) | Reagent for measuring immunological composite and measurement of composite using the same | |
| JPH0746106B2 (en) | Immunoassay method | |
| JPS63290963A (en) | Latex sensitized with enzyme labeled antibody and enzyme immunoassay using said latex | |
| JPH0682446A (en) | Diagnostic method for kidney disease | |
| JPS5926065A (en) | Reagent for measuring human urinary kallikrein | |
| JP2022094146A (en) | Sensitizer and measurement method for immuno-chromatography measurement method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20050811 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20070618 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20070626 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20070821 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20080311 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20080805 |
|
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20080818 |
|
| R150 | Certificate of patent or registration of utility model |
Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20110905 Year of fee payment: 3 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20110905 Year of fee payment: 3 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20140905 Year of fee payment: 6 |
|
| S533 | Written request for registration of change of name |
Free format text: JAPANESE INTERMEDIATE CODE: R313533 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| LAPS | Cancellation because of no payment of annual fees |