JP4318092B2 - Measuring reagent for measuring substance and method for stabilizing measuring reagent for measuring substance - Google Patents

Description

【０２００】
３．考察
表１より、陽イオン及び陰イオンが各々１００ｍＭ未満である〔測定試薬Ａ〕において、３０℃での保存５０日後に得た吸光度差の値は、保存開始前の吸光度差の値の５０％まで減少してしまっていることが分かる。すなわち、シグナルの生成が大きく減少し、測定の感度が低下してしまっている。
【０２０１】
これに対して、１００ｍＭの塩化ナトリウムを含有して、陽イオン及び陰イオンが各々１００ｍＭ以上存在することになる〔測定試薬Ｂ〕において、３０℃での保存５０日後に得た吸光度差の値は、保存開始前の吸光度差の値の８３％であり、生成するシグナルの減少、すなわち測定の感度の低下が抑制されていることが分かる。
【０２０２】
更に、５００ｍＭの塩化ナトリウムを含有して、陽イオン及び陰イオンが各々５００ｍＭ以上存在することになる〔測定試薬Ｃ〕において、３０℃での保存５０日後に得た吸光度差の値は、保存開始前の吸光度差の値の９６％であり、生成するシグナルの減少、すなわち測定の感度の低下をほとんど抑制できていることが分かる。
【０２０３】
従って、陽イオン及び陰イオンが各々１００ｍＭ以上存在することにより、測定対象物質の測定試薬を長期間安定に保つことができ、例え、３０℃で５０日間という測定試薬にとって過酷な保存条件においても、生成するシグナルの減少を防ぎ、測定の感度の低下を抑制することができることが確かめられた。
【０２０４】
〔実施例３〕（トレポネーマ・パリダムに対する抗体の測定試薬の保存安定性の確認）
【０２０５】
前記実施例１で調製した、トレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子を含む測定試薬を、３０℃で保存し、保存時の安定性の確認を行った。
【０２０６】
１．測定試薬の保存
前記実施例１の１の（２）で調製した、〔測定試薬Ｄ〕、〔測定試薬Ｅ〕、〔測定試薬Ｆ〕、及び〔測定試薬Ｇ〕の４種類の測定試薬を、各々３０℃で６０日間保存した。
【０２０７】
２．測定試薬の安定性の確認
保存前、保存中及び保存後の前記測定試薬の各々を用いて試料中の測定対象物質（トレポネーマ・パリダムに対する抗体）の測定を行い、前記測定試薬の保存時の安定性の確認を行った。
【０２０８】
（１） 試薬
【０２０９】
▲１▼ 第１試薬
前記実施例１の２で調製した試料の希釈液である〔第１試薬〕を、第１試薬として用いた。
【０２１０】
▲２▼ 第２試薬
下記の測定試薬をそれぞれ第２試薬として用いた。
【０２１１】
・ 前記１における３０℃での保存６０日後の〔測定試薬Ｄ〕、〔測定試薬Ｅ〕、〔測定試薬Ｆ〕、若しくは〔測定試薬Ｇ〕。
【０２１２】
・ 前記１における３０℃での保存３０日後の〔測定試薬Ｄ〕、〔測定試薬Ｅ〕、〔測定試薬Ｆ〕、若しくは〔測定試薬Ｇ〕。 又は、
【０２１３】
・ 測定試薬調製時すなわち保存開始前の〔測定試薬Ｄ〕、〔測定試薬Ｅ〕、〔測定試薬Ｆ〕、若しくは〔測定試薬Ｇ〕。
【０２１４】
（２） 試料
トレポネーマ・パリダムに対する抗体を含むヒト血清を試料として用いた。
【０２１５】
（３） 試料中の測定対象物質（トレポネーマ・パリダムに対する抗体）の測定
【０２１６】
▲１▼ 測定は、日立−７１７０形自動分析装置（日立製作所社製）を使用して行った。
【０２１７】
まず、測定用セル（キュベット）に、前記の試料の２０μＬを添加した。
次に、この測定用セル（キュベット）に、前記の第１試薬の１７５μＬを添加し、混合した。
【０２１８】
そして、この測定用セル（キュベット）を、３７℃で静置した。
【０２１９】
▲２▼ 前記の第１試薬の添加後４分１４秒目（１５ポイント目）に、この測定用セル（キュベット）内の混合液の吸光度（波長：７００ｎｍ）を試料盲検として測定した。
【０２２０】
▲３▼ 前記の第１試薬の添加後４分３０秒目（１６ポイント目）に、この測定用セル（キュベット）内の混合液に、更に、前記の第２試薬の２５μＬを添加し、混合した。
【０２２１】
そして、この測定用セル（キュベット）を、３７℃で静置して、反応を行わせた。
これにより、前記のラテックス粒子に固定化されたトレポネーマ・パリダムの４７Ｋダルトン抗原と、前記の試料に含まれていたトレポネーマ・パリダムに対する抗体との抗原抗体反応を行わせ、ラテックス粒子の凝集塊を生成させた。
【０２２２】
▲４▼ 前記の第１試薬の添加後９分４７秒目（３４ポイント目）に、この測定用セル（キュベット）内の反応混合液の吸光度（波長：７００ｎｍ）を、前記試料の測定値として測定した。
【０２２３】
▲５▼ 前記▲４▼において測定した吸光度（測定値）から前記▲２▼において測定した吸光度（試料盲検）を差し引き、吸光度差を得た。
なお、この吸光度差は試料に含まれる測定対象物質（トレポネーマ・パリダムに対する抗体）の量（濃度）に比例したものである。
【０２２４】
（４） 測定結果
前記（３）において、試料中の測定対象物質（トレポネーマ・パリダムに対する抗体）の測定を行って得られた吸光度差を、表２に示した。
【０２２５】
【表２】

【０２２６】
３．考察
表２より、陽イオン及び陰イオンが各々１００ｍＭ未満である〔測定試薬Ｄ〕において、３０℃での保存３０日後に得た吸光度差の値は、保存開始前の吸光度差の値の７３％まで減少してしまっており、更に、３０℃での保存６０日後に得た吸光度差の値は、保存開始前の吸光度差の値の５６％にまで減少してしまっていることが分かる。すなわち、シグナルの生成が大きく減少し、測定の感度が低下してしまっている。
【０２２７】
これに対して、１００ｍＭの塩化ナトリウムを含有して、陽イオン及び陰イオンが各々１００ｍＭ以上存在することになる〔測定試薬Ｅ〕において、３０℃での保存３０日後に得た吸光度差の値は減少しておらず、更に、３０℃での保存６０日後に得た吸光度差の値は、保存開始前の吸光度差の値の９２％であり、生成するシグナルの減少、すなわち測定の感度の低下が抑制されていることが分かる。
【０２２８】
また、３００ｍＭの塩化ナトリウムを含有して、陽イオン及び陰イオンが各々３００ｍＭ以上存在することになる〔測定試薬Ｆ〕において、３０℃での保存３０日後に得た吸光度差の値は減少しておらず、更に、３０℃での保存６０日後に得た吸光度差の値は、保存開始前の吸光度差の値の９６％であり、生成するシグナルの減少、すなわち測定の感度の低下をほとんど抑制できていることが分かる。
【０２２９】
そして、５００ｍＭの塩化ナトリウムを含有して、陽イオン及び陰イオンが各々５００ｍＭ以上存在することになる〔測定試薬Ｇ〕においても、３０℃での保存３０日後に得た吸光度差の値は減少しておらず、更に、３０℃での保存６０日後に得た吸光度差の値は、保存開始前の吸光度差の値の９５％であり、生成するシグナルの減少、すなわち測定の感度の低下をほとんど抑制できていることが分かる。
【０２３０】
従って、陽イオン及び陰イオンが各々１００ｍＭ以上存在することにより、測定対象物質の測定試薬を長期間安定に保つことができ、例え、３０℃で６０日間という測定試薬にとって過酷な保存条件においても、生成するシグナルの減少を防ぎ、測定の感度の低下を抑制することができることを、再度確かめることができた。
【０２３１】
〔実施例４〕（測定対象物質の測定試薬における測定の感度低下の作用機序に関する確認）
【０２３２】
測定対象物質の測定試薬において、保存することにより測定の感度が低下するようになる作用機序について、前記実施例１で調製した、トレポネーマ・パリダムの抗原を固定化したラテックス粒子を含む測定試薬を使用して確認を行った。
【０２３３】
Ｉ．トレポネーマ・パリダムの１７Ｋダルトン抗原を使用した測定試薬での確認
【０２３４】
１．測定試薬の保存
前記実施例１の１の（１）で調製した〔測定試薬Ａ〕を、３０℃で６０日間保存した。
【０２３５】
２．保存後の測定試薬による測定
保存１日後及び保存６０日後の前記測定試薬を用いて試料中の測定対象物質（トレポネーマ・パリダムに対する抗体）の測定を行い、吸光度差の値を得た。
【０２３６】
（１） 試薬
【０２３７】
▲１▼ 第１試薬
前記実施例１の２で調製した試料の希釈液である〔第１試薬〕を、第１試薬として用いた。
【０２３８】
▲２▼ 第２試薬
下記の測定試薬をそれぞれ第２試薬として用いた。
【０２３９】
・ 前記１における３０℃での保存６０日後の〔測定試薬Ａ〕。 又は、
【０２４０】
・ 前記１における３０℃での保存１日後の〔測定試薬Ａ〕。
【０２４１】
（２） 試料
トレポネーマ・パリダムに対する抗体を含むヒト血清を試料として用いた。
【０２４２】
（３） 試料中の測定対象物質（トレポネーマ・パリダムに対する抗体）の測定前記試薬及び前記試料を使用し、前記実施例３の２の（３）の記載の通りに各々操作を行い、試料中の測定対象物質（トレポネーマ・パリダムに対する抗体）の測定を行った。
【０２４３】
（４）測定結果
これらの測定により得られた吸光度差を、表３に示した。
【０２４４】
３．分散媒を交換した測定試薬による測定
前記保存を行った測定試薬の分散媒の交換（置換）を行い、この測定試薬を用いて試料中の測定対象物質（トレポネーマ・パリダムに対する抗体）の測定を行って、吸光度差の値を得た。
【０２４５】
（１） 試薬
【０２４６】
▲１▼ 第１試薬
前記実施例１の２で調製した試料の希釈液である〔第１試薬〕を、第１試薬として用いた。
【０２４７】
▲２▼ 第２試薬
前記１における３０℃での保存６０日後の〔測定試薬Ａ〕の０．４ｍＬについて、回転数１４，０００ｒｐｍでの遠心分離を、１０℃にて３０分間行った。
【０２４８】
遠心分離後、デカンテーションにより上清（分散媒）を除き、沈殿物であるラテックス粒子（トレポネーマ・パリダムの１７Ｋダルトン抗原を固定化したラテックス粒子）のみを分取した。
【０２４９】
このラテックス粒子（トレポネーマ・パリダムの１７Ｋダルトン抗原を固定化したラテックス粒子）を、１％（ｗ／ｖ）ＢＳＡ及び１５ｍＭアジ化ナトリウムを含有する５０ｍＭトリス（ヒドロキシメチル）アミノメタン−塩酸緩衝液〔ｐＨ７．６〕よりなる分散媒の０．４ｍＬに、０．２５％（ｗ／ｖ）となるように懸濁させて、再度、〔測定試薬Ａ〕を調製した。
【０２５０】
すなわち、以上の操作により、３０℃で６０日間保存した〔測定試薬Ａ〕について、ラテックス粒子（トレポネーマ・パリダムの１７Ｋダルトン抗原を固定化したラテックス粒子）はそのままで、分散媒のみを調製直後のものに交換して、〔測定試薬Ａ〕の再構成を行ったことになる。
【０２５１】
（２） 試料
トレポネーマ・パリダムに対する抗体を含むヒト血清を試料として用いた。
【０２５２】
（３） 試料中の測定対象物質（トレポネーマ・パリダムに対する抗体）の測定前記の分散媒の交換を行った〔測定試薬Ａ〕及び前記試料を使用し、前記実施例３の２の（３）の記載の通りに各々操作を行い、試料中の測定対象物質（トレポネーマ・パリダムに対する抗体）の測定を行った。
【０２５３】
（４）測定結果
これらの測定により得られた吸光度差をも、表３に示した。
【０２５４】
【表３】

【０２５５】
ＩＩ．トレポネーマ・パリダムの４７Ｋダルトン抗原を使用した測定試薬での確認
【０２５６】
１．測定試薬の保存
前記実施例１の１の（２）で調製した〔測定試薬Ｄ〕を、３０℃で６０日間保存した。
【０２５７】
２．保存後の測定試薬による測定
保存１日後及び保存６０日後の前記測定試薬を用いて試料中の測定対象物質（トレポネーマ・パリダムに対する抗体）の測定を行い、吸光度差の値を得た。
【０２５８】
（１） 試薬
【０２５９】
▲１▼ 第１試薬
前記実施例１の２で調製した試料の希釈液である〔第１試薬〕を、第１試薬として用いた。
【０２６０】
▲２▼ 第２試薬
下記の測定試薬をそれぞれ第２試薬として用いた。
【０２６１】
・ 前記１における３０℃での保存６０日後の〔測定試薬Ｄ〕。 又は、
【０２６２】
・ 前記１における３０℃での保存１日後の〔測定試薬Ｄ〕。
【０２６３】
（２） 試料
トレポネーマ・パリダムに対する抗体を含むヒト血清を試料として用いた。
【０２６４】
（３） 試料中の測定対象物質（トレポネーマ・パリダムに対する抗体）の測定前記試薬及び前記試料を使用し、前記実施例３の２の（３）の記載の通りに各々操作を行い、試料中の測定対象物質（トレポネーマ・パリダムに対する抗体）の測定を行った。
【０２６５】
これらの測定により得られた吸光度差を、表４に示した。
【０２６６】
３．分散媒を交換した測定試薬による測定
前記保存を行った測定試薬の分散媒の交換（置換）を行い、この測定試薬を用いて試料中の測定対象物質（トレポネーマ・パリダムに対する抗体）の測定を行って、吸光度差の値を得た。
【０２６７】
（１） 試薬
【０２６８】
▲１▼ 第１試薬
前記実施例１の２で調製した試料の希釈液である〔第１試薬〕を、第１試薬として用いた。
【０２６９】
▲２▼ 第２試薬
前記１における３０℃での保存６０日後の〔測定試薬Ｄ〕の０．４ｍＬについて、回転数１４，０００ｒｐｍでの遠心分離を、１０℃にて３０分間行った。
【０２７０】
遠心分離後、デカンテーションにより上清（分散媒）を除き、沈殿物であるラテックス粒子（トレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子）のみを分取した。
【０２７１】
このラテックス粒子（トレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子）を、１％（ｗ／ｖ）ＢＳＡ及び１５ｍＭアジ化ナトリウムを含有する５０ｍＭトリス（ヒドロキシメチル）アミノメタン−塩酸緩衝液〔ｐＨ７．２〕よりなる分散媒の０．４ｍＬに、０．２５％（ｗ／ｖ）となるように懸濁させて、再度、〔測定試薬Ｄ〕を調製した。
【０２７２】
すなわち、以上の操作により、３０℃で６０日間保存した〔測定試薬Ｄ〕について、ラテックス粒子（トレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子）はそのままで、分散媒のみを調製直後のものに交換して、〔測定試薬Ｄ〕の再構成を行ったことになる。
【０２７３】
（２） 試料
トレポネーマ・パリダムに対する抗体を含むヒト血清を試料として用いた。
【０２７４】
（３） 試料中の測定対象物質（トレポネーマ・パリダムに対する抗体）の測定前記の分散媒の交換を行った〔測定試薬Ｄ〕及び前記試料を使用し、前記実施例３の２の（３）の記載の通りに各々操作を行い、試料中の測定対象物質（トレポネーマ・パリダムに対する抗体）の測定を行った。
【０２７５】
これらの測定により得られた吸光度差をも、表４に示した。
【０２７６】
【表４】

【０２７７】
ＩＩＩ．考察
【０２７８】
表３により、陽イオン及び陰イオンが各々１００ｍＭ未満である〔測定試薬Ａ〕において、３０℃での保存６０日後に得た吸光度差の値は、３０℃での保存１日後に得た吸光度差の値の５４％まで減少してしまっていることが分かる。すなわち、シグナルの生成が大きく減少し、測定の感度が低下してしまっている。
【０２７９】
また、表４により、陽イオン及び陰イオンが各々１００ｍＭ未満である〔測定試薬Ｄ〕においても、３０℃での保存６０日後に得た吸光度差の値は、３０℃での保存１日後に得た吸光度差の値の７３％まで減少してしまっていることが分かる。すなわち、これもシグナルの生成が大きく減少し、測定の感度が低下してしまっている。
【０２８０】
これに対して、前記の３０℃で６０日間保存した〔測定試薬Ａ〕の分散媒を交換（置換）して、再構成した〔測定試薬Ａ〕により得た吸光度差の値は、先の３０℃での保存１日後に得た吸光度差の値の８６％であり、分散媒交換前に比べて３２％も増加、回復していることが分かる。
【０２８１】
また、前記の３０℃で６０日間保存した〔測定試薬Ｄ〕の分散媒を交換（置換）して、再構成した〔測定試薬Ｄ〕により得た吸光度差の値は、先の３０℃での保存１日後に得た吸光度差の値の９２％であり、分散媒交換前に比べて１９％も増加、回復していることが分かる。
【０２８２】
以上の測定結果より、「トレポネーマ・パリダムの１７Ｋダルトン抗原を固定化したラテックス粒子を含む測定試薬」、及び「トレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子を含む測定試薬」において、つまり、「測定対象物質に対する特異的結合物質を固定化した担体を含む測定対象物質の測定試薬」において、その保存中に測定試薬が劣化してしまい、試料中の測定対象物質（トレポネーマ・パリダムに対する抗体）の測定の感度が低下してしまうようになる原因が、「測定対象物質に対する特異的結合物質を固定化した担体」から「測定対象物質に対する特異的結合物質」が剥離（解離）してしまうため、すなわち、「トレポネーマ・パリダムの抗原を固定化したラテックス粒子」から「トレポネーマ・パリダムの抗原」が剥離（解離）してしまうためであることが分かる。
【０２８３】
これを以下、より詳細に説明する。
【０２８４】
測定対象物質の測定試薬（〔測定試薬Ａ〕、〔測定試薬Ｄ〕）の保存により、測定によって得られる吸光度差すなわちシグナルは大きく減少し、測定の感度が低下してしまう。
【０２８５】
しかし、保存を行った前記測定試薬の「測定対象物質に対する特異的結合物質を固定化した担体以外の成分」（分散媒）を分離・除去して、残った「測定対象物質に対する特異的結合物質を固定化した担体」（トレポネーマ・パリダムの抗原を固定化したラテックス粒子）と、新たに調製した「測定対象物質に対する特異的結合物質を固定化した担体以外の成分」（分散媒）とより再構成を行った測定試薬（〔測定試薬Ａ〕、〔測定試薬Ｄ〕）においては、測定によって得られる吸光度差すなわちシグナルは回復する。
【０２８６】
すなわち、「測定対象物質に対する特異的結合物質を固定化した担体」を含む測定対象物質の測定試薬においては、保存により前記測定試薬が劣化し、測定反応において生成するシグナルは減少し、測定の感度は低下するようになる。
そして、「測定対象物質に対する特異的結合物質を固定化した担体以外の成分」（分散媒）を分離・除去して、新たに調製した前記成分と交換（置換）することにより、前記の阻害物質が、測定試薬ひいては測定反応系（反応混合液）から取り除かれるので、吸光度差（シグナル）が回復する。
【０２８７】
これにより、保存を行った前記測定試薬の「測定対象物質に対する特異的結合物質を固定化した担体以外の成分」（分散媒）の中に、「測定対象物質」（トレポネーマ・パリダムに対する抗体）と「測定対象物質に対する特異的結合物質」（トレポネーマ・パリダムの抗原）との特異的結合反応（抗原抗体反応）を阻害する物質が含まれていることが分かる。
【０２８８】
更に、保存を行った前記測定試薬より、「測定対象物質に対する特異的結合物質を固定化した担体以外の成分」（分散媒）を分離・除去して、新たに調製した前記成分と交換（置換）した場合でも、吸光度差（シグナル）が完全には回復しないことより、前記の阻害物質が「測定対象物質に対する特異的結合物質」であること、そして、その「測定対象物質に対する特異的結合物質」が、前記測定試薬の保存中に、「測定対象物質に対する特異的結合物質を固定化した担体」から剥離（解離）し、「測定対象物質に対する特異的結合物質を固定化した担体以外の成分」（分散媒）の中に遊離状態で存在するようになったものであることが分かる。
【０２８９】
すなわち、本実施例における検討により、「測定対象物質に対する特異的結合物質を固定化した担体」を含む測定対象物質の測定試薬においては、保存により以上のようなことが起こり、そのため下記のようなことが起こって、測定によって得られるシグナルが減少し、測定の感度が低下するようになることが確かめられた。
【０２９０】
▲１▼ 測定対象物質の測定試薬の保存中に、「測定対象物質に対する特異的結合物質を固定化した担体」から「測定対象物質に対する特異的結合物質」が剥離（解離）することにより、「担体に固定化された測定対象物質に対する特異的結合物質」の数（量）は、前記測定試薬の調製時に比べて減少してしまい、これにより、この「担体に固定化された測定対象物質に対する特異的結合物質」に結合することができる「測定対象物質」の数（量）も減ってしまう。
【０２９１】
そうすると、「…〔測定対象物質に対する特異的結合物質＝担体＝測定対象物質に対する特異的結合物質〕−〔測定対象物質〕−〔測定対象物質に対する特異的結合物質＝担体＝測定対象物質に対する特異的結合物質〕…」等の架橋の形成も減少してしまうので、生成するシグナルの量も減少してしまい、これにより測定の感度が低下してしまう。
【０２９２】
▲２▼ 「測定対象物質に対する特異的結合物質を固定化した担体」から剥離（解離）し遊離状態となった「測定対象物質に対する特異的結合物質」は、測定時、試料と前記測定試薬が混合された反応混合液中において、試料中に含まれていた「測定対象物質」の一部又は全部と結合してしまう。
この結合した「測定対象物質」は、もう担体に固定化された「測定対象物質に対する特異的結合物質」とは結合できなくなる。
【０２９３】
すなわち、遊離状態となった「測定対象物質に対する特異的結合物質」と結合した「測定対象物質」は、「…〔測定対象物質に対する特異的結合物質＝担体＝測定対象物質に対する特異的結合物質〕−〔測定対象物質〕−〔測定対象物質に対する特異的結合物質＝担体＝測定対象物質に対する特異的結合物質〕…」等の架橋の形成に参加できなくなる。
これにより、生成するシグナルの量が減少してしまい、測定の感度が低下してしまう。
【０２９４】
そして、前記▲２▼のため、保存を行った前記測定試薬より「測定対象物質に対する特異的結合物質を固定化した担体以外の成分」（分散媒）を取り去って、剥離（解離）した「測定対象物質に対する特異的結合物質」を分離・除去することにより、測定によって得られる吸光度差すなわちシグナルは回復するものの、前記▲１▼のため、完全には回復しないのである。
【０２９５】
〔実施例５〕（トレポネーマ・パリダムに対する抗体の測定試薬の保存安定性の確認）
トレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子を含む測定試薬を調製し、これを３０℃で保存して、保存時の安定性の確認を行った。
【０２９６】
Ｉ．トレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子を含む測定試薬の調製
【０２９７】
１．トレポネーマ・パリダムの抗原を固定化したラテックス粒子を含む測定試薬の調製（第２試薬の調製）
【０２９８】
遺伝子組み換え法により調製されたトレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子を含む測定試薬（第２試薬）を以下の通り調製した。
【０２９９】
▲１▼ トレポネーマ・パリダムの４７Ｋダルトン抗原の調製から、ラテックス粒子（トレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子）の取得までの過程は、前記実施例１の１の（２）における▲１▼〜▲６▼の記載の通りに行った。
【０３００】
▲２▼ 次の５種類のラテックス粒子分散媒の調製を行った。
・ １％（ｗ／ｖ）ＢＳＡ及び１５ｍＭアジ化ナトリウムを含有する５０ｍＭトリス（ヒドロキシメチル）アミノメタン−塩酸緩衝液〔ｐＨ７．２〕
・ ３００ｍＭ塩化ナトリウム（ＮａＣｌ）、１％（ｗ／ｖ）ＢＳＡ及び１５ｍＭアジ化ナトリウムを含有する５０ｍＭトリス（ヒドロキシメチル）アミノメタン−塩酸緩衝液〔ｐＨ７．２〕
・ ３００ｍＭ塩化カリウム（ＫＣｌ）、１％（ｗ／ｖ）ＢＳＡ及び１５ｍＭアジ化ナトリウムを含有する５０ｍＭトリス（ヒドロキシメチル）アミノメタン−塩酸緩衝液〔ｐＨ７．２〕
・ ３００ｍＭ臭化ナトリウム（ＮａＢｒ）、１％（ｗ／ｖ）ＢＳＡ及び１５ｍＭアジ化ナトリウムを含有する５０ｍＭトリス（ヒドロキシメチル）アミノメタン−塩酸緩衝液〔ｐＨ７．２〕
・ １５０ｍＭ塩化マグネシウム（ＭｇＣｌ_２）、１％（ｗ／ｖ）ＢＳＡ及び１５ｍＭアジ化ナトリウムを含有する５０ｍＭトリス（ヒドロキシメチル）アミノメタン−塩酸緩衝液〔ｐＨ７．２〕
【０３０１】
▲３▼ 前記▲２▼で調製した５種類のラテックス粒子分散媒の各々の０．８ｍＬに、前記の取得したラテックス粒子（トレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子）をそれぞれ０．１２５％（ｗ／ｖ）となるように懸濁させて、下記の５種類の測定試薬（第２試薬）を調製した。
【０３０２】
〔測定試薬Ｈ〕： ０．１２５％（ｗ／ｖ）のラテックス粒子（トレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子）を含む、１％（ｗ／ｖ）ＢＳＡ及び１５ｍＭアジ化ナトリウムを含有する５０ｍＭトリス（ヒドロキシメチル）アミノメタン−塩酸緩衝液〔ｐＨ７．２〕。
【０３０３】
〔測定試薬Ｉ〕： ０．１２５％（ｗ／ｖ）のラテックス粒子（トレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子）を含む、３００ｍＭ塩化ナトリウム（ＮａＣｌ）、１％（ｗ／ｖ）ＢＳＡ及び１５ｍＭアジ化ナトリウムを含有する５０ｍＭトリス（ヒドロキシメチル）アミノメタン−塩酸緩衝液〔ｐＨ７．２〕。
【０３０４】
〔測定試薬Ｊ〕： ０．１２５％（ｗ／ｖ）のラテックス粒子（トレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子）を含む、３００ｍＭ塩化カリウム（ＫＣｌ）、１％（ｗ／ｖ）ＢＳＡ及び１５ｍＭアジ化ナトリウムを含有する５０ｍＭトリス（ヒドロキシメチル）アミノメタン−塩酸緩衝液〔ｐＨ７．２〕。
【０３０５】
〔測定試薬Ｋ〕： ０．１２５％（ｗ／ｖ）のラテックス粒子（トレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子）を含む、３００ｍＭ臭化ナトリウム（ＮａＢｒ）、１％（ｗ／ｖ）ＢＳＡ及び１５ｍＭアジ化ナトリウムを含有する５０ｍＭトリス（ヒドロキシメチル）アミノメタン−塩酸緩衝液〔ｐＨ７．２〕。
【０３０６】
〔測定試薬Ｌ〕： ０．１２５％（ｗ／ｖ）のラテックス粒子（トレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子）を含む、１５０ｍＭ塩化マグネシウム（ＭｇＣｌ _２ ）、１％（ｗ／ｖ）ＢＳＡ及び１５ｍＭアジ化ナトリウムを含有する５０ｍＭトリス（ヒドロキシメチル）アミノメタン−塩酸緩衝液〔ｐＨ７．２〕。
【０３０７】
２．試料の希釈液である試薬の調製（第１試薬の調製）
【０３０８】
前記実施例１の２の記載の通りに、試料の希釈液（第１試薬）を調製した。
【０３０９】
ＩＩ．トレポネーマ・パリダムに対する抗体の測定試薬の保存安定性の確認
前記Ｉで調製した、トレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子を含む測定試薬を、３０℃で保存し，保存時の安定性の確認を行った。
【０３１０】
１．測定試薬の保存
【０３１１】
前記Ｉの１で調製した、〔測定試薬Ｈ〕、〔測定試薬Ｉ〕、〔測定試薬Ｊ〕、〔測定試薬Ｋ〕、及び〔測定試薬Ｌ〕の５種類の測定試薬を、各々３０℃で５６日間保存した。
【０３１２】
２．測定試薬の安定性の確認
【０３１３】
保存前、保存中及び保存後の前記測定試薬の各々を用いて試料中の測定対象物質（トレポネーマ・パリダムに対する抗体）の測定を行い、前記測定試薬の保存時の安定性の確認を行った。
【０３１４】
（１） 試薬
【０３１５】
▲１▼ 第１試薬
前記Ｉの２で調製した試料の希釈液である〔第１試薬〕を、第１試薬として用いた。
【０３１６】
▲２▼ 第２試薬
下記の測定試薬をそれぞれ第２試薬として用いた。
【０３１７】
・ 前記１における３０℃での保存５６日後の〔測定試薬Ｈ〕、〔測定試薬Ｉ〕、〔測定試薬Ｊ〕、〔測定試薬Ｋ〕、若しくは〔測定試薬Ｌ〕。
【０３１８】
・ 前記１における３０℃での保存２８日後の〔測定試薬Ｈ〕、〔測定試薬Ｉ〕、〔測定試薬Ｊ〕、〔測定試薬Ｋ〕、若しくは〔測定試薬Ｌ〕。
【０３１９】
・ 前記１における３０℃での保存１４日後の〔測定試薬Ｈ〕、〔測定試薬Ｉ〕、〔測定試薬Ｊ〕、〔測定試薬Ｋ〕、若しくは〔測定試薬Ｌ〕。
【０３２０】
・ 前記１における３０℃での保存７日後の〔測定試薬Ｈ〕、〔測定試薬Ｉ〕、〔測定試薬Ｊ〕、〔測定試薬Ｋ〕、若しくは〔測定試薬Ｌ〕。 又は、
【０３２１】
・ 測定試薬調製時すなわち保存開始前の〔測定試薬Ｈ〕、〔測定試薬Ｉ〕、〔測定試薬Ｊ〕、〔測定試薬Ｋ〕、若しくは〔測定試薬Ｌ〕。
【０３２２】
（２） 試料
トレポネーマ・パリダムに対する抗体を含むヒト血清を試料として用いた。
【０３２３】
（３） 試料中の測定対象物質（トレポネーマ・パリダムに対する抗体）の測定
【０３２４】
▲１▼ 測定は、日立−７１７０Ｓ形自動分析装置（日立製作所社製）を使用して行った。
【０３２５】
まず、測定用セル（キュベット）に、前記の試料の１５μＬを添加した。
次に、この測定用セル（キュベット）に、前記の第１試薬の１５０μＬを添加し、混合した。
【０３２６】
そして、この測定用セル（キュベット）を、３７℃で静置した。
【０３２７】
▲２▼ 前記の第１試薬の添加後４分１４秒目（１５ポイント目）に、この測定用セル（キュベット）内の混合液の吸光度（波長：７００ｎｍ）を試料盲検として測定した。
【０３２８】
▲３▼ 前記の第１試薬の添加後４分３０秒目（１６ポイント目）に、この測定用セル（キュベット）内の混合液に、更に、前記の第２試薬の５０μＬを添加し、混合した。
【０３２９】
そして、この測定用セル（キュベット）を、３７℃で静置して、反応を行わせた。
これにより、前記のラテックス粒子に固定化されたトレポネーマ・パリダムの４７Ｋダルトン抗原と、前記の試料に含まれていたトレポネーマ・パリダムに対する抗体との抗原抗体反応を行わせ、ラテックス粒子の凝集塊を生成させた。
【０３３０】
▲４▼ 前記の第１試薬の添加後９分４７秒目（３４ポイント目）に、この測定用セル（キュベット）内の反応混合液の吸光度（波長：７００ｎｍ）を、前記試料の測定値として測定した。
【０３３１】
▲５▼ 前記▲４▼において測定した吸光度（測定値）から前記▲２▼において測定した吸光度（試料盲検）を差し引き、吸光度差を得た。
なお、この吸光度差は試料に含まれる測定対象物質（トレポネーマ・パリダムに対する抗体）の量（濃度）に比例したものである。
【０３３２】
（４） 測定結果
前記（３）において、試料中の測定対象物質（トレポネーマ・パリダムに対する抗体）の測定を行って得られた吸光度差を、表５に示した。
【０３３３】
【表５】

【０３３４】
ＩＩＩ．考察
表５より、陽イオン及び陰イオンが各々１００ｍＭ未満である〔測定試薬Ｈ〕において、３０℃での保存１４日後に得た吸光度差の値は、保存開始前の吸光度差の値の７７％まで減少してしまっており、そして、３０℃での保存２８日後に得た吸光度差の値は、保存開始前の吸光度差の値の６６％まで減少してしまっており、更に、３０℃での保存５６日後に得た吸光度差の値は、保存開始前の吸光度差の値の４９％にまで減少してしまっていることが分かる。すなわち、シグナルの生成が大きく減少し、測定の感度が低下してしまっている。
【０３３５】
これに対して、３００ｍＭの塩化ナトリウム（ＮａＣｌ）を含有して、陽イオン及び陰イオンが各々３００ｍＭ以上存在することになる〔測定試薬Ｉ〕において、３０℃での保存１４日後、３０℃での保存２８日後、及び３０℃での保存５６日後に得た吸光度差の値はいずれも減少しておらず、生成するシグナルの減少、すなわち測定の感度の低下を抑制できていることが分かる。
【０３３６】
また、３００ｍＭの塩化カリウム（ＫＣｌ）を含有して、陽イオン及び陰イオンが各々３００ｍＭ以上存在することになる〔測定試薬Ｊ〕において、３０℃での保存１４日後、３０℃での保存２８日後、及び３０℃での保存５６日後に得た吸光度差の値はいずれも減少しておらず、生成するシグナルの減少、すなわち測定の感度の低下を抑制できていることが分かる。
【０３３７】
そして、３００ｍＭの臭化ナトリウム（ＮａＢｒ）を含有して、陽イオン及び陰イオンが各々３００ｍＭ以上存在することになる〔測定試薬Ｋ〕において、３０℃での保存１４日後、及び３０℃での保存２８日後に得た吸光度差の値は減少しておらず、更に、３０℃での保存５６日後に得た吸光度差の値は、保存開始前の吸光度差の値の９８％であり、生成するシグナルの減少、すなわち測定の感度の低下をほとんど抑制できていることが分かる。
【０３３８】
更に、１５０ｍＭの塩化マグネシウム（ＭｇＣｌ_２）を含有して、陽イオン及び陰イオンが各々１５０ｍＭ以上存在することになる〔測定試薬Ｌ〕において、３０℃での保存１４日後に得た吸光度差の値は、保存開始前の吸光度差の値の９６％であり、また、３０℃での保存２８日後に得た吸光度差の値は、保存開始前の吸光度差の値の９４％であり、そして、３０℃での保存５６日後に得た吸光度差の値も、保存開始前の吸光度差の値の９４％であり、生成するシグナルの減少、すなわち測定の感度の低下をほとんど抑制できていることが分かる。
【０３３９】
以上のことより、種々の陽イオン及び陰イオンにおいても、各々１００ｍＭ以上存在することにより、測定対象物質の測定試薬を長期間安定に保つことができ、例え、３０℃で５６日間という測定試薬にとって過酷な保存条件においても、生成するシグナルの減少を防ぎ、測定の感度の低下を抑制することができることを、確かめることができた。
【０３４０】
〔実施例６〕（トレポネーマ・パリダムに対する抗体の測定試薬の保存安定性の確認）
トレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子を含む測定試薬を調製し、これを３０℃で保存して、保存時の安定性の確認を行った。
【０３４１】
Ｉ．トレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子を含む測定試薬の調製
【０３４２】
１．トレポネーマ・パリダムの抗原を固定化したラテックス粒子を含む測定試薬の調製（第２試薬の調製）
【０３４３】
遺伝子組み換え法により調製されたトレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子を含む測定試薬（第２試薬）を以下の通り調製した。
【０３４４】
▲１▼ トレポネーマ・パリダムの４７Ｋダルトン抗原の調製から、ラテックス粒子（トレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子）の取得までの過程は、前記実施例１の１の（２）における▲１▼〜▲６▼の記載の通りに行った。
【０３４５】
▲２▼ 次の３種類のラテックス粒子分散媒の調製を行った。
・ １％（ｗ／ｖ）ＢＳＡ及び１５ｍＭアジ化ナトリウムを含有する５０ｍＭトリス（ヒドロキシメチル）アミノメタン−塩酸緩衝液〔ｐＨ７．２〕
・ １５０ｍＭ硫酸ナトリウム（Ｎａ_２ＳＯ_４）、１％（ｗ／ｖ）ＢＳＡ及び１５ｍＭアジ化ナトリウムを含有する５０ｍＭトリス（ヒドロキシメチル）アミノメタン−塩酸緩衝液〔ｐＨ７．２〕
・ １５０ｍＭ硫酸マグネシウム（ＭｇＳＯ_４）、１％（ｗ／ｖ）ＢＳＡ及び１５ｍＭアジ化ナトリウムを含有する５０ｍＭトリス（ヒドロキシメチル）アミノメタン−塩酸緩衝液〔ｐＨ７．２〕
【０３４６】
▲３▼ 前記▲２▼で調製した３種類のラテックス粒子分散媒の各々の０．８ｍＬに、前記の取得したラテックス粒子（トレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子）をそれぞれ０．１２５％（ｗ／ｖ）となるように懸濁させて、下記の３種類の測定試薬（第２試薬）を調製した。
【０３４７】
〔測定試薬Ｍ〕： ０．１２５％（ｗ／ｖ）のラテックス粒子（トレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子）を含む、１％（ｗ／ｖ）ＢＳＡ及び１５ｍＭアジ化ナトリウムを含有する５０ｍＭトリス（ヒドロキシメチル）アミノメタン−塩酸緩衝液〔ｐＨ７．２〕。
【０３４８】
〔測定試薬Ｎ〕： ０．１２５％（ｗ／ｖ）のラテックス粒子（トレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子）を含む、１５０ｍＭ硫酸ナトリウム（Ｎａ _２ ＳＯ _４ ）、１％（ｗ／ｖ）ＢＳＡ及び１５ｍＭアジ化ナトリウムを含有する５０ｍＭトリス（ヒドロキシメチル）アミノメタン−塩酸緩衝液〔ｐＨ７．２〕。
【０３４９】
〔測定試薬Ｐ〕： ０．１２５％（ｗ／ｖ）のラテックス粒子（トレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子）を含む、１５０ｍＭ硫酸マグネシウム（ＭｇＳＯ _４ ）、１％（ｗ／ｖ）ＢＳＡ及び１５ｍＭアジ化ナトリウムを含有する５０ｍＭトリス（ヒドロキシメチル）アミノメタン−塩酸緩衝液〔ｐＨ７．２〕。
【０３５０】
２．試料の希釈液である試薬の調製（第１試薬の調製）
【０３５１】
前記実施例１の２の記載の通りに、試料の希釈液（第１試薬）を調製した。
【０３５２】
ＩＩ．トレポネーマ・パリダムに対する抗体の測定試薬の保存安定性の確認
前記Ｉで調製した、トレポネーマ・パリダムの４７Ｋダルトン抗原を固定化したラテックス粒子を含む測定試薬を、３０℃で保存し，保存時の安定性の確認を行った。
【０３５３】
１．測定試薬の保存
【０３５４】
前記Ｉの１で調製した、〔測定試薬Ｍ〕、〔測定試薬Ｎ〕、及び〔測定試薬Ｐ〕の３種類の測定試薬を、各々３０℃で２８日間保存した。
【０３５５】
２．測定試薬の安定性の確認
【０３５６】
保存前、保存中及び保存後の前記測定試薬の各々を用いて試料中の測定対象物質（トレポネーマ・パリダムに対する抗体）の測定を行い、前記測定試薬の保存時の安定性の確認を行った。
【０３５７】
（１） 試薬
【０３５８】
▲１▼ 第１試薬
前記Ｉの２で調製した試料の希釈液である〔第１試薬〕を、第１試薬として用いた。
【０３５９】
▲２▼ 第２試薬
下記の測定試薬をそれぞれ第２試薬として用いた。
【０３６０】
・ 前記１における３０℃での保存２８日後の〔測定試薬Ｍ〕、〔測定試薬Ｎ〕、若しくは〔測定試薬Ｐ〕。
【０３６１】
・ 前記１における３０℃での保存１４日後の〔測定試薬Ｍ〕、〔測定試薬Ｎ〕、若しくは〔測定試薬Ｐ〕。
【０３６２】
・ 前記１における３０℃での保存７日後の〔測定試薬Ｍ〕、〔測定試薬Ｎ〕、若しくは〔測定試薬Ｐ〕。 又は、
【０３６３】
・ 測定試薬調製時すなわち保存開始前の〔測定試薬Ｍ〕、〔測定試薬Ｎ〕、若しくは〔測定試薬Ｐ〕。
【０３６４】
（２） 試料
トレポネーマ・パリダムに対する抗体を含むヒト血清を試料として用いた。
【０３６５】
（３） 試料中の測定対象物質（トレポネーマ・パリダムに対する抗体）の測定
【０３６６】
測定は、日立−７１７０Ｓ形自動分析装置（日立製作所社製）を使用し、前記実施例５のＩＩの２の（３）における▲１▼〜▲５▼の記載の通りに行った。
【０３６７】
（４） 測定結果
前記（３）において、試料中の測定対象物質（トレポネーマ・パリダムに対する抗体）の測定を行って得られた吸光度差を、表６に示した。
【０３６８】
【表６】

【０３６９】
ＩＩＩ．考察
表６より、陽イオン及び陰イオンが各々１００ｍＭ未満である〔測定試薬Ｍ〕において、３０℃での保存１４日後に得た吸光度差の値は、保存開始前の吸光度差の値の７７％まで減少してしまっており、更に、３０℃での保存２８日後に得た吸光度差の値は、保存開始前の吸光度差の値の６６％にまで減少してしまっていることが分かる。すなわち、シグナルの生成が大きく減少し、測定の感度が低下してしまっている。
【０３７０】
これに対して、１５０ｍＭの硫酸ナトリウム（Ｎａ_２ＳＯ_４）を含有して、陽イオン及び陰イオンが各々１５０ｍＭ以上存在することになる〔測定試薬Ｎ〕において、３０℃での保存１４日後に得た吸光度差の値は減少しておらず、更に、３０℃での保存２８日後に得た吸光度差の値は、保存開始前の吸光度差の値の９１％であり、生成するシグナルの減少、すなわち測定の感度の低下が抑制されていることが分かる。
【０３７１】
また、１５０ｍＭの硫酸マグネシウム（ＭｇＳＯ_４）を含有して、陽イオン及び陰イオンが各々１５０ｍＭ以上存在することになる〔測定試薬Ｐ〕において、３０℃での保存１４日後に得た吸光度差の値は減少しておらず、更に、３０℃での保存２８日後に得た吸光度差の値は、保存開始前の吸光度差の値の７３％であり、生成するシグナルの減少、すなわち測定の感度の低下が抑制されていることが分かる。
【０３７２】
以上のことより、更に異なる陽イオン及び陰イオンの組合せにおいても、各々１００ｍＭ以上存在することにより、測定対象物質の測定試薬を長期間安定に保つことができ、例え、３０℃で２８日間という測定試薬にとって過酷な保存条件においても、生成するシグナルの減少を防ぎ、測定の感度の低下を抑制することができることを、確かめることができた。
【０３７３】
〔実施例の結果のまとめ〕
以上の実施例の結果をまとめると、陽イオン及び陰イオンが、塩化ナトリウム、塩化カリウム、臭化ナトリウム、塩化マグネシウム、硫酸ナトリウム、又は硫酸マグネシウムのいずれの場合においても、１００ｍＭ以上存在することにより、「測定対象物質に対する特異的結合物質」が担体から剥離（解離）することが抑制され、これにより測定の感度の低下を抑制することができた。
【０３７４】
すなわち、種々の陽イオン及び陰イオンにおいて、１００ｍＭ以上存在することにより、測定対象物質に対する特異的結合物質を固定化した担体を含む測定対象物質の測定試薬を長期間安定に保ち、正確な測定対象物質の測定結果を得ることができることが確かめられた。
【０３７５】
【発明の効果】
本発明の測定対象物質の測定試薬、及び本発明の安定化された測定対象物質の測定試薬は、測定対象物質に対する特異的結合物質を固定化した担体を含む測定対象物質の測定試薬において、測定対象物質に対する特異的結合物質の担体からの剥離を抑制するために（当該剥離の抑制剤として）、陽イオン及び陰イオンを各々１００ｍＭ以上存在させることを特徴とするものであって、これにより前記測定試薬の保存中に、「測定対象物質に対する特異的結合物質を固定化した担体」から「測定対象物質に対する特異的結合物質」が剥離（解離）してしまうことを防止し、これにより前記測定試薬の測定の感度の低下を防いで前記測定試薬の安定化が図られ、長期間、正確な測定対象物質の測定結果を得ることができるという効果を有するものである。
【０３７６】
また、本発明の測定対象物質の測定試薬の安定化方法は、測定対象物質に対する特異的結合物質を固定化した担体を含む測定対象物質の測定試薬において、測定対象物質に対する特異的結合物質を固定化した担体とともに、陽イオン及び陰イオンを各々１００ｍＭ以上存在させることを特徴とするものであって、これにより前記測定対象物質の測定試薬の保存中に、「測定対象物質に対する特異的結合物質を固定化した担体」から「測定対象物質に対する特異的結合物質」が剥離（解離）してしまうことを防止し、これにより前記測定試薬の測定の感度の低下を防いで前記測定試薬の安定化が図られ、長期間、正確な測定対象物質の測定結果を得ることができるという効果を有するものである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a stabilized measurement reagent for a measurement target substance capable of preventing the specific binding substance for the measurement target substance from peeling (dissociating) from the carrier on which the specific binding substance for the measurement target substance is immobilized. Is.
[0002]
The present invention is useful in the field of life science such as clinical examination, immunology, and medicine, the field of chemistry such as analytical chemistry, the field of food hygiene, and the field of environmental health.
[0003]
[Prior art]
Reagents for measuring small amounts of target substances in samples using reactions between substances with specific affinity such as antigen and antibody, sugar and lectin, nucleotide chain and complementary nucleotide chain, and ligand and receptor Various measurement methods are known.
[0004]
This is the presence or absence of binding between the measurement target substance contained in the sample and the specific binding substance (specific binding substance for the measurement target substance) that specifically binds to the measurement target substance. By measuring, the presence or absence of a measurement target substance contained in a sample is measured [qualitative measurement], or the content (concentration) thereof is measured [quantitative measurement].
[0005]
In particular, a method of performing measurement using a substance in which a specific binding substance for a substance to be measured is immobilized on a carrier, or a measurement reagent is frequently used.
[0006]
For example, in an immunological measurement reagent (immunological measurement method) using an antigen-antibody reaction (immune reaction, immunological reaction) between an antigen and an antibody, latex turbidimetry using latex particles as a carrier; Indirect agglutination measurement using latex particles, organic polymer particles, inorganic particles, metal particles, or red blood cells as a carrier; enzyme immunoassay using microtiter plates, beads, particles, test tubes, or containers as a carrier Labeled immunoassay methods such as immunoassay, luminescence immunoassay method, or immunochromatography method using cellulose or non-woven fabric as a carrier is frequently used.
[0007]
A specific explanation will be given below, taking as an example the measurement of antibodies against Treponema pallidum contained in a sample such as serum.
[0008]
Treponema Paris Dam (Treponema  Pallidum) Is a pathogen of syphilis, and the presence of antibodies against this treponema pallidum (anti-treponema pallidum antibody) in a sample such as serum means that it currently suffers from syphilis or has been affected by syphilis in the past. Evidence that it was.
Therefore, for the diagnosis of syphilis, antibodies against Treponema pallidum are being measured.
[0009]
In the measurement of antibodies (measurement substances) against Treponema pallidum by latex turbidimetry using latex particles as a carrier, components derived from Treponema pallidum, that is, antigens of Treponema pallidum, are used as latex particles as a carrier. Those immobilized by physical adsorption or by chemical bonding are used as measurement reagents for antibodies against Treponema pallidum.
[0010]
As antigens of Treponema pallidum, 15K dalton antigen, 17K dalton antigen, 47K dalton antigen, etc., which are antigens on the surface of Treponema pallidum, are known. Used.
[0011]
In addition, a recombinant antigen prepared by gene recombination of an antigen of Treponema pallidum such as the 15 KDalton antigen, 17 KDalton antigen, or 47 KDalton antigen is also used as a reagent for measuring an antibody against Treponema pallidum.
[0012]
Usually, in the measurement of antibodies against Treponema pallidum by latex turbidimetry, a reagent comprising a buffer or physiological saline is used as a first reagent for measuring antibodies against Treponema pallidum, and the latex particles as a carrier are treponema. Measurement is performed by a two-step method (two-reagent method) using a measurement reagent containing an immobilized Paridam antigen as a second reagent for measuring antibodies against Treponema pallidum.
[0013]
Alternatively, measurement is also performed by a one-step method (one-reagent method) using a measurement reagent including a latex particle as a carrier immobilized with an antigen of Treponema pallidum as an antibody measurement reagent against Treponema pallidum. .
[0014]
The measurement operation in the two-step method (two-reagent method) is as follows. First, a sample and the first reagent are mixed, and after a predetermined time, the second reagent is added to the mixed solution of the sample and the first reagent. Mix and allow reaction to occur.
[0015]
In the one-step method (one-reagent method), a sample and the measurement reagent are mixed and reacted.
[0016]
When an antibody against Treponema pallidum is contained in the sample, this antibody reacts with and binds to the antigen of Treponema pallidum immobilized on latex particles, and through this antibody, “… [ Cross-linking of “antigen = latex particle = antigen of treponema pallidum] − [antibody against treponema pallidum] − [antigen of treponema pallidum = latex particle = antigen of treponema pallidum]...” Is formed.
[0017]
As a result, aggregates of “latex particles immobilizing the antigen of Treponema pallidum” via the antibody against Treponema pallidum are generated.
[0018]
The amount of agglomerates formed between the latex particles increases in a certain range in accordance with the amount of antibody against Treponema pallidum contained in the sample.
[0019]
Then, the reaction mixture liquid in which the sample, the first reagent, and the second reagent are mixed (or the reaction mixture liquid in which the sample and the measurement reagent are mixed) is irradiated with light, and agglomerates of the generated latex particles are aggregated. By measuring the decrease in transmitted light (increase in absorbance) or the increase in scattered light, which is a signal generated by the above, the amount of the aggregate produced, that is, the amount of antibody against Treponema pallidum contained in the sample is determined. .
[0020]
In addition, although it is calculation of a quantitative value, the sample containing the antibody with respect to the decrease of the transmitted light of the reaction liquid mixture which performed the said measurement (increase in light absorbency) or the increase in scattered light, and a known concentration of Treponema paridam The amount (concentration) of the antibody against Treponema pallidum contained in the sample is calculated by comparing the decrease in transmitted light (increase in absorbance) or the value of increase in scattered light.
[0021]
[Problems to be solved by the invention]
As described above, the measurement reagent for the measurement target substance including the carrier on which the specific binding substance for the measurement target substance is immobilized, and the measurement method for the measurement target substance using the measurement reagent for the measurement target substance are frequently used. The present inventors have found that there are the following problems.
[0022]
That is, in the measurement reagent of the measurement target substance including the carrier on which the specific binding substance for the measurement target substance is immobilized, during the storage, from the “carrier on which the specific binding substance for the measurement target substance is immobilized” to the “measurement target” Since the “specific binding substance to the substance” is peeled (dissociated), the following (1) and (2) reduce the amount of signal generated, which reduces the sensitivity of the measurement, and the measurement target Even in the measurement of a sample containing a substance, an erroneous measurement result indicating that the measurement target substance is not contained may be obtained. (False negative)
[0023]
(1) The “specific binding substance for the measurement target substance” is peeled off (dissociated) from the “carrier having the specific binding substance for the measurement target substance immobilized”, so that “specificity for the measurement target substance immobilized on the carrier is determined”. The number (amount) of “chemically binding substances” has decreased compared to the preparation of the measurement reagent.
[0024]
Then, at the time of measurement, in the reaction mixture in which the sample and the measurement reagent are mixed, the “measurement substance” contained in the sample can bind to “specificity to the measurement substance immobilized on the carrier”. The number (quantity) of “substances to be measured” that can bind to “specific binding substances for the target substance immobilized on the carrier” has decreased since the number (quantities) of “substances to be bound” has decreased. Will also decrease.
[0025]
Therefore, “... [specific binding substance for the measurement target substance = carrier = specific binding substance for the measurement target substance] − [measurement target substance] − [specific binding substance for the measurement target substance = carrier = measurement target substance” Specific binding substance for ...] or "... (carrier = specific binding substance for measurement target substance)-[measurement target substance]" because the number (amount) of crosslinks formed decreases. The amount is reduced, which reduces the sensitivity of the measurement. That is, even in the measurement of the sample containing the measurement target substance, an erroneous measurement result that the measurement target substance is not included may be obtained. (False negative)
[0026]
(2) At the time of measurement, in the reaction mixture in which the sample and the measurement reagent are mixed, a part or all of the “measurement target substance” contained in the sample is “fixes a specific binding substance to the measurement target substance. “Specific binding substance for the measurement target substance” immobilized on the carrier by binding with the “specific binding substance for the measurement target substance” released (dissociated) from the “supported carrier” and released into the free state. Can no longer be combined.
[0027]
In other words, the “measurement target substance” bound to the “specific binding substance for the measurement target substance” in the free state is “... [specific binding substance for the measurement target substance = carrier = specific binding substance for the measurement target substance]”. -[Measurement substance]-[Specific binding substance for measurement substance = carrier = Specific binding substance for measurement substance] ... or "... [Carrier = Specific binding substance for measurement substance]-[Measurement substance] ] Cannot be participated in the formation of a cross-linkage, so that the amount of signal generated is reduced, resulting in a decrease in measurement sensitivity. That is, in the measurement of the sample containing the measurement target substance, an erroneous measurement result that the measurement target substance is not included may be obtained. (False negative)
[0028]
As described above, in the conventional measurement reagent for a measurement target substance including a carrier on which a specific binding substance for the measurement target substance is immobilized, during the storage, “specific binding substance for the measurement target substance is immobilized. When the “specific binding substance for the substance to be measured” is peeled (dissociated) from the “carrier”, the amount of signal generated in the measurement reaction is reduced, which may reduce the sensitivity of the measurement. Even in the measurement of a sample containing a measurement target substance, there is a possibility that an erroneous measurement result (false negative) that the measurement target substance is not included may be obtained. It included danger.
[0029]
The present invention provides a measurement reagent for a measurement target substance including a carrier on which a specific binding substance for the measurement target substance is immobilized, and a method for measuring the measurement target substance using the measurement reagent for the measurement target substance. Prevents the “specific binding substance for the measurement target substance” from peeling (dissociating) from the “carrier having the specific binding substance for the measurement target substance immobilized”, thereby reducing the sensitivity of the measurement reagent. The purpose and problem of the present invention are to provide a measurement reagent capable of preventing the above-mentioned problems and stabilizing the measurement reagent and obtaining an accurate measurement result of the measurement target substance for a long period of time.
[0030]
Similarly, in the present invention, during storage of the measurement reagent of the measurement target substance, the “specific binding substance for the measurement target substance” is detached (dissociated) from the “carrier having the specific binding substance for the measurement target substance immobilized”. ), Thereby preventing a decrease in sensitivity of the measurement reagent, and obtaining an accurate measurement result of the measurement target substance over a long period of time. It is another object of the present invention to provide a stable measurement reagent for a substance to be measured.
[0031]
[Means for Solving the Problems]
  The present invention relates to a measurement reagent for a measurement target substance comprising a carrier on which a specific binding substance for the measurement target substance is immobilized,A peeling inhibitor from a carrier of a specific binding substance for a substance to be measured, each comprising a cation and an anion of 100 mM or more,It is a measuring reagent for the substance to be measured.
[0032]
In the measurement reagent for the substance to be measured, the carrier is preferably latex particles.
[0033]
In the measurement reagent for the measurement target substance, it is preferable that the measurement target substance is an antibody against Treponema pallidum and the specific binding substance for the measurement target substance is an antigen of Treponema pallidum.
[0034]
Further, the present invention provides a measurement reagent for a measurement target substance including a carrier on which a specific binding substance for the measurement target substance is immobilized, together with a carrier on which the specific binding substance for the measurement target substance is immobilized, and a cation and an anion. It is a method for stabilizing a measurement reagent of a substance to be measured, characterized in that each is present in an amount of 100 mM or more.
[0035]
In the method for stabilizing the measurement reagent for the substance to be measured, the carrier is preferably latex particles.
[0036]
In the method for stabilizing the measurement reagent of the measurement target substance, it is preferable that the measurement target substance is an antibody against Treponema paridum, and the specific binding substance for the measurement target substance is an antigen of Treponema paridum. .
[0037]
  Further, the present invention provides a measurement reagent for a measurement target substance comprising a carrier on which a specific binding substance for the measurement target substance is immobilized,In order to suppress the separation of the specific binding substance for the measurement target substance from the carrier,Cations and anionsTheMore than 100 mM eachLetA stabilized reagent for measuring a substance to be measured.
[0038]
In the above-described stabilized measurement reagent for the substance to be measured, the carrier is preferably latex particles.
[0039]
In the above-described stabilized measurement reagent for the measurement target substance, it is preferable that the measurement target substance is an antibody against Treponema paridum and the specific binding substance for the measurement target substance is an antigen of Treponema paridum. .
[0040]
DETAILED DESCRIPTION OF THE INVENTION
(1) Basic requirements of the invention
  In the “measurement reagent for the measurement target substance” and the “measurement reagent for the stabilized measurement target substance” of the present invention, in the measurement reagent for the measurement target substance including a carrier on which a specific binding substance for the measurement target substance is immobilized,In order to suppress peeling of the specific binding substance for the measurement target substance from the carrier (as a peeling inhibitor),Cations and anionsTheMore than 100 mM eachLetIt is essential.
[0041]
In the “method for stabilizing a measurement reagent for a measurement target substance” of the present invention, a specific binding substance for a measurement target substance in a measurement reagent for the measurement target substance including a carrier on which a specific binding substance for the measurement target substance is immobilized. It is essential that the cation and the anion be present at 100 mM or more together with the carrier on which is immobilized.
[0042]
As described above, in the measurement reagent of the measurement target substance including the carrier on which the specific binding substance to the measurement target substance is immobilized, during the storage of the measurement reagent, “the carrier on which the specific binding substance to the measurement target substance is immobilized” It is possible to prevent the “specific binding substance for the substance to be measured” from peeling (dissociating).
[0043]
(2) Cation and anion concentrations
  In the present invention, in the measurement reagent of the measurement target substance containing a carrier on which a specific binding substance for the measurement target substance is immobilized, the cation and the anion are:In order to suppress peeling of the specific binding substance for the measurement target substance from the carrier (as a peeling inhibitor),Each is present at 100 mM or more.
[0044]
And it is preferable to make the said cation and an anion each exist 150 mM or more, since the said peeling (dissociation) can be prevented more strongly. For the same reason, it is more preferable that each of the cation and the anion be present at 300 mM or more, and it is particularly preferable that each of the cation and the anion be present at 500 mM or more.
[0045]
In addition, if the concentration at which the cation and the anion are present is less than 100 mM, the effect of preventing the separation (dissociation) is weakened.
[0046]
In addition, the upper limit of the concentration at which the cation and the anion are present is not particularly limited. Generally, when the concentration exceeds 2,000 mM, the specific binding substance to the measurement target substance immobilized on the carrier is denatured. Since the possibility of being deactivated or spontaneous aggregation of the carriers increases, it is preferable that each be 2,000 mM or less.
[0047]
In addition, although it is the density | concentration to which the said cation and an anion exist, if each of a cation and an anion is more than a regular density | concentration, each may be a different density | concentration.
[0048]
(3) Cation and anion
In the present invention, the cation and the anion present in the measurement reagent of the substance to be measured will be described below.
[0049]
▲ 1 ▼ Cation
The cation in the cation and the anion is not particularly limited as long as it is an ion having a positive charge.
[0050]
The cation may be a monovalent cation or a divalent or higher valent cation.
[0051]
And as this cation, a metal ion, an ammonium ion, or another cation etc. can be mentioned, for example.
[0052]
Examples of the metal ions include alkali metal ions, alkaline earth metal ions, and other metal ions.
[0053]
Examples of alkali metal ions include lithium ions, sodium ions, or potassium ions.
[0054]
Examples of alkaline earth metal ions include beryllium ions, magnesium ions, calcium ions, and the like.
[0055]
Examples of other metal ions include manganese ions, iron ions, cobalt ions, nickel ions, copper ions, zinc ions, and aluminum ions.
[0056]
Examples of ammonium ions include primary ammonium ions, secondary ammonium ions, tertiary ammonium ions, and quaternary ammonium ions.
[0057]
As other cations, for example, carbon atoms, silicon atoms, boron atoms, nitrogen atoms (in cases other than ammonium ions), phosphorus atoms, sulfur atoms, etc. are positively charged atoms or atomic groups Can be mentioned.
Specific examples thereof include a choline ion in which a carbon atom has a positive charge.
[0058]
In addition, as this cation, only one type may be used, or a plurality of types may be used simultaneously.
[0059]
▲ 2 ▼ Anion
The anion in the cation and the anion is not particularly limited as long as it is an ion having a negative charge.
[0060]
The anion may be a monovalent anion or a divalent or higher polyvalent anion.
[0061]
And as this anion, the acid group which consists of an organic compound, a halogen ion, or another inorganic compound etc. can be mentioned, for example.
[0062]
Examples of the acid group made of this organic compound include acetate ion, citrate ion, gluconate ion, or oxalate ion.
[0063]
As a halogen ion, a fluorine ion, a chlorine ion, a bromine ion, an iodine ion etc. can be mentioned, for example.
[0064]
Examples of acid groups composed of other inorganic compounds include sulfate ion, sulfite ion, pyrosulfite ion, dithionite ion, thiosulfite ion, nitrate ion, nitrite ion, hyponitrite ion, peroxonitrite ion, Examples include phosphate ion, phosphite ion, pyrophosphite ion, hypophosphite ion, diphosphate ion, borate ion, carbonate ion, cyanate ion, isocyanate ion, or silicate ion. it can.
[0065]
In addition, as this anion, a monovalent anion is preferable.
And as this monovalent anion, a halogen ion is preferable. In particular, chlorine ion and bromine ion are preferable.
[0066]
Moreover, as this anion, only one type may be used, or a plurality of types may be used simultaneously.
[0067]
(3) Cation and anion
The cation and the anion are present in the measurement reagent of the measurement target substance. Any method may be used as long as the cation and the anion can be present in the measurement reagent of the measurement target substance at 100 mM or more. .
[0068]
For example, a compound containing the cation and a compound containing the anion are added separately, and a specific binding substance for the substance to be measured is present together with an immobilized carrier to prepare a measurement reagent for the substance to be measured. May be.
[0069]
Alternatively, a measurement reagent for a measurement target substance may be prepared by adding a compound containing both the cation and the anion and allowing a specific binding substance for the measurement target substance to exist together with an immobilized carrier.
[0070]
As a result, it is sufficient that the cations and the anions are present in the measurement reagent of the measurement target substance at 100 mM or more.
[0071]
In addition, as a compound containing both the said cation and an anion, the salt etc. which consist of this cation and an anion can be mentioned, for example.
[0072]
(4) Substances to be measured
(1) Substance to be measured
In the present invention, the substance to be measured is a substance whose presence or content (concentration) is to be measured in a sample.
[0073]
Any substance can be used as the measurement target substance as long as the measurement target substance can be measured using a measurement reagent of the measurement target substance including a carrier on which a specific binding substance for the measurement target substance is immobilized.
[0074]
Examples of the measurement target substance include organic substances such as proteins, carbohydrates, lipids, and nucleic acids, or inorganic substances such as metals.
[0075]
More specifically, for example, virus-related antigens or antibodies such as HBs antigen, anti-HBs antibody, HBe antigen, anti-HBe antibody, anti-HBc antibody, anti-HCV antibody, anti-HIV antibody, anti-ATLV antibody; E. coli O157 antigen, Bacteria-related antigens or antibodies such as anti-Treponema pallidum antibody, anti-mycoplasma antibody, anti-streptridine O antibody (ASO); immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), or immunity Immunoglobulins such as globulin E (IgE); C-reactive protein (CRP), α1-acid glycoprotein, haptoglobin, complement C3, complement C4, inflammation markers such as rheumatoid factor; α-fetoprotein, CEA, CA19-9 Tumor markers such as: human placental chorionic gonadotropin and other hormones; alleles Allergen-related antigens or antibodies such as antigens, allergen-specific IgE antibodies; blood coagulation-related substances such as antithrombin III (ATIII); fibrinolysis products (FDP), fibrinolytic substances such as D-dimer; ABO blood group Blood group-related antigens or antibodies such as antibodies and irregular antibodies; viral DNA or RNA; bacterial DNA or RNA; animal or plant DNA or RNA such as humans; other diseases such as lipoprotein (a) and ferritin Substances related to the above; drugs; metals; poisonous or deleterious substances.
[0076]
As the substance to be measured, the virus-related antigen or antibody, or the bacteria-related antigen or antibody, etc. are preferable, and in particular, the antibody against Treponema paridum (anti-treponema paridum antibody) is suitable. .
[0077]
(2) Sample
In the present invention, the sample refers to a sample in which there is a possibility that the above-described measurement target substance is present, and the presence / absence of the measurement target substance or content (concentration) is to be measured.
[0078]
For example, human or animal blood, serum, plasma, urine, semen, spinal fluid, saliva, sweat, tears, ascites, amniotic fluid, or other body fluids; human or animal brain or other organs, hair, skin, nails, muscles, or Extracts or suspensions of stool from human or animals; extracts of cells or cells; extracts of plants; extracts of plants; foods such as grains, vegetables, fruits, seafood, meat or processed foods; Examples include beverages such as water, tea, coffee, milk, or fruit juice; reagents or pharmaceuticals; and samples for environmental analysis such as drinking water, river water, lake water, seawater, or soil suspensions.
[0079]
(5) Specific binding substance for the substance to be measured
In the present invention, the specific binding substance for the substance to be measured is a substance that has a specific affinity and can bind to the substance to be measured.
[0080]
Specific binding substances for this measurement target substance include, for example, an antibody against this antigen when the measurement target substance is an antigen, an antigen against this antibody or an antibody against this antibody when the measurement target substance is an antibody, measurement Ligand when the target substance is a substance made of protein, etc., lectin that binds to this sugar when the target substance is sugar, sugar that binds to this lectin when the target substance is lectin, In the case where the substance to be measured is a nucleotide chain, a nucleotide chain complementary to this nucleotide chain, or in the case where the substance to be measured is a receptor, a substance for this receptor can be mentioned.
[0081]
That is, any substance can be used in the present invention as a specific binding substance for a measurement target substance without particular limitation as long as it is a substance that has a specific affinity for and can bind to the measurement target substance.
[0082]
In the present invention, it is preferable that the measurement target substance is an antigen and the specific binding substance for the measurement target substance is an antibody against the antigen.
[0083]
Here, the antibody may be either a polyclonal antibody or a monoclonal antibody, and these fragments [F (ab) '₂Or Fab ′ etc.].
[0084]
In the present invention, it is preferable that the substance to be measured is an antibody and the specific binding substance for the substance to be measured is an antigen against the antibody or an antibody against the antibody.
[0085]
When the measurement target substance is an antibody and the specific binding substance for the measurement target substance is an antigen against the antibody, the “antigen against the antibody” may be a natural substance derived from bacteria or viruses, Alternatively, it may be artificially prepared by a genetic recombination method or the like, but it is particularly preferable that it is artificially prepared by a genetic recombination method or the like.
[0086]
And when it is artificially prepared by this genetic recombination method or the like, the “antigen against the antibody” may be fused with other proteins, and as other proteins to be fused, For example, glutathione-S-transferase (GST), maltose binding protein (MBP), thioredoxin, β-galactosidase, lactase, signal peptide, biotinylated protein, protein A, or gene 10 can be used.
[0087]
More specifically, for example, when the measurement target substance is an antibody against Treponema paridum, maltose binding protein (MBP) is fused to the 17 K dalton antigen of Treponema paridum as a specific binding substance to the measurement target substance. And those in which glutathione-S-transferase (GST) is fused to the 47 K Dalton antigen of Treponema paridam.
[0088]
In addition, when prepared artificially by this genetic recombination method or the like, the “antigen against the antibody” may be a fusion of a plurality of types of antigens.
[0089]
More specifically, for example, when the substance to be measured is an antibody against Treponema pallidum, the specific binding substance for the substance to be measured includes 17K dalton antigen of Treponema pallidum and 47K dalton antigen of Treponema pallidum. Examples thereof include those fused, or those obtained by fusing 17K Dalton antigen of Treponema pallidum, 47K Dalton antigen of Treponema pallidum, and glutathione-S-transferase (GST).
[0090]
In the present invention, it is preferable that the substance to be measured is an antibody against Treponema pallidum and the specific binding substance to the substance to be measured is an antigen of Treponema pallidum.
[0091]
Examples of the antigen of Treponema pallidum include 15K dalton antigen, 17K dalton antigen, or 47K dalton antigen.
[0092]
These antigens of Treponema pallidum may be naturally derived from Treponema pallidum cells or artificially prepared by genetic recombination methods, etc. It is preferable that the material is artificially prepared.
[0093]
When the antigen of Treponema pallidum is artificially prepared by a genetic recombination method or the like, for example, glutathione-S-transferase (GST), maltose binding protein (MBP), thioredoxin, β-galactosidase, It may be fused with other proteins such as lactase, signal peptide, biotinylated protein, protein A, or gene 10.
[0094]
(6) Carrier
(1) In the present invention, the carrier can be used without particular limitation as long as it can immobilize a specific binding substance for the substance to be measured.
[0095]
That is, a carrier used in a method and a measuring reagent for measuring a measurement target substance in a sample using a specific binding reaction between a “specific binding substance for a measurement target substance” and a “specific binding substance” Or any carrier that can be used.
[0096]
(2) Examples of the carrier in the present invention include latex particles used in latex turbidimetry, or latex particles that can be used in latex turbidimetry.
[0097]
Examples of such latex particles include polystyrene / latex particles, styrene / styrene sulfonate copolymer / latex particles, acrylonitrile / butadiene / styrene copolymer / latex particles, vinyl chloride / acrylate copolymer / Latex particles, vinyl acetate-acrylic acid copolymer / latex particles, polyacrolein / latex particles, styrene-methacrylic acid copolymer / latex particles, styrene-glycidyl (meth) acrylic acid copolymer / latex particles, heavy methacrylate Examples thereof include latex particles in which synthetic polymer particles such as coalescence / latex particles or acrylic acid polymer / latex particles are uniformly suspended.
[0098]
As the latex particles, polystyrene / latex particles or styrene / methacrylic acid copolymer / latex particles are preferable, and polystyrene / latex particles are particularly preferable.
[0099]
There is no restriction | limiting in particular about the particle size of this latex particle.
When measuring a measurement target substance using the latex turbidimetric method as a measurement principle, the degree to which latex particles are bonded via the measurement target substance to form an aggregate, the ease of measurement of the generated aggregate, etc. For this reason, the average particle diameter of the latex particles is preferably 0.04 to 1 μm.
[0100]
In addition, in the measurement reagent for the measurement target substance, the concentration includes “latex particles with a specific binding substance immobilized on the measurement target substance”. This is the concentration of the measurement target substance, the concentration in the sample, the measurement target The optimum concentration differs depending on various conditions such as the kind of specific binding substance to the substance and the distribution density on the surface of the latex particles, the particle size of the latex particles, the mixing ratio of the sample and the measuring reagent, etc.
Yes.
[0101]
However, normally, the sample and the measurement reagent of the measurement target substance are mixed, and the “specific binding substance for the measurement target substance” immobilized on the latex particles and the “measurement target substance” contained in the sample are specific. During the measurement reaction in which a typical binding reaction is performed, the concentration of “latex particles with a specific binding substance immobilized on the substance to be measured” is 0.005 to 1% (w / v) in the reaction mixture. In this case, the measurement target substance is measured with “latex particles having a specific binding substance immobilized on the target substance to be measured” having such a concentration in the reaction mixture. Include in reagent.
[0102]
(3) Further, as the carrier in the present invention, for example, particles used in an indirect agglutination reaction measurement method such as a particle agglutination reaction measurement method or a latex agglutination reaction measurement method using gelatin particles, or the indirect agglutination reaction measurement Examples thereof include particles that can be used in the method.
[0103]
Examples of such particles include particles made of an organic polymer material such as liposome, latex, gelatin, polyacrylamide, microcapsule or emulsion, particles made of an inorganic polymer material such as glass, silica gel, carbon or bentonite, and others. And the like.
[0104]
It should be noted that magnetic particles prepared by coating the polymer particles with a ferromagnetic material or dispersing the ferromagnetic material at the time of particle molding may be used as a carrier.
[0105]
There are no particular restrictions on the particle size of these particles.
However, the average particle size of these particles is preferably in the range of 0.01 to 100 μm, and more preferably in the range of 0.5 to 10 μm.
[0106]
Further, the specific gravity of these particles is preferably in the range of 1 to 10, more preferably in the range of 1 to 2.
[0107]
(4) Further, examples of the carrier in the present invention include a container used in the indirect agglutination reaction measurement method or a container that can be used in the indirect agglutination reaction measurement method.
[0108]
Examples of such containers include test tubes, microplates (microtiter plates) or trays made of glass, polystyrene, vinyl chloride polymer, methacrylic acid polymer, or the like.
[0109]
In addition, it is preferable that the bottom surface of the solution storage portion (such as a well of a microplate) of the container has an inclined shape from the center to the periphery of the bottom surface such as a U shape, a V shape, or a UV shape.
[0110]
(5) The carrier in the present invention is, for example, an immunoassay using a labeling substance such as an enzyme immunoassay, a fluorescence immunoassay, a radioimmunoassay, or a luminescence immunoassay, that is, a labeled immunoassay. Examples include carriers that are used or carriers that can be used in this labeled immunoassay.
[0111]
Examples of such carriers include polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, vinyl chloride polymer, nylon, methacrylic acid polymer, polyacrylamide, latex, liposome, gelatin, agarose, cellulose, sepharose, glass, metal And particles made of materials such as ceramics or magnetic material, microcapsules, beads, microplates (microtiter plates), test tubes, sticks or test pieces.
[0112]
(6) Further, as the carrier in the present invention, for example, a specific binding substance for a measurement target substance (test substance) described in JP-A-9-229936 and JP-A-10-132919 is immobilized. A carrier having a coated surface and a carrier used in a measurement method using particles in which a specific binding substance for a measurement target substance (test substance) is immobilized, or a carrier that can be used in this measurement method Can be mentioned.
[0113]
(7) Also, a magnetic carrier prepared by coating the above-described carrier with a ferromagnetic material or containing a ferromagnetic material at the time of carrier molding can be used.
[0114]
(8) In the present invention, a known method such as physical adsorption can be used as a method for immobilizing a specific binding substance for the substance to be measured on the carrier.
[0115]
In the case of the physical adsorption method, according to a known method, a specific binding substance for the measurement target substance dissolved in a buffer solution or the like is added to the solution storage portion of the container and brought into contact with the inner wall surface, or specific for the measurement target substance. By immobilizing the binding substance and particles in a solution such as a buffer solution and bringing them into contact, it can be immobilized on a carrier.
[0116]
For example, a specific binding substance for a measurement target substance dissolved in a buffer solution or the like is added to the solution storage part of the container and brought into contact with the inner wall surface, or the specific binding substance and particles for the measurement target substance are added to a solution such as a buffer solution. Mixing and contacting at about 2 ° C. to about 40 ° C. for about 10 minutes to about 1 day.
[0117]
Further, if it is necessary to carry out treatment to suppress non-specific reaction or spontaneous aggregation of the carrier, the inner wall surface or particles of the solution storage part of the container in which the specific binding substance for the measurement target substance is immobilized is used. Specific to the substance to be measured by treatment by known methods such as bovine serum albumin (BSA), protein such as casein, gelatin, ovalbumin or its salt, surfactant or nonfat dry milk You may perform the blocking process (masking process) of the support | carrier which fix | immobilized the binding substance.
[0118]
(7) Measuring reagent
(1) Measuring principle of measuring reagent (measuring method)
In the present invention, the measurement reagent for the measurement target substance uses a specific binding reaction between the “specific binding substance for the measurement target substance” immobilized on the carrier and the “measurement target substance” contained in the sample. Thus, the measurement reagent is based on the measurement principle of a method of measuring a measurement target substance in a sample.
[0119]
The specific binding reaction between the “specific binding substance for the measurement target substance” and the “measurement target substance” includes, for example, an antigen-antibody reaction (immune reaction) between an antigen and an antibody, a protein, and the like. Examples include a reaction with a ligand, a reaction between a sugar and a lectin, a reaction between a nucleotide chain and a complementary nucleotide chain, or a reaction between a receptor and a substance corresponding thereto.
[0120]
Examples of a method for measuring a measurement target substance in a sample using such a binding reaction include, for example, an immunological measurement method, a gene measurement method, and the like.
[0121]
In addition, as this immunological measurement method, for example, latex turbidimetry; indirect aggregation reaction measurement method such as latex agglutination measurement method; enzyme immunoassay, fluorescence immunoassay, radioimmunoassay, or luminescence immunoassay Immunoassay using a labeling substance such as a labeling method, ie, labeled immunoassay; Western blotting; ELSA method (enzyme-linked ligand assay) shown by Dahlbeack et al. [Thromb. Haemost. 79, 767-772, 1998, and WO 98/23963]; or JP-A-9-229936 and JP-A-10-132919, etc. Examples thereof include a carrier having a surface on which a specific binding substance is fixed and coated, and a measurement method using particles on which a specific binding substance for a measurement target substance (test substance) is fixed.
[0122]
The labeled immunoassay is the above-described method, and the present invention can be applied to any method such as the sandwich method, the competitive method, or the homogeneous method (homogeneous method).
[0123]
The gene measurement method includes, for example, a sandwich method, a branched probe method, or a method in which measurement is performed by agglutination of a carrier such as latex particles on which a polynucleotide probe (specific binding substance to a measurement target substance) is immobilized. Can be mentioned.
[0124]
(2) Measuring reagent
  The measurement reagent of the measurement target substance in the present invention can be based on the measurement method described above, and in the measurement reagent of the measurement target substance including a carrier on which a specific binding substance for the measurement target substance is immobilized. ,In order to suppress peeling of the specific binding substance for the measurement target substance from the carrier (as a peeling inhibitor),Cations and anionsTheMore than 100 mM eachLetIt is characterized by that.
[0125]
That is, the measurement reagent for the substance to be measured in the present invention includes a carrier on which a specific binding substance for the substance to be measured is immobilized, and each contains 100 mM or more of a cation and an anion.
[0126]
As a result, in the measurement reagent of the measurement target substance including the carrier on which the specific binding substance to the measurement target substance is immobilized, during the storage of the measurement reagent of the measurement target substance, the “specific binding substance to the measurement target substance is immobilized. It is possible to prevent separation (dissociation) of the “specific binding substance for the substance to be measured” from the “carrier”, it is possible to keep the measurement reagent of the substance to be measured stable for a long period of time, and it is stable for a long time. A measurement reagent for the substance to be measured can be obtained.
[0127]
In addition, the measurement reagent of the measurement target substance in the present invention can be sold by itself or used for measurement of the measurement target substance in a sample.
In addition, the measurement reagent for the measurement target substance in the present invention can be sold in combination with other reagents or used for measurement of the measurement target substance in the sample.
[0128]
Examples of the other reagent include a buffer solution, a sample diluent, a reagent diluent, a reagent containing a labeling substance, a reagent containing a substance that generates a signal such as color development, or calibration (calibration). The reagent of the substance containing these substances can be mentioned.
[0129]
The other reagent is the first reagent, the measurement reagent of the measurement target substance in the present invention is the second reagent, or the measurement reagent of the measurement target substance in the present invention is the first reagent, and the other reagent Can be sold and used in various combinations as appropriate.
[0130]
In addition, various aqueous solvents can be used as the solvent for the measurement reagent of the substance to be measured in the present invention.
[0131]
Examples of the aqueous solvent include purified water, physiological saline, or various buffer solutions such as tris (hydroxymethyl) aminomethane buffer, phosphate buffer, or phosphate buffered saline. .
[0132]
About the pH of this buffer solution, an appropriate pH may be appropriately selected and used. Although there is no particular limitation, it is general to select and use a pH within the range of pH 3-12.
[0133]
In the present invention, it is preferable to use a pH in the neutral region rather than an alkaline region or an acidic region because the measurement reagent can have a higher stabilizing effect than the alkaline region or acidic region. For example, the pH is more preferably pH 7.0 to pH 7.3.
[0134]
In addition, the measurement reagent of the measurement target substance in the present invention includes a carrier on which a specific binding substance for the measurement target substance is immobilized, bovine serum albumin (BSA), human in addition to the cation and anion. Proteins such as serum albumin (HSA), casein or salts thereof; various salts other than the above cations and anions; various sugars; skim milk powder; various animal sera such as normal rabbit serum; various kinds such as sodium azide or antibiotics Preservatives; activators; reaction accelerators; sensitivity-enhancing substances such as polyethylene glycol; nonspecific reaction inhibitors; or nonionic surfactants, amphoteric surfactants or anionic surfactants You may make 1 type or 2 types or more of various surfactants etc. contain suitably.
[0135]
And the density | concentration at the time of making these contain in the measuring reagent of a measuring object substance is although it does not specifically limit, 0.001-10% (W / V) is preferable, Especially 0.01-5% (W / V) V) is preferred.
[0136]
Examples of the surfactant include sorbitan fatty acid ester, glycerin fatty acid ester, decaglycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene alkyl ether, Nonionic surfactants such as polyoxyethylene phytosterol, phytostanol, polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene alkylphenyl ether, polyoxyethylene castor oil, hydrogenated castor oil or polyoxyethylene lanolin; betaine acetate, etc. Amphoteric surfactants; or polyoxyethylene alkyl ether sulfate or polyoxyethylene alkyl ether acetic acid Anionic surfactants, such as and the like.
[0137]
In the present invention, the stabilizing effect of preventing the specific binding substance for the measurement target substance from peeling (dissociating) from the carrier on which the specific binding substance for the measurement target substance is immobilized is the specific binding substance for the measurement target substance. This is particularly remarkable in the stabilization when the carrier on which is immobilized is in contact with the liquid. (Note that the carrier on which the specific binding substance for the substance to be measured is in contact with the liquid means that the carrier that is not in contact with the liquid is brought into contact with water or other liquid during use. (Including those that are included)
[0138]
(8) Measurement of substances to be measured in samples
In order to measure a measurement target substance in a sample using the measurement reagent of the measurement target substance in the present invention, the measurement operation may be performed according to the operation method of the measurement method which is the measurement principle of the measurement reagent of the measurement target substance. .
[0139]
This measurement may be performed by a method or using an apparatus such as an analyzer.
[0140]
In addition, this measurement may be performed by a one-step method (one-reagent method) or by a method performed by a plurality of operation steps such as a two-step method (two-reagent method).
[0141]
In the following, a specific explanation will be given, taking as an example the case of measuring an antibody against Treponema pallidum (anti-Treponema pallidum antibody) in a sample using a measurement reagent for the substance to be measured based on the latex turbidimetric method. Do.
[0142]
(1) First, the following are prepared as measurement reagents for the substance to be measured.
First reagent: Buffer solution containing a buffer and adjusting the pH to a constant value
Second reagent: Buffer containing latex particles on which antigens of Treponema pallidum are immobilized and containing 100 mM or more of cations and anions.
[0143]
(2) A fixed amount of a sample such as serum and a fixed amount of the first reagent are mixed and allowed to stand.
[0144]
(3) After a certain period of time, a certain amount of the second reagent is added to and mixed with the mixture of the sample and the first reagent, and left as a reaction mixture at a constant temperature for a certain period of time.
[0145]
Here, when an antibody against Treponema pallidum is present in the sample, the antibody against Treponema pallidum is bound by an antigen-antibody reaction with the antigen of Treponema pallidum immobilized on latex particles.
[0146]
Furthermore, “... [antigen of treponema pallidum = latex particle = antigen of treponema pallidum] − [antibody against treponema pallidum] − [antigen of treponema pallidum = latex particle = antigen of treponema pallidum] ...” Are formed, and aggregates of latex particles to which the antigen of Treponema pallidum is immobilized are generated via antibodies against Treponema pallidum.
[0147]
(4) Next, after a certain period of time, in an analyzer or a spectrophotometer, the reaction mixture is irradiated with light, and the transmitted light intensity of an appropriate wavelength, which is a signal generated by agglomerates of the produced latex particles, is observed. By measuring the decrease (increase in absorbance) or the increase in scattered light intensity, the amount of the generated aggregate, that is, the amount of antibody against Treponema pallidum contained in the sample is determined.
[0148]
(5) Then, “a value of decrease in transmitted light intensity (increase in absorbance) or increase in scattered light intensity of the reaction mixture subjected to the measurement” and “sample containing an antibody against Treponema pallidum whose concentration is known (standard) By comparing the value of decrease in transmitted light intensity (increase in absorbance) or increase in scattered light intensity when measuring liquid, standard serum, etc.) with respect to Treponema Paridam contained in the measured sample The amount (concentration) of the antibody is calculated.
[0149]
【Example】
The present invention will be further described below with reference to examples.
In addition, this invention is not limited by these.
[0150]
[Example 1] (Preparation of antibody measurement reagent against Treponema pallidum)
The measurement reagent of the measurement target substance of the present invention was prepared when the measurement target substance was Treponema pallidum.
[0151]
1. Preparation of measuring reagent containing latex particles with immobilized antigen of Treponema pallidum (Preparation of second reagent)
[0152]
(1) A measuring reagent (second reagent) using a 17 K Dalton antigen of Treponema paridum prepared by a genetic recombination method.
[0153]
(1) 17K Dalton antigen (Biotech Corporation) expressed from Escherichia coli by a genetic recombination method was used as the 17K Dalton antigen of Treponema pallidum.
[0154]
(2) The 17 K Dalton antigen of Treponema pallidum described in (1) above was added and dissolved in a 50 mM glycine buffer (pH 9.0) so that the final concentration was 50 μg / mL.
[0155]
(3) Latex particles (manufactured by Sekisui Chemical Co., Ltd., average particle size 0.4 μm, polystyrene) are added to 50 mM glycine buffer (pH 9.0) so as to be 0.5% (w / v). Made cloudy.
[0156]
(4) 0.2 mL of the 17 K Dalton antigen solution of Treponema Paridam of the above (2) and 0.2 mL of the latex particle suspension of the above (3) were mixed.
Then, the mixture was allowed to stand at 37 ° C. for 3 hours to immobilize the Treponema pallidum 17K Dalton antigen to the latex particles.
[0157]
(5) 50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer containing 1% (w / v) BSA in a suspension of latex particles on which the 17 K Dalton antigen of Treponema pallidum of (4) is immobilized 1 mL of [pH 7.6] was added and allowed to stand at 37 ° C. for 2 hours to perform blocking treatment (masking treatment) on the surface of latex particles as a carrier.
[0158]
(6) After the blocking treatment (masking treatment) in (5) above, centrifugation at 14,000 rpm was performed at 10 ° C. for 20 minutes.
After centrifugation, the supernatant was removed by decantation to obtain latex particles (latex particles immobilizing the 17K Dalton antigen of Treponema Paridam) as precipitates.
[0159]
(7) Three types of latex particle dispersion media differing only in the concentration of sodium chloride were prepared.
That is, 50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.6] containing 1% (w / v) BSA and 15 mM sodium azide; 100 mM sodium chloride, 1% (w / v) BSA and 15 mM 50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.6] containing sodium azide; and 50 mM Tris (hydroxy) containing 500 mM sodium chloride, 1% (w / v) BSA and 15 mM sodium azide. A methyl) aminomethane-hydrochloric acid buffer solution [pH 7.6] was prepared.
[0160]
(8) Latex particles obtained in (6) above (latex particles with immobilized 17K Dalton antigen of Treponema Paridam) are added to 0.4 mL of each of the three types of latex particle dispersion media prepared in (7) above. The following three types of measurement reagents (second reagents) were prepared by suspending each so as to be 0.25% (w / v).
[0161]
[Measurement reagent A]: containing 1% (w / v) BSA and 15 mM sodium azide containing 0.25% (w / v) latex particles (latex particles having immobilized 17 K Dalton antigen of Treponema paridam) Containing 50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.6].
[0162]
[Measurement reagent B]: containing 0.25% (w / v) latex particles (latex particles having immobilized 17 K Dalton antigen of Treponema pallidum),100 mM sodium chloride,50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.6] containing 1% (w / v) BSA and 15 mM sodium azide.
[0163]
[Measurement Reagent C]: containing 0.25% (w / v) latex particles (latex particles having immobilized 17 K Dalton antigen of Treponema Paridam),500 mM sodium chloride,50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.6] containing 1% (w / v) BSA and 15 mM sodium azide.
[0164]
(2) A measuring reagent (second reagent) using the 47 K Dalton antigen of Treponema pallidum prepared by a genetic recombination method.
[0165]
(1) The 47K Dalton antigen of Treponema Paridam Weigel, M .; It was prepared by a gene recombination method according to the method of Norgard et al. [“INFECTION AND IMMUNITY”, Vol. 60, No. 4, pp. 1568 to 1576, 1992].
[0166]
(2) The 47 K Dalton antigen of Treponema pallidum prepared in (1) above was added and dissolved in a 50 mM glycine buffer (pH 9.0) so that the final concentration was 50 μg / mL.
[0167]
(3) Latex particles (manufactured by Sekisui Chemical Co., Ltd., average particle size 0.4 μm, polystyrene) are added to 50 mM glycine buffer (pH 9.0) so as to be 0.5% (w / v). Made cloudy.
[0168]
(4) 0.2 mL of the 47 K Dalton antigen solution of Treponema Paridam in (2) above and 0.2 mL of the latex particle suspension in (3) above were mixed.
Then, the solution was allowed to stand at 37 ° C. for 3 hours to immobilize the Treponema pallidum 47K Dalton antigen on the latex particles.
[0169]
(5) 50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution containing 1% (w / v) BSA in a suspension of latex particles on which 47 K Dalton antigen of Treponema pallidam of (4) is immobilized 1 mL of [pH 7.2] was added and allowed to stand at 37 ° C. for 2 hours to perform blocking treatment (masking treatment) on the surface of latex particles as a carrier.
[0170]
(6) After the blocking treatment (masking treatment) in (5) above, centrifugation at 14,000 rpm was performed at 10 ° C. for 20 minutes.
After centrifugation, the supernatant was removed by decantation to obtain latex particles (latex particles immobilizing the 47K Dalton antigen of Treponema Paridam) as precipitates.
[0171]
(7) Four types of latex particle dispersion media differing only in the concentration of sodium chloride were prepared.
That is, 50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.2] containing 1% (w / v) BSA and 15 mM sodium azide; 100 mM sodium chloride, 1% (w / v) BSA and 15 mM 50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.2] containing sodium azide; 50 mM Tris (hydroxymethyl) containing 300 mM sodium chloride, 1% (w / v) BSA and 15 mM sodium azide Aminomethane-hydrochloric acid buffer [pH 7.2]; and 50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.2] containing 500 mM sodium chloride, 1% (w / v) BSA and 15 mM sodium azide. Was prepared.
[0172]
(8) Latex particles obtained in (6) above (latex particles with 47K Dalton antigen of Treponema Paridam immobilized) are added to 0.4 mL of each of the four types of latex particle dispersion media prepared in (7) above. The following four types of measurement reagents (second reagents) were prepared by suspending each so as to be 0.25% (w / v).
[0173]
[Measurement reagent D]: containing 1% (w / v) BSA and 15 mM sodium azide containing 0.25% (w / v) latex particles (latex particles to which 47K Dalton antigen of Treponema Paridam was immobilized) Contains 50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.2].
[0174]
[Measurement reagent E]: containing 0.25% (w / v) latex particles (latex particles having immobilized 47 K Dalton antigen of Treponema paridam)100m M sodium chloride,50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.2] containing 1% (w / v) BSA and 15 mM sodium azide.
[0175]
[Measurement reagent F]: containing 0.25% (w / v) latex particles (latex particles having immobilized 47 K Dalton antigen of Treponema paridam)300 mM sodium chloride,50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.2] containing 1% (w / v) BSA and 15 mM sodium azide.
[0176]
[Measurement reagent G]: containing 0.25% (w / v) latex particles (latex particles to which 47 K Dalton antigen of Treponema paridum is immobilized),500 mM sodium chloride,50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.2] containing 1% (w / v) BSA and 15 mM sodium azide.
[0177]
2. Preparation of reagent as sample dilution (preparation of first reagent)
[0178]
The following first reagent was prepared as a sample diluent.
[0179]
[First Reagent]: 50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer containing 4% (w / v) BSA, 0.2% (w / v) polyethylene glycol and 15 mM sodium azide [pH 8.5 ].
[0180]
[Example 2] (Confirmation of storage stability of reagent for measuring antibody against Treponema pallidum)
[0181]
The measurement reagent containing latex particles immobilized with Treponema pallidum 17 K Dalton antigen prepared in Example 1 was stored at 30 ° C., and stability during storage was confirmed.
[0182]
1. Storage of measurement reagent
[0183]
The three types of measurement reagents [Measurement reagent A], [Measurement reagent B], and [Measurement reagent C] prepared in (1) of Example 1 were each stored at 30 ° C. for 50 days.
[0184]
2. Confirmation of stability of measuring reagent
[0185]
Using each of the measurement reagents before and after storage, the measurement target substance (antibody against Treponema pallidum) in the sample was measured, and the stability of the measurement reagent during storage was confirmed.
[0186]
(1) Reagent
[0187]
(1) First reagent
[1st reagent], which is a diluted solution of the sample prepared in 2 of Example 1, was used as the first reagent.
[0188]
(2) Second reagent
The following measuring reagents were used as the second reagents.
[0189]
[Measurement reagent A], [Measurement reagent B], or [Measurement reagent C] after 50 days of storage at 30 ° C. in 1 above. Or
[0190]
[Measurement reagent A], [Measurement reagent B], or [Measurement reagent C] at the time of preparation of the measurement reagent, that is, before the start of storage.
[0191]
(2) Sample
Human serum containing an antibody against Treponema pallidum was used as a sample.
[0192]
(3) Measurement of substances to be measured (antibodies against Treponema pallidum) in samples
[0193]
(1) 87.5 μL of the first reagent was added to a well of a 96-well microtiter plate (manufactured by NUNK), and then 10 μL of the sample was added thereto and mixed.
[0194]
(2) After adding 12.5 μL of the second reagent to the well and mixing it, the absorbance (main wavelength: 490 nm, subwavelength: 655 nm) of the mixed solution in the well was immediately measured. (3350 type, manufactured by Bio-Rad).
[0195]
(3) Immediately after the measurement of the absorbance, the microtiter plate was allowed to stand at 37 ° C.
As a result, an antigen-antibody reaction between the 17 K Dalton antigen of Treponema pallidum immobilized on the latex particles and an antibody against Treponema pallidum contained in the sample is performed, and an aggregate of latex particles is generated. I let you.
[0196]
(4) Ten minutes after the measurement of the absorbance in (2) above, the absorbance of the reaction mixture in the well was measured in the same manner as in (2) above.
[0197]
(5) The difference in absorbance was obtained by subtracting the absorbance measured in (2) from the absorbance measured in (4).
This difference in absorbance is proportional to the amount (concentration) of the substance to be measured (antibody against Treponema pallidum) contained in the sample.
[0198]
(4) Measurement results
Table 1 shows the difference in absorbance obtained by measuring the substance to be measured (antibody against Treponema pallidum) in the sample in (3).
[0199]
[Table 1]

[0200]
3. Consideration
From Table 1, the value of the difference in absorbance obtained after 50 days of storage at 30 ° C. is up to 50% of the value of the difference in absorbance before the start of storage when the cation and the anion are each less than 100 mM. It turns out that it has decreased. That is, the signal generation is greatly reduced, and the sensitivity of the measurement is reduced.
[0201]
On the other hand, the value of the difference in absorbance obtained after 50 days of storage at 30 ° C. in which 100 mM sodium chloride is contained and cations and anions are each present in an amount of 100 mM or more is This is 83% of the value of the difference in absorbance before the start of storage, indicating that a decrease in the generated signal, that is, a decrease in the sensitivity of the measurement is suppressed.
[0202]
Furthermore, in the case of containing 500 mM sodium chloride and having a cation and an anion of 500 mM or more respectively, the value of the absorbance difference obtained after 50 days of storage at 30 ° C. is the start of storage. It is 96% of the value of the previous absorbance difference, and it can be seen that the decrease in the generated signal, that is, the decrease in the sensitivity of the measurement can be almost suppressed.
[0203]
Therefore, the presence of 100 mM or more of each cation and anion makes it possible to keep the measurement reagent of the measurement target substance stable for a long period of time. For example, even under harsh storage conditions for the measurement reagent at 30 ° C. for 50 days, It was confirmed that it was possible to prevent a decrease in the generated signal and to suppress a decrease in measurement sensitivity.
[0204]
[Example 3] (Confirmation of storage stability of reagent for measuring antibody against Treponema pallidum)
[0205]
The measurement reagent containing latex particles immobilized with Treponema pallidum 47 K Dalton antigen prepared in Example 1 was stored at 30 ° C., and stability during storage was confirmed.
[0206]
1. Storage of measurement reagent
Four types of measurement reagents [Measurement Reagent D], [Measurement Reagent E], [Measurement Reagent F], and [Measurement Reagent G] prepared in 1 of (2) of Example 1 above were prepared at 30 ° C., respectively. Stored for 60 days.
[0207]
2. Confirmation of stability of measuring reagent
The measurement target substance (antibody against Treponema pallidum) in the sample was measured using each of the measurement reagents before, during and after storage, and the stability of the measurement reagent during storage was confirmed.
[0208]
(1) Reagent
[0209]
(1) First reagent
[1st reagent], which is a diluted solution of the sample prepared in 2 of Example 1, was used as the first reagent.
[0210]
(2) Second reagent
The following measuring reagents were used as the second reagents.
[0211]
[Measurement reagent D], [Measurement reagent E], [Measurement reagent F], or [Measurement reagent G] after 60 days of storage at 30 ° C. in 1 above.
[0212]
[Measuring reagent D], [Measuring reagent E], [Measuring reagent F], or [Measuring reagent G] 30 days after storage at 30 ° C. in 1 above. Or
[0213]
[Measurement reagent D], [Measurement reagent E], [Measurement reagent F], or [Measurement reagent G] when preparing the measurement reagent, that is, before starting storage.
[0214]
(2) Sample
Human serum containing an antibody against Treponema pallidum was used as a sample.
[0215]
(3) Measurement of substances to be measured (antibodies against Treponema pallidum) in samples
[0216]
(1) The measurement was performed using a Hitachi-7170 type automatic analyzer (manufactured by Hitachi, Ltd.).
[0217]
First, 20 μL of the sample was added to a measurement cell (cuvette).
Next, 175 μL of the first reagent was added to the measurement cell (cuvette) and mixed.
[0218]
The measurement cell (cuvette) was allowed to stand at 37 ° C.
[0219]
(2) At 4 minutes and 14 seconds (15th point) after the addition of the first reagent, the absorbance (wavelength: 700 nm) of the mixed solution in the measurement cell (cuvette) was measured as a sample blind test.
[0220]
(3) At 4 minutes 30 seconds (16th point) after addition of the first reagent, 25 μL of the second reagent is further added to the mixed solution in the measurement cell (cuvette) and mixed. did.
[0221]
The measurement cell (cuvette) was allowed to stand at 37 ° C. for reaction.
As a result, an antigen-antibody reaction between the 47 K Dalton antigen of Treponema pallidum immobilized on the latex particles and an antibody against Treponema pallidum contained in the sample is performed, and an aggregate of latex particles is generated. I let you.
[0222]
(4) At 9 minutes and 47 seconds (34 points) after the addition of the first reagent, the absorbance (wavelength: 700 nm) of the reaction mixture in the measurement cell (cuvette) was used as the measurement value of the sample. It was measured.
[0223]
(5) The difference in absorbance was obtained by subtracting the absorbance (sample blind test) measured in (2) above from the absorbance (measured value) measured in (4) above.
This difference in absorbance is proportional to the amount (concentration) of the substance to be measured (antibody against Treponema pallidum) contained in the sample.
[0224]
(4) Measurement results
Table 2 shows the difference in absorbance obtained by measuring the substance to be measured (antibody against Treponema pallidum) in the sample in (3).
[0225]
[Table 2]

[0226]
3. Consideration
From Table 2, when the cation and the anion are each less than 100 mM [measurement reagent D], the value of the absorbance difference obtained after 30 days of storage at 30 ° C. is up to 73% of the value of the absorbance difference before the start of storage. It can be seen that the absorbance difference value obtained after 60 days of storage at 30 ° C. has decreased to 56% of the absorbance difference value before the start of storage. That is, the signal generation is greatly reduced, and the sensitivity of the measurement is reduced.
[0227]
On the other hand, in the case of containing 100 mM sodium chloride and having a cation and an anion of 100 mM or more [measurement reagent E], the value of the absorbance difference obtained after 30 days of storage at 30 ° C. is Further, the absorbance difference value obtained after 60 days of storage at 30 ° C. is 92% of the absorbance difference value before the start of storage, and the generated signal decreases, that is, the sensitivity of the measurement decreases. It can be seen that is suppressed.
[0228]
In addition, in the case of containing 300 mM sodium chloride and having a cation and an anion of 300 mM or more respectively, the value of the absorbance difference obtained after 30 days of storage at 30 ° C. decreased. Furthermore, the value of the difference in absorbance obtained after 60 days of storage at 30 ° C. is 96% of the value of the difference in absorbance before the start of storage, and almost suppresses the decrease in the generated signal, that is, the decrease in the sensitivity of the measurement. You can see that it is made.
[0229]
Even in the case of containing 500 mM sodium chloride and having a cation and an anion of 500 mM or more [measurement reagent G], the value of the absorbance difference obtained after 30 days of storage at 30 ° C. decreases. Furthermore, the value of the difference in absorbance obtained after 60 days of storage at 30 ° C. is 95% of the value of the difference in absorbance before the start of storage, and almost no decrease in the generated signal, that is, a decrease in the sensitivity of the measurement. It turns out that it can suppress.
[0230]
Therefore, the presence of 100 mM or more of each cation and anion makes it possible to keep the measurement reagent of the measurement target substance stable for a long period of time. For example, even under harsh storage conditions for the measurement reagent at 30 ° C. for 60 days, It was confirmed again that it was possible to prevent a decrease in the generated signal and to suppress a decrease in the sensitivity of the measurement.
[0231]
[Example 4] (Confirmation of action mechanism of sensitivity reduction in measurement reagent of measurement target substance)
[0232]
Regarding the mechanism of action in which the measurement sensitivity of the measurement target substance is reduced by storing the measurement reagent, the measurement reagent containing latex particles immobilized with the antigen of Treponema pallidum prepared in Example 1 above is used. Used to confirm.
[0233]
I. Confirmation with the measurement reagent using 17K Dalton antigen of Treponema Paridam
[0234]
1. Storage of measurement reagent
[Measurement reagent A] prepared in (1) of Example 1 was stored at 30 ° C. for 60 days.
[0235]
2. Measurement with measurement reagent after storage
The measurement target substance (antibodies against Treponema pallidum) in the sample was measured using the measurement reagent 1 day after storage and 60 days after storage, and the value of the difference in absorbance was obtained.
[0236]
(1) Reagent
[0237]
(1) First reagent
[1st reagent], which is a diluted solution of the sample prepared in 2 of Example 1, was used as the first reagent.
[0238]
(2) Second reagent
The following measuring reagents were used as the second reagents.
[0239]
[Measurement reagent A] after 60 days of storage at 30 ° C. in 1 above. Or
[0240]
[Measurement reagent A] after 1 day of storage at 30 ° C. in 1 above.
[0241]
(2) Sample
Human serum containing an antibody against Treponema pallidum was used as a sample.
[0242]
(3) Measurement of a substance to be measured (antibody against Treponema pallidum) in a sample Using the reagent and the sample, each operation was performed as described in (3) of Example 3-2, Measurement of the substance to be measured (antibodies against Treponema pallidum) was performed.
[0243]
(4) Measurement results
The absorbance difference obtained by these measurements is shown in Table 3.
[0244]
3. Measurement with a measuring reagent with a different dispersion medium
The storage medium of the measurement reagent that was stored was exchanged (replaced), and the measurement target substance (antibody against Treponema pallidum) in the sample was measured using this measurement reagent to obtain the value of the difference in absorbance. .
[0245]
(1) Reagent
[0246]
(1) First reagent
[1st reagent], which is a diluted solution of the sample prepared in 2 of Example 1, was used as the first reagent.
[0247]
(2) Second reagent
Centrifugation at 14,000 rpm was performed for 30 minutes at 10 ° C. for 0.4 mL of [Measurement reagent A] 60 days after storage at 30 ° C. in 1 above.
[0248]
After centrifugation, the supernatant (dispersion medium) was removed by decantation, and only latex particles (latex particles immobilizing the 17K Dalton antigen of Treponema Paridam) were collected.
[0249]
The latex particles (latex particles to which the 17 K Dalton antigen of Treponema pallidum was immobilized) were mixed with 50 mM tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7 containing 1% (w / v) BSA and 15 mM sodium azide. .6] was suspended in 0.4 mL of a dispersion medium consisting of 0.25% (w / v), and [Measurement reagent A] was prepared again.
[0250]
That is, with respect to [Measurement Reagent A] stored for 60 days at 30 ° C. by the above operation, the latex particles (latex particles with immobilized 17 K Dalton antigen of Treponema pallidum) remained as they were, and only the dispersion medium was prepared. The measurement reagent A was reconstituted.
[0251]
(2) Sample
Human serum containing an antibody against Treponema pallidum was used as a sample.
[0252]
(3) Measurement of measurement target substance (antibodies against Treponema pallidum) in a sample [Measurement reagent A] in which the above-mentioned dispersion medium was exchanged and the sample were used. Each operation was performed as described, and the measurement target substance (antibody against Treponema pallidum) in the sample was measured.
[0253]
(4) Measurement results
The absorbance difference obtained by these measurements is also shown in Table 3.
[0254]
[Table 3]

[0255]
II. Confirmation with a measuring reagent using the 47K Dalton antigen of Treponema Paridam
[0256]
1. Storage of measurement reagent
[Measuring reagent D] prepared in (2) of Example 1 was stored at 30 ° C. for 60 days.
[0257]
2. Measurement with measurement reagent after storage
The measurement target substance (antibodies against Treponema pallidum) in the sample was measured using the measurement reagent 1 day after storage and 60 days after storage, and the value of the difference in absorbance was obtained.
[0258]
(1) Reagent
[0259]
(1) First reagent
[1st reagent], which is a diluted solution of the sample prepared in 2 of Example 1, was used as the first reagent.
[0260]
(2) Second reagent
The following measuring reagents were used as the second reagents.
[0261]
-[Measurement reagent D] after 60 days of storage at 30 ° C in 1 above. Or
[0262]
-[Measurement reagent D] one day after storage at 30 ° C in 1 above.
[0263]
(2) Sample
Human serum containing an antibody against Treponema pallidum was used as a sample.
[0264]
(3) Measurement of a substance to be measured (antibody against Treponema pallidum) in a sample Using the reagent and the sample, each operation was performed as described in (3) of Example 3-2, Measurement of the substance to be measured (antibodies against Treponema pallidum) was performed.
[0265]
The absorbance difference obtained by these measurements is shown in Table 4.
[0266]
3. Measurement using a measuring reagent with a different dispersion medium
The storage medium of the measurement reagent that was stored was exchanged (replaced), and the measurement target substance (antibody against Treponema pallidum) in the sample was measured using this measurement reagent to obtain the value of the difference in absorbance. .
[0267]
(1) Reagent
[0268]
(1) First reagent
[1st reagent], which is a diluted solution of the sample prepared in 2 of Example 1, was used as the first reagent.
[0269]
(2) Second reagent
Centrifugation at 14,000 rpm was performed for 30 minutes at 10 ° C. for 0.4 mL of [Measurement reagent D] 60 days after storage at 30 ° C. in 1 above.
[0270]
After centrifugation, the supernatant (dispersion medium) was removed by decantation, and only the latex particles (latex particles immobilizing the 47 K Dalton antigen of Treponema pallidum) were collected.
[0271]
The latex particles (latex particles immobilizing the 47K Dalton antigen of Treponema pallidum) were mixed with 50 mM tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution [pH 7 containing 1% (w / v) BSA and 15 mM sodium azide. .2] was suspended in 0.4 mL of the dispersion medium consisting of 0.25% (w / v), and [Measurement Reagent D] was prepared again.
[0272]
That is, with respect to [Measurement Reagent D] stored for 60 days at 30 ° C. by the above operation, the latex particles (latex particles immobilizing the 47 K Dalton antigen of Treponema pallidum) remain as they are, and only the dispersion medium is prepared. The measurement reagent D was reconstituted.
[0273]
(2) Sample
Human serum containing an antibody against Treponema pallidum was used as a sample.
[0274]
(3) Measurement of measurement target substance (antibody against Treponema pallidum) in sample [Measurement reagent D] in which the above-mentioned dispersion medium was exchanged and the sample were used. Each operation was performed as described, and the measurement target substance (antibodies against Treponema pallidum) in the sample was measured.
[0275]
The absorbance difference obtained by these measurements is also shown in Table 4.
[0276]
[Table 4]

[0277]
III. Consideration
[0278]
According to Table 3, the value of the difference in absorbance obtained after 60 days of storage at 30 ° C. is the difference in absorbance obtained after 1 day of storage at 30 ° C. when the cation and the anion are each less than 100 mM. It turns out that it has decreased to 54% of the value of. That is, the signal generation is greatly reduced, and the sensitivity of the measurement is reduced.
[0279]
Moreover, according to Table 4, also in [Measurement reagent D] in which the cation and the anion are each less than 100 mM, the value of the absorbance difference obtained after 60 days of storage at 30 ° C. is obtained after 1 day of storage at 30 ° C. It can be seen that the value has decreased to 73% of the value of the absorbance difference. That is, the signal generation is greatly reduced, and the sensitivity of the measurement is lowered.
[0280]
On the other hand, the value of the absorbance difference obtained by replacing (substituting) the dispersion medium of [measurement reagent A] stored at 30 ° C. for 60 days and reconstructing [measurement reagent A] is 30 It is 86% of the value of the difference in absorbance obtained after 1 day of storage at 0 ° C., indicating that it has increased and recovered by 32% compared to before the dispersion medium exchange.
[0281]
In addition, the value of the absorbance difference obtained by reconstituting [measuring reagent D] after replacing (substituting) the dispersion medium of [measuring reagent D] stored at 30 ° C. for 60 days is the same as that at 30 ° C. It is 92% of the value of the difference in absorbance obtained after 1 day of storage, and it can be seen that it has increased and recovered by 19% compared to before dispersion medium exchange.
[0282]
From the above measurement results, in "Measurement reagent containing latex particles immobilized with 17K Dalton antigen of Treponema pallidum" and "Measurement reagent comprising latex particles immobilized with 47K Dalton antigen of Treponema pallidum", that is, In "Measurement reagent for measurement target substance containing carrier with specific binding substance immobilized on measurement target substance", the measurement reagent deteriorates during storage, and the measurement target substance in the sample (antibodies against Treponema pallidum) The reason why the sensitivity of the measurement is reduced is that the “specific binding substance for the measurement target substance” is peeled off (dissociated) from the “carrier in which the specific binding substance for the measurement target substance is immobilized”. In other words, from “latex particles with immobilized antigen of Treponema pallidum” to “Treponema It can be seen dam antigen "is because peeled off (dissociation).
[0283]
This will be described in more detail below.
[0284]
By storing the measurement reagent ([measurement reagent A], [measurement reagent D]) of the substance to be measured, the difference in absorbance, that is, the signal obtained by the measurement is greatly reduced, and the sensitivity of the measurement is lowered.
[0285]
However, after separating and removing the “component other than the carrier on which the specific binding substance for the measurement target substance is immobilized” (dispersion medium) of the measurement reagent stored, the remaining “specific binding substance for the measurement target substance is removed. And the newly prepared “component other than the carrier on which the specific binding substance for the substance to be measured is immobilized” (dispersion medium). In the measurement reagents ([Measurement reagent A] and [Measurement reagent D]) that have been configured, the difference in absorbance, that is, the signal obtained by the measurement is recovered.
[0286]
That is, in the measurement reagent of the measurement target substance including “a carrier on which a specific binding substance for the measurement target substance is immobilized”, the measurement reagent deteriorates due to storage, and the signal generated in the measurement reaction decreases, and the measurement sensitivity Will begin to decline.
Then, by separating / removing “components other than the carrier on which the specific binding substance for the substance to be measured is immobilized” (dispersion medium) and replacing (substituting) with the newly prepared component, the inhibitory substance is obtained. However, since it is removed from the measurement reagent and thus from the measurement reaction system (reaction mixture), the difference in absorbance (signal) is recovered.
[0287]
As a result, the “measurement substance” (antibody against Treponema pallidum) and the “component other than the carrier on which the specific binding substance for the measurement target substance is immobilized” (dispersion medium) of the measurement reagent that has been stored It can be seen that substances that inhibit the specific binding reaction (antigen-antibody reaction) with the “specific binding substance for the substance to be measured” (antigen of Treponema paridam) are included.
[0288]
Furthermore, “components other than the carrier on which the specific binding substance for the measurement target substance is immobilized” (dispersion medium) is separated and removed from the stored measurement reagent, and replaced (replaced) with the newly prepared component. ), The difference in absorbance (signal) is not completely recovered, so that the inhibitory substance is a “specific binding substance for the measurement target substance” and the “specific binding substance for the measurement target substance”. ”Is peeled (dissociated) from“ the carrier on which the specific binding substance for the measurement target substance is immobilized ”during storage of the measurement reagent, and“ components other than the carrier on which the specific binding substance for the measurement target substance is immobilized ” "(Dispersion medium) is found to exist in a free state.
[0289]
That is, as a result of the examination in this example, in the measurement reagent of the measurement target substance containing “the carrier on which the specific binding substance to the measurement target substance is immobilized”, the above-described phenomenon occurs due to storage, and therefore the following This has been confirmed to reduce the signal obtained by the measurement and reduce the sensitivity of the measurement.
[0290]
(1) During the storage of the measurement reagent for the measurement target substance, the “specific binding substance for the measurement target substance” is peeled (dissociated) from the “carrier on which the specific binding substance for the measurement target substance is immobilized”. The number (amount) of the “specific binding substance to the measurement target substance immobilized on the carrier” is reduced as compared with the time of preparation of the measurement reagent. The number (amount) of “substances to be measured” that can be bound to “specific binding substances” is also reduced.
[0291]
Then, “... [specific binding substance for the measurement target substance = carrier = specific binding substance for the measurement target substance] − [measurement target substance] − [specific binding substance for the measurement target substance = carrier = specific for the measurement target substance” Since the formation of cross-links such as "binding substance] ..." is also reduced, the amount of signal generated is also reduced, thereby reducing the sensitivity of the measurement.
[0292]
(2) The “specific binding substance for the measurement target substance” released (dissociated) from the “carrier on which the specific binding substance for the measurement target substance is immobilized” is in a free state. In the mixed reaction mixture, it is combined with a part or all of the “substance to be measured” contained in the sample.
The bound “measurement target substance” can no longer bind to the “specific binding substance for the measurement target substance” immobilized on the carrier.
[0293]
That is, the “measurement substance” bound to the “specific binding substance for the measurement target substance” in the free state is “... [specific binding substance for the measurement target substance = carrier = specific binding substance for the measurement target substance]”. -[Measurement target substance]-[Specific binding substance for the measurement target substance = carrier = Specific binding substance for the measurement target substance].
This reduces the amount of signal generated and reduces the sensitivity of the measurement.
[0294]
For the above (2), “component other than the carrier on which the specific binding substance for the substance to be measured is immobilized” (dispersion medium) is removed from the stored measurement reagent, and separated (dissociated). By separating and removing the “specific binding substance for the target substance”, the difference in absorbance obtained by measurement, that is, the signal is recovered, but is not completely recovered due to the above (1).
[0295]
[Example 5] (Confirmation of storage stability of reagent for measuring antibody against Treponema pallidum)
A measurement reagent containing latex particles on which 47K Dalton antigen of Treponema pallidum was immobilized was prepared and stored at 30 ° C. to confirm stability during storage.
[0296]
I. Preparation of measurement reagent containing latex particles immobilized with 47K Dalton antigen of Treponema pallidum
[0297]
1. Preparation of measuring reagent containing latex particles with immobilized antigen of Treponema pallidum (Preparation of second reagent)
[0298]
A measuring reagent (second reagent) containing latex particles on which 47K Dalton antigen of Treponema paridum prepared by genetic recombination was immobilized was prepared as follows.
[0299]
(1) The process from the preparation of the 47 K Dalton antigen of Treponema pallidum to the acquisition of latex particles (latex particles immobilizing the 47 K Dalton antigen of Treponema pallidum) is the same as in (1) (2) of Example 1 above. The procedure was as described in 1 ▼ to 6).
[0300]
(2) The following five types of latex particle dispersion media were prepared.
-50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.2] containing 1% (w / v) BSA and 15 mM sodium azide
50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.2] containing 300 mM sodium chloride (NaCl), 1% (w / v) BSA and 15 mM sodium azide
50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.2] containing 300 mM potassium chloride (KCl), 1% (w / v) BSA and 15 mM sodium azide
50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.2] containing 300 mM sodium bromide (NaBr), 1% (w / v) BSA and 15 mM sodium azide
150 mM magnesium chloride (MgCl₂) 50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.2] containing 1% (w / v) BSA and 15 mM sodium azide
[0301]
(3) 0.8 ml of each of the five types of latex particle dispersion media prepared in (2) above was added to each of the obtained latex particles (latex particles with 47 K Dalton antigen of Treponema Paridam immobilized). The following five types of measurement reagents (second reagents) were prepared by suspending to 125% (w / v).
[0302]
[Measurement Reagent H]: 1% (w / v) BSA and 15 mM sodium azide containing 0.125% (w / v) latex particles (latex particles to which 47K Dalton antigen of Treponema Paridam was immobilized) Contains 50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.2].
[0303]
[Measurement reagent I]: 0.125% (w / v) of latex particles (latex particles on which 47 K Dalton antigen of Treponema pallidum is immobilized),300 mM sodium chloride (NaCl),50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.2] containing 1% (w / v) BSA and 15 mM sodium azide.
[0304]
[Measurement reagent J]: 0.125% (w / v) of latex particles (latex particles on which 47 K Dalton antigen of Treponema Paridam was immobilized),300 mM potassium chloride (KCl),50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.2] containing 1% (w / v) BSA and 15 mM sodium azide.
[0305]
[Measurement reagent K]: 0.125% (w / v) of latex particles (latex particles on which 47K Dalton antigen of Treponema pallidum is immobilized),300 mM sodium bromide (NaBr),50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.2] containing 1% (w / v) BSA and 15 mM sodium azide.
[0306]
[Measurement reagent L]: 0.125% (w / v) of latex particles (latex particles on which 47 K Dalton antigen of Treponema Paridam was immobilized),150 mM magnesium chloride (MgCl ₂ ),50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.2] containing 1% (w / v) BSA and 15 mM sodium azide.
[0307]
2. Preparation of reagent as sample dilution (preparation of first reagent)
[0308]
As described in Example 1-2, a sample dilution (first reagent) was prepared.
[0309]
II. Confirmation of storage stability of antibody measurement reagent against Treponema pallidum
The measurement reagent containing latex particles immobilized with Treponema pallidum 47 K Dalton antigen prepared in I above was stored at 30 ° C., and stability during storage was confirmed.
[0310]
1. Storage of measurement reagent
[0311]
Five measurement reagents prepared in 1 of 1 above, [Measuring reagent H], [Measuring reagent I], [Measuring reagent J], [Measuring reagent K], and [Measuring reagent L], were each at 30 ° C. Stored for 56 days.
[0312]
2. Confirmation of stability of measuring reagent
[0313]
The measurement target substance (antibody against Treponema pallidum) in the sample was measured using each of the measurement reagents before, during and after storage, and the stability of the measurement reagent during storage was confirmed.
[0314]
(1) Reagent
[0315]
(1) First reagent
[1st reagent], which is a diluted solution of the sample prepared in I-2 above, was used as the first reagent.
[0316]
(2) Second reagent
The following measuring reagents were used as the second reagents.
[0317]
[Measuring reagent H], [Measuring reagent I], [Measuring reagent J], [Measuring reagent K], or [Measuring reagent L] 56 days after storage at 30 ° C. in 1 above.
[0318]
[Measuring reagent H], [Measuring reagent I], [Measuring reagent J], [Measuring reagent K], or [Measuring reagent L] after 28 days of storage at 30 ° C. in 1 above.
[0319]
[Measuring reagent H], [Measuring reagent I], [Measuring reagent J], [Measuring reagent K], or [Measuring reagent L] after 14 days of storage at 30 ° C. in 1 above.
[0320]
[Measuring reagent H], [Measuring reagent I], [Measuring reagent J], [Measuring reagent K], or [Measuring reagent L] 7 days after storage at 30 ° C. in 1 above. Or
[0321]
[Measurement reagent H], [Measurement reagent I], [Measurement reagent J], [Measurement reagent K], or [Measurement reagent L] at the time of preparation of the measurement reagent, that is, before the start of storage.
[0322]
(2) Sample
Human serum containing an antibody against Treponema pallidum was used as a sample.
[0323]
(3) Measurement of substances to be measured (antibodies against Treponema pallidum) in samples
[0324]
(1) The measurement was performed using a Hitachi-7170S type automatic analyzer (manufactured by Hitachi, Ltd.).
[0325]
First, 15 μL of the sample was added to a measurement cell (cuvette).
Next, 150 μL of the first reagent was added to the measurement cell (cuvette) and mixed.
[0326]
The measurement cell (cuvette) was allowed to stand at 37 ° C.
[0327]
(2) At 4 minutes and 14 seconds (15th point) after the addition of the first reagent, the absorbance (wavelength: 700 nm) of the mixed solution in the measurement cell (cuvette) was measured as a sample blind test.
[0328]
(3) At 4 minutes and 30 seconds (16th point) after addition of the first reagent, 50 μL of the second reagent is further added to the mixed solution in the measurement cell (cuvette) and mixed. did.
[0329]
The measurement cell (cuvette) was allowed to stand at 37 ° C. for reaction.
As a result, an antigen-antibody reaction between the 47 K Dalton antigen of Treponema pallidum immobilized on the latex particles and an antibody against Treponema pallidum contained in the sample is performed, and an aggregate of latex particles is generated. I let you.
[0330]
(4) At 9 minutes and 47 seconds (34 points) after the addition of the first reagent, the absorbance (wavelength: 700 nm) of the reaction mixture in the measurement cell (cuvette) was used as the measurement value of the sample. It was measured.
[0331]
(5) The difference in absorbance was obtained by subtracting the absorbance (sample blind test) measured in (2) above from the absorbance (measured value) measured in (4) above.
This difference in absorbance is proportional to the amount (concentration) of the substance to be measured (antibody against Treponema pallidum) contained in the sample.
[0332]
(4) Measurement results
Table 5 shows the difference in absorbance obtained by measuring the measurement target substance (antibody against Treponema pallidum) in the sample in (3).
[0333]
[Table 5]

[0334]
III. Consideration
From Table 5, the value of the absorbance difference obtained after 14 days of storage at 30 ° C. is 77% of the value of the absorbance difference before the start of storage when the cation and the anion are each less than 100 mM [measurement reagent H]. The value of the absorbance difference obtained after 28 days of storage at 30 ° C. has decreased to 66% of the value of the absorbance difference before the start of storage, and further at 30 ° C. It can be seen that the value of the absorbance difference obtained 56 days after storage has decreased to 49% of the value of the absorbance difference before the start of storage. That is, the signal generation is greatly reduced, and the sensitivity of the measurement is reduced.
[0335]
On the other hand, it contains 300 mM sodium chloride (NaCl), and each cation and anion are present at 300 mM or more [Measurement reagent I], after 14 days of storage at 30 ° C., at 30 ° C. The absorbance difference values obtained after 28 days of storage and after 56 days of storage at 30 ° C. are not decreased, indicating that the decrease in the generated signal, that is, the decrease in the sensitivity of the measurement can be suppressed.
[0336]
Further, it contains 300 mM potassium chloride (KCl), and each cation and anion are present in an amount of 300 mM or more [Measurement reagent J], 14 days after storage at 30 ° C., 28 days after storage at 30 ° C. , And the absorbance difference value obtained after 56 days of storage at 30 ° C. did not decrease, indicating that the decrease in the generated signal, that is, the decrease in the sensitivity of the measurement could be suppressed.
[0337]
Then, it contains 300 mM sodium bromide (NaBr), and cations and anions each exist in an amount of 300 mM or more [Measurement reagent K], after 14 days of storage at 30 ° C. and at 30 ° C. The absorbance difference value obtained after 28 days has not decreased, and the absorbance difference value obtained after 56 days of storage at 30 ° C. is 98% of the value of the absorbance difference before the start of storage. It can be seen that the decrease of the signal, that is, the decrease of the sensitivity of the measurement can be almost suppressed.
[0338]
In addition, 150 mM magnesium chloride (MgCl₂In the case of [Measurement reagent L], the absorbance difference value obtained after 14 days of storage at 30 ° C. is the difference in absorbance before the start of storage. The value of the absorbance difference obtained after 28 days of storage at 30 ° C. is 94% of the value of the absorbance difference before the start of storage and obtained after 56 days of storage at 30 ° C. The value of the difference in absorbance was 94% of the value of the difference in absorbance before the start of storage, indicating that a decrease in the generated signal, that is, a decrease in the sensitivity of the measurement was almost suppressed.
[0339]
From the above, even when various cations and anions are present at 100 mM or more, the measurement reagent of the measurement target substance can be kept stable for a long period of time, for example, for a measurement reagent of 56 days at 30 ° C. It was confirmed that even under severe storage conditions, it was possible to prevent a decrease in the generated signal and suppress a decrease in the sensitivity of the measurement.
[0340]
[Example 6] (Confirmation of storage stability of reagent for measuring antibody against Treponema pallidum)
A measurement reagent containing latex particles on which 47K Dalton antigen of Treponema pallidum was immobilized was prepared and stored at 30 ° C. to confirm stability during storage.
[0341]
I. Preparation of measurement reagent containing latex particles immobilized with 47K Dalton antigen of Treponema pallidum
[0342]
1. Preparation of measuring reagent containing latex particles with immobilized antigen of Treponema pallidum (Preparation of second reagent)
[0343]
A measuring reagent (second reagent) containing latex particles on which 47K Dalton antigen of Treponema paridum prepared by genetic recombination was immobilized was prepared as follows.
[0344]
(1) The process from the preparation of the 47 K Dalton antigen of Treponema pallidum to the acquisition of latex particles (latex particles immobilizing the 47 K Dalton antigen of Treponema pallidum) is the same as in (1) (2) of Example 1 above. The procedure was as described in 1 ▼ to 6).
[0345]
(2) The following three types of latex particle dispersion media were prepared.
-50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.2] containing 1% (w / v) BSA and 15 mM sodium azide
150 mM sodium sulfate (Na₂SO₄) 50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.2] containing 1% (w / v) BSA and 15 mM sodium azide
150 mM magnesium sulfate (MgSO₄) 50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.2] containing 1% (w / v) BSA and 15 mM sodium azide
[0346]
(3) 0.8 ml of each of the three types of latex particle dispersion media prepared in (2) above was added to each of the obtained latex particles (latex particles having 47 K Dalton antigen of Treponema Paridam immobilized) in an amount of 0. The following three types of measurement reagents (second reagents) were prepared by suspending to 125% (w / v).
[0347]
[Measurement reagent M]: 1% (w / v) BSA and 15 mM sodium azide containing 0.125% (w / v) latex particles (latex particles on which 47K Dalton antigen of Treponema paridam was immobilized) Contains 50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.2].
[0348]
[Measurement reagent N]: 0.125% (w / v) of latex particles (latex particles on which 47 K Dalton antigen of Treponema Paridam was immobilized),150 mM sodium sulfate (Na ₂ SO ₄ ),50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.2] containing 1% (w / v) BSA and 15 mM sodium azide.
[0349]
[Measurement Reagent P]: 0.125% (w / v) of latex particles (latex particles on which 47K Dalton antigen of Treponema Paridam was immobilized),150 mM magnesium sulfate (MgSO ₄ ),50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer [pH 7.2] containing 1% (w / v) BSA and 15 mM sodium azide.
[0350]
2. Preparation of reagent as sample dilution (preparation of first reagent)
[0351]
As described in Example 1-2, a sample dilution (first reagent) was prepared.
[0352]
II. Confirmation of storage stability of antibody measurement reagent against Treponema pallidum
The measurement reagent containing latex particles immobilized with Treponema pallidum 47 K Dalton antigen prepared in I above was stored at 30 ° C., and stability during storage was confirmed.
[0353]
1. Storage of measurement reagent
[0354]
The three types of measurement reagents [Measurement Reagent M], [Measurement Reagent N], and [Measurement Reagent P] prepared in 1 of 1 above were each stored at 30 ° C. for 28 days.
[0355]
2. Confirmation of stability of measuring reagent
[0356]
The measurement target substance (antibody against Treponema pallidum) in the sample was measured using each of the measurement reagents before, during and after storage, and the stability of the measurement reagent during storage was confirmed.
[0357]
(1) Reagent
[0358]
(1) First reagent
[1st reagent], which is a diluted solution of the sample prepared in I-2 above, was used as the first reagent.
[0359]
(2) Second reagent
The following measuring reagents were used as the second reagents.
[0360]
[Measuring reagent M], [Measuring reagent N], or [Measuring reagent P] after 28 days of storage at 30 ° C. in 1 above.
[0361]
[Measuring reagent M], [Measuring reagent N], or [Measuring reagent P] after 14 days of storage at 30 ° C. in 1 above.
[0362]
[Measuring reagent M], [Measuring reagent N], or [Measuring reagent P] after 7 days of storage at 30 ° C. in 1 above. Or
[0363]
[Measurement reagent M], [Measurement reagent N], or [Measurement reagent P] at the time of preparation of the measurement reagent, that is, before the start of storage.
[0364]
(2) Sample
Human serum containing an antibody against Treponema pallidum was used as a sample.
[0365]
(3) Measurement of substances to be measured (antibodies against Treponema pallidum) in samples
[0366]
The measurement was performed using a Hitachi-7170S type automatic analyzer (manufactured by Hitachi, Ltd.) as described in (1) to (5) in II-2 (3) of Example 5.
[0367]
(4) Measurement results
Table 6 shows the difference in absorbance obtained by measuring the substance to be measured (antibody against Treponema pallidum) in the sample in (3).
[0368]
[Table 6]

[0369]
III. Consideration
From Table 6, the value of the absorbance difference obtained after 14 days of storage at 30 ° C. is up to 77% of the value of the absorbance difference before the start of storage when the cation and the anion are less than 100 mM each. Further, it can be seen that the absorbance difference value obtained after 28 days of storage at 30 ° C. has decreased to 66% of the absorbance difference value before the start of storage. That is, the signal generation is greatly reduced, and the sensitivity of the measurement is reduced.
[0370]
In contrast, 150 mM sodium sulfate (Na₂SO₄), And a cation and an anion each exist in an amount of 150 mM or more, the value of the difference in absorbance obtained after 14 days of storage at 30 ° C. is not decreased. The absorbance difference value obtained after 28 days of storage at 30 ° C. is 91% of the absorbance difference value before the start of storage, indicating that the decrease in the generated signal, that is, the decrease in the sensitivity of the measurement is suppressed. .
[0371]
In addition, 150 mM magnesium sulfate (MgSO₄In the case of [Measurement Reagent P] in which cation and anion are present in an amount of 150 mM or more, the absorbance difference value obtained after 14 days of storage at 30 ° C. is not decreased, The value of the absorbance difference obtained after 28 days of storage at 30 ° C. is 73% of the value of the absorbance difference before the start of storage, indicating that the decrease in the generated signal, that is, the decrease in the sensitivity of the measurement is suppressed. .
[0372]
From the above, even in the combination of different cations and anions, the presence of 100 mM or more makes it possible to keep the measurement reagent of the measurement target substance stable for a long period of time, for example, measurement at 30 ° C. for 28 days. It was confirmed that even under harsh storage conditions for the reagent, it was possible to prevent a decrease in the generated signal and to suppress a decrease in measurement sensitivity.
[0373]
[Summary of Example Results]
Summarizing the results of the above examples, the cation and the anion are present in 100 mM or more in any case of sodium chloride, potassium chloride, sodium bromide, magnesium chloride, sodium sulfate, or magnesium sulfate. Peeling (dissociation) of the “specific binding substance for the substance to be measured” from the carrier was suppressed, thereby suppressing a decrease in measurement sensitivity.
[0374]
That is, the presence of 100 mM or more in various cations and anions ensures that the measurement reagent for the measurement target substance including the carrier on which the specific binding substance for the measurement target substance is immobilized is stable for a long period of time, and is an accurate measurement target. It was confirmed that the measurement result of the substance can be obtained.
[0375]
【The invention's effect】
  The measurement reagent of the measurement target substance of the present invention and the measurement reagent of the stabilized measurement target substance of the present invention are the measurement reagent of the measurement target substance including a carrier on which a specific binding substance for the measurement target substance is immobilized.In order to suppress peeling of the specific binding substance for the measurement target substance from the carrier (as a peeling inhibitor),Cations and anionsTheMore than 100 mM eachLetThus, during storage of the measurement reagent, the “specific binding substance for the measurement target substance” is peeled off from the “carrier having the specific binding substance for the measurement target substance immobilized” ( Dissociation), thereby preventing a decrease in measurement sensitivity of the measurement reagent, stabilizing the measurement reagent, and obtaining an accurate measurement result of the measurement target substance for a long period of time. It has the effect.
[0376]
In addition, the method for stabilizing a measurement reagent of a measurement target substance of the present invention immobilizes a specific binding substance for a measurement target substance in the measurement reagent of the measurement target substance including a carrier on which a specific binding substance for the measurement target substance is immobilized. The cation and the anion are each present in an amount of 100 mM or more together with the cationized carrier, so that during the storage of the measurement reagent of the measurement target substance, a “specific binding substance for the measurement target substance is added”. This prevents the “specific binding substance for the substance to be measured” from peeling (dissociated) from the “immobilized carrier”, thereby preventing the measurement reagent from decreasing in sensitivity and stabilizing the measurement reagent. It has an effect that an accurate measurement result of the measurement target substance can be obtained for a long time.

Claims (3)

In the measurement reagent of the analyte comprising a specific binding substance immobilized carrier for analyte, with the specific binding substance immobilized carrier for analyte, the presence of each 3 100 mM or more cations and anions By this, the method for stabilizing the measurement reagent of the measurement target substance is characterized in that the specific binding substance for the measurement target substance is prevented from peeling from the carrier . The carrier is a latex particle, a method of stabilizing reagent for measuring analyte in claim 1, wherein. The method for stabilizing a measurement reagent for a measurement target substance according to claim 1 or 2 , wherein the measurement target substance is an antibody against Treponema paridum, and the specific binding substance for the measurement target substance is an antigen of Treponema paridum.