JP3616454B2 - Reagent for measuring biological components - Google Patents

Description

【００２３】
化合物２
【化８】

【００２４】
【発明の実施の形態】
本発明に用いるキノロン系抗生物質は、それぞれ前記のような構造と次のような一般名を持つ化合物である。本発明に用いるキノロン系抗生物質の多くは構造中にフッ素を含む、いわゆるフルオロキノロン系抗生物質である。これらのキノロン系抗生物質は、もともと各種感染症の治療を目的として開発されたものであるが、本発明者らは、これらの抗菌剤が試薬に添加した時に先に述べたような多くの課題を解決する抗菌剤として有用性の高い化合物であることを見出し、本発明を完成した。以下、各抗菌剤について説明する。
【００３０】
化合物１：スパルフロキサシン(sparfloxacin)
スパルフロキサシン(sparfloxacin;5-amino-1-cyclopropyl-6、8-difluoro-7-(cis-3、5-dimethyl-1-piperazinyl)-4(1H)-oxoquinoline-3-carboxylic acid)[12]は、オキソキノリン骨格の５位にアミノ基を、６位と８位にフッ素を、７位に３，５−ジメチルピペラジニル基を有する３環式のキノロン系合成抗菌剤である。肺炎球菌、ＭＲＳＡを含む黄色ブドウ球菌、グラム陽性菌、グラム陰性菌、マイコプラズマ、クラミジア、結核菌等、広範囲の微生物に対する抗菌活性を備えており、その作用は殺菌的である。キノロン剤の中でもその抗菌活性は最も強い部類に属すると言われている。
【００３５】
化合物２：ロメフロキサシン(lomefloxacin)
ロメフロキサシン(lomefloxacin;(±)-1-ethyl-6、8-difluoro-1、4-dihydro-7-(3-methyl-1-piperazinyl)-4-oxo-3-quinolinecarboxylic acid)[20]は、３環式キノロンに属する合成キノロン系抗菌剤である。グラム陽性細菌、陰性細菌、および嫌気性細菌に対して広い抗菌スペクトルを示し、その作用は殺菌的である。酸性側では抗菌活性が低下する傾向が有る。主に塩酸塩として流通している。
【００３６】
以上のような化合物は、市販されているものであれば入手が容易である。また市販されていないものであっても、類似の構造を持つ化合物の製造法を基に合成することが可能である。
【００３７】
これらの抗菌剤は単独で添加しても良いし、何種類かを混合して添加しても良い。何種類かを混合して用いれば耐性菌が出現しにくくなるという効果を期待できる。また単独で用いるにしろ、混合する場合にしろ、これらのキノロン系抗生物質の試薬中における好ましい使用濃度は、０．０００５％Ｗ／Ｖ（５μｇ／ｍｌ）以上である。この濃度よりも低い場合には、特に血清をベースとした微生物の繁殖し易い試薬における抗菌力が不十分になる場合が有る。またより広範囲の微生物に対して十分な抗菌活性を保証し、しかも耐性菌の出現しにくい環境を実現するためには、０．００１−０．０１％Ｗ／Ｖ（１０−１００μｇ／ｍｌ）の範囲で用いると良い。本発明の抗菌剤はこのような非常に高い濃度で用いても、免疫反応や酵素反応に影響を与える可能性が低い。たとえばロメフロキサシンの場合は、ＲＩＡにおいて０．０５％Ｗ／Ｖ（５００μｇ／ｍｌ）まで反応に影響を与えないことを確認しているが、一部の酵素反応には阻害的に働くことも有るのであまりにも高い濃度は限定された反応系でしか利用できないものと思われる。
基本的には本発明で用いるキノロン系抗菌剤は、高い濃度で用いても各種反応系に対して影響の出にくいものである。したがって高い濃度で使ってはならないということではない。ただ、不必要に高い濃度は原料コストの上昇につながるので好ましくない。また溶解度を越える濃度で用いても添加量に応じた効果は期待できないので、この程度の濃度で用いるのが効率的である
【００３８】
本発明において、前記抗菌剤を適用することができる試薬は、微生物の繁殖から保護すべき成分を含む試薬である。具体的には、酵素を含むもの、抗原や抗体のような免疫学的活性物質を含むもの、血清蛋白を含むもの等をあげることができる。
抗原や抗体のような免疫学的活性物質を含む試薬には、トリヨードサイロニン、サイロキシン、甲状腺刺激ホルモン、卵巣刺激ホルモン、黄体ホルモン、絨毛性ゴナドトロピン、インスリンといった各種ホルモン、インターフェロン、インターロイキン、マクロファージ遊走阻止因子、顆粒球コロニー刺激因子といったリンホカイン、ウイルス粒子やその断片、細菌、毒素、ＣＥＡやＡＦＰのような腫瘍マーカーを分析するための抗体を含む試薬を示すことができる。あるいはウイルス粒子、細菌、あるいは毒素等に対する抗体を測定するための抗原を含む試薬をこの免疫学的活性成分を含む試薬の範疇に入る。これら免疫学的な活性成分は、ラジオアイソトープ、酵素、補酵素、酵素基質、発光物質、あるいは蛍光物質等の標識成分と結合していても良い。またラテックス粒子、金属コロイド粒子、試験管の内壁やビーズ等の固相に結合した状態であっても良い。
更にこれらの測定対象成分の含有量をあらかじめ検定した標準物質も含めて、本発明における試薬とする。標準物質は、微生物の繁殖し易い血清をベースとすることが多く特に抗菌剤の添加が重要である。血清をベースとする試薬では、蛋白濃度が高いうえに微生物の繁殖に必要なその他の栄養素も豊富に存在しているので微生物にとってはかっこうの繁殖場所になる。それだけに、この種の抗菌剤の効果が大きく現れる。
【００３９】
免疫学的活性物質を含む試薬の中でも、トリヨードサイロニンやサイロキシンは本発明によって特別な効果が期待できる測定項目の一つである。これらの甲状腺ホルモンは、血中でＴＢＧ（Ｔｈｙｒｏｘｉｎｅ Ｂｉｎｄｉｎｇ Ｇｌｏｂｕｌｉｎ）やアルブミン等の血清蛋白と結合した状態にあるものと、遊離の状態で存在するものとが知られている。両者は生体内で微妙な平衡状態にあり、このうち遊離の状態にあるものがホルモンとしての生理活性を持っている。そしてこの遊離型のホルモンの測定が、内分泌機能の診断において有用な情報となる。遊離の状態にあるホルモンは、結合状態にあるものとの平衡をくずさないように測定しなければ臨床的に意味のある測定値とならない。本発明者らが確認したところによれば、いくつかの抗生物質はこの平衡状態に影響を与え、遊離分画の測定値を実際よりも大きくしてしまう。本発明が提案する抗菌剤は、このような測定系において測定値に与える影響が小さく、遊離の状態にあるホルモンの免疫学的な測定用試薬や、標準物質を微生物の繁殖から保護するために有用である。
【００４０】
本発明者らは、このような特殊な測定項目において血清蛋白との結合率が５０％以下である抗菌剤が有用であることを見出した。したがって、本発明は前記のような特定構造のキノロン系抗生物質の試薬用防腐剤としての用途を提供するのみならず、血清蛋白との結合率が５０％以下である抗菌剤の、遊離型リガンド測定用試薬のための抗菌剤としての新しい用途を提供するものである。血清蛋白との結合率が５０％以下である抗菌剤の代表的なものがロメフロキサシンで、ヒト血清蛋白に対する結合率は２４．４％である。本発明における結合率が５０％以下の抗菌剤としては、次のような抗菌剤を挙げられる［２１］［２２］。これら血清蛋白との結合率が５０％以下である抗菌剤は、遊離型リガンドの測定値に影響を与えにくい優れた化合物である。

なお薬剤の血清蛋白との結合率とは、薬物動態を把握する上で重要な情報である。そのため抗菌剤としてヒトへの投与を目的とする化合物であれば少なくともヒト血清蛋白に対する結合率が公知となっている。試薬用の抗菌剤として用いるときには、ヒト以外の動物血液が試料となる可能性、あるいは測定試薬成分や標準試薬の媒体としてヒト以外の血清を含む可能性等が考えられる。このような場合には、ヒト血清蛋白との結合率ではなく想定される血液蛋白の結合率を求めることも可能である。血清蛋白結合率を求める方法は公知である［２２］。
【００４１】
なおこのような公知の方法によって求めることができる血清蛋白結合率は、血清中に含まれる多様な蛋白質のどの蛋白質と結合するのかは問題としておらず、単にある化合物がどのていど血清蛋白と結合するのかを求めているに過ぎない。したがって、特定の遊離型リガンドの測定値に与える影響をこのような薬物動態を示す一般的な数値から推測することは困難である。本発明者らは、新たに見出した遊離リガンドの測定値に影響を与えにくい抗菌剤の血清蛋白結合率を公知の抗菌剤のものと比較分析し、特定の血清蛋白結合率を境に測定値への影響が無視できるていどに抑制できることを確認し本発明を完成した。
【００４２】
一方、酵素を含む試薬とは、グルコース、コレステロール、トリグリセライド、クレアチニンといった各種酵素基質を酵素反応によって測定するための試薬や、アミラーゼ、ＬＤＨ、γＧＴＰ、ＧＯＴ、ＧＰＴといった酵素の活性を測定するためにその基質や共役酵素を含む試薬等を例示できる。酵素や基質についても免疫学的な活性物質と同じように含有量をあらかじめ検定した標準物質が求められるので、抗菌剤の添加が必要なことに変わりはない。
【００４３】
ところで微生物学的な分析技術の一つに、薬剤感受性試験と呼ばれる分析方法が有る。単離した微生物を抗菌剤の存在下で培養し、その培養成績を基に抗菌剤に対する感受性を決定するための分析技術である。この分析技術においては培養用の栄養素や増殖指示薬といった試薬成分と抗菌剤の組み合わせが利用されている。しかしこれらの試薬成分は微生物によって消費される成分であって、微生物の増殖から保護すべき成分とは言えないので、本発明における試薬とは明瞭に区別される。
【００４４】
本発明が提供する試薬は、抗菌力が優れているので従来用いられていた抗菌成分の添加を省略することができる。たとえば製造工程において爆発性の金属アジドを生成するおそれの有るアジ化ナトリウム、有機水銀を含むため利用を避けたいチメロサールのような公知の抗菌剤はもはや添加の必要はないし、本発明の利点を生かすために添加するべきではない。
また試薬の反応を妨害するおそれの有る抗菌剤も不要とする。たとえば遊離サイロキシンの測定を大きく妨害するＰＳＦは、本発明の試薬によりもはや添加の必要はない。
【００４５】
本発明で抗菌剤として添加する特定のキノロン系製剤は、主として細菌やマイコプラズマに対して有効な薬剤である。したがって糸状菌に対しては抗菌活性が不足する場合が考えられる。このような場合には、アンホテリシンＢ（以下ＡＭＰＨ−Ｂと省略する）の添加が有効である。ＡＭＰＨ−Ｂはファンギゾンとも呼ばれＰＳＦに含有されている抗真菌性の化合物であるが、本発明による特定のキノロン系製剤と組み合わせた場合には遊離サイロキシン測定の妨害作用を示すこともなく、好ましい組み合わせとして示すことができる。ＡＭＰＨ−Ｂは、およそ０．５〜３０μｇ／ｍｌの濃度で添加すると十分な抗真菌活性を期待できる。
ＡＭＰＨ−Ｂの他に同様の抗真菌活性を期待できる化合物として、ナイスタチン、フルシトシン、ミコナゾール、およびイトラコナゾール等を示すことができる。これらの抗真菌活性を持つ化合物のうち、フルシトシンは血清蛋白とほとんど結合せず本発明によって提供される遊離型リガンド測定用試薬に添加する場合に有利である。
【００４６】
【作用】
本発明に用いる特定のキノロン系抗菌剤は、本来試薬に期待されている反応性に有害な影響を与えることなく、微生物に分解されやすい試薬成分を微生物の繁殖から守る。しかも非常に微生物の繁殖し易い血清ベースの試薬においてさえ十分な抗菌活性をもたらす。これらの抗菌剤は、免疫学的活性成分、酵素的に活性な成分といった試薬中に含まれる各種の活性成分を保存中に変性させる事もない。
【００４７】
キノロン系の抗生物質はＤＮＡジャイレースという核酸合成に関与する酵素を阻害する事によって抗菌活性を示す化合物である。この酵素は現在利用されている臨床検査用試薬には使われておらず、また分析対象ともなっていないので反応系に影響の無い抗菌剤として利用する事ができる。
ただし、同様の機構によって抗菌活性を示す化合物であれば、試薬用の抗菌剤として何でも応用可能ということではない。薬剤の生理活性は非常に複雑であり一面的に捉える事はできない。実際、同じキノロン系の薬剤であっても副作用や抗菌性能、あるいは溶解性といった本来の抗菌活性以外の特性も総合的に考慮したうえで開発が進められている事は言うまでもない。このような配慮は試薬用の抗菌剤として利用するときにも当然配慮されるべき事柄である。もちろん、治療用の判断基準と試薬用の抗菌剤として用いるときに配慮すべき事項とは全く異なる。たとえば治療用に用いるときにもっとも配慮の必要な点は安全性である。しかし試薬においては、安全性よりもむしろ試薬の反応に影響を与えない事が最優先されるべき事項である。本発明者らは治療用に開発されている各種キノロン系薬剤について、試薬用の抗菌剤としての有用性を見出したのである。
【００４８】
【発明の効果】
本発明は、新しい抗菌剤を含む試薬を提供する。本発明における抗菌剤は、試薬中の微生物の繁殖を効果的に抑制する。また免疫学的な試薬から酵素学的な試薬にいたる幅広い種類の反応性を持つ様々な試薬において、本来の反応性に大きな影響を与えることがない。更に、製造上も、環境衛生上も問題となりにくい安全性の高い抗菌剤である。本発明のキノロン剤によって期待できる利点について、いくつかの例を具体的に示す。
まず、アジ化物によって酵素活性を阻害されるペルオキシダーゼは本発明の抗菌剤によって影響を受けない。アジ化ナトリウムのように製造工程で爆発性の化合物を生じない。ＰＳＦによって測定値に影響の出る遊離サイロキシンは、本発明のキノロン剤の存在下では測定値にほとんど影響を受けない。
【００４９】
加えて本発明に用いる特定のキノロン系抗菌剤は、これまでに用いられていた各種抗菌剤に比べて高い濃度で用いても反応性に影響しにくいので、高濃度で用いる事が可能であり、耐性菌の出現防止に有効である。特に抗菌剤によって誤差を生じやすい遊離の状態にあるホルモンの免疫学的な反応に基づく測定系において、本発明による抗菌剤は測定値に影響を与えにくい優れた抗菌剤と言うことができる。以下、実施例によって本発明をより具体的に説明する。
【００５０】
【実施例】
１．キノロン系抗菌剤のヒト血清中における抗菌効果
塩酸ロメフロキサシン（北陸製薬製、商品名「バレオン」、以下ＬＦＬＸと記す）、およびスパルフロキサシン（大日本製薬製、商品名「スパラ」、以下ＳＰＦＸと記す）を用い、ヒト血清中における抗菌効果について調査した。ＬＦＬＸは１ＮのＮａＯＨでいったん溶解後に、ＳＰＦＸはそのまま蒸留水で溶解して目的の濃度とした。比較対照として、ストレプトマイシン硫酸塩（和光純薬工業製、以下ＳＭと記す）、およびペニシリンＧカリウム（和光純薬工業製、以下ＰＣＧと記す）を蒸留水で目的濃度に希釈して用いた。
【００５１】
以下の濃度（最終濃度）になるように、２００μｌのヒトプール血清に５０μｌの各種抗菌剤溶液、および５０μｌの微生物混濁液を添加した。実験に用いた微生物は次の３菌種である。各菌数を測定後、トータルで１，０００，０００ Ｃｅｌｌｓ／ｍｌとなるように等菌数混合して微生物混濁液とした。抗菌剤を添加した６種類の試験材料（プール血清）は、３７℃のインキュベーター中で１週間保存した。保存後の各試験材料について、６５０ｎｍにおける吸光度を測定し無菌の状態の同じプール血清の吸光度を０としたときの吸光度が０．１以上のものを白濁と判定し、抗菌剤の効果を確認した。結果は表１に示した。

【００５２】
【表１】

【００５３】
表１から、抗菌剤を加えない場合２４時間で（条件１）、アジ化ナトリウム（条件２）、ＳＭ（条件３）、ＰＣＧ（条件４）をそれぞれ加えた場合４８時間で血清の白濁が認められ、細菌の増殖が確認された。これに対して本発明による抗菌剤を加えた場合（条件５、６）では、３７℃で６日間放置後も血清の白濁は認められず、この時点で細菌の増殖していないことが確認された。
【００５４】
２．各種測定系への本抗菌剤の影響
各抗菌剤の、ＲＩＡへの影響について調査した。測定項目としてはＩｇＥを選び、実験に用いた抗菌剤と使用濃度は以下のとおりである。

本実施例では、標識抗体、および検体に抗菌剤を添加した。試験管に、抗菌剤をこの濃度となるように添加した血清検体２０μｌ（または標準物質）、抗ヒトＩｇＥ抗体固相化ビーズ、やはりこの濃度で各抗菌剤を添加した^１２５Ｉ標識抗ヒトＩｇＥ抗体（３００，０００ｃｐｍ）１００μｌを混和し、２５℃で２時間インキュベーション後に試験管中の未反応標識抗体を生理食塩水で洗浄した。最終的に固相化ビーズに結合した^１２５Ｉ標識抗ヒトＩｇＥ抗体の放射活性をガンマーカウンターで測定した。結果は表２に示した。この値は二重測定で１回測定してデータの平均値をとったものである。ＲＩＡの場合にはこれらの抗菌剤が測定系に影響を与えないことが確認できた。
【００５５】
【表２】

【００５６】
３．化学発光分析系
本実施例では、検体中に抗菌剤を添加した。抗ＣＥＡ抗体を固定した反応カップに血清検体６０μｌ、希釈緩衝液２００μｌ、７分反応後ＰＯＤ標識抗ヒトＣＥＡモノクローナル抗体４０μｌを添加して２５℃で７分間インキュベートし、反応液を除去した。反応カップを希釈緩衝液で５回洗浄し、Ｈ_２Ｏ_２と発光試薬を各１００μｌ加えて発光値（３秒間の積算値）を計測した。Ｈ_２Ｏ_２は０．１Ｍのリン酸緩衝液（ｐＨ８．０）で３０％Ｗ／Ｖに希釈したものを用いた。また発光試薬は、ルミノール２２０ｍｇ、ｐ−ヨードフェノール１１ｍｇを同じリン酸緩衝液に溶解したものを用いた。測定結果を表３に示した。この測定値は二重測定で１回測定してデータの平均値をとったものである。
化学発光分析系では、本発明による抗菌剤を検体に添加する限り測定値に影響を与えないことが確認できた。一方これまで用いられていたアジ化ナトリウムでは測定値の低下が観察された。ＰＯＤ標識抗体が検体と接触している間に酵素活性が影響を受け、検体を除いた後に酵素反応を行わせているのにも関らず測定値が低下しているものと考えられる。
【００５７】
【表３】

【００５８】
４．遊離サイロキシン測定系への影響
遊離サイロキシンの免疫学的な測定に与える各種抗菌剤の影響を調査するために、以下のような実験を行った。利用した抗菌剤と使用濃度（検体中の濃度）は次のとおりである。

反応は、上記濃度の抗菌剤を含む検体２０μｌ、^１２５Ｉ標識ＨＣＧ結合サイロキシン溶液５０μｌ、抗サイロキシン・ウサギ血清含有抗血清５０μｌ、抗ウサギＩｇＧ抗体結合ビーズ１コを混和し、３７℃水浴中で２時間反応させた。反応後に未反応試薬を洗浄後、γカウンタ−でビーズに結合した標識抗原のカウントを測定した。
ＨＣＧ結合サイロキシンは、抗サイロキシン抗体との免疫学的な結合活性を持つ一方で、生体内における結合パートナーであるＴＢＧ等とはＨＣＧによる修飾のために結合できない。このような特殊な標識抗原を利用することによって遊離サイロキシンと、ＴＢＧ等と結合した状態に有るサイロキシンとの平衡を乱すことなく遊離サイロキシンの測定が可能となる。各検体は１０重測定を行い、その測定値の平均値を表４に示す。
ＳＭやＰＣＧを加えた場合に測定値が大きくなり、抗菌剤の添加によって誤差が生じる場合のあることが確認された。本発明の抗菌剤であるＬＦＬＸではこのような大きな誤差は生じておらず、測定系への影響が小さいことは明らかである。このような現象は、ＰＣＧ等の抗菌剤がＴＢＧやアルブミンと結合したサイロキシン（本実施例では測定対象ではない）と、遊離の状態で存在するサイロキシン（本実施例における測定対象）との間に成立している微妙な平衡関係に影響を与えたためと推測される。ＰＣＧは血清蛋白との結合率が５３％（超遠心法）〜５６％（透析平衡法）の化合物［２２］であるのに対して、ＬＦＬＸは２４．４％であり結合率が５０％を越える場合に無視できない大きさの誤差の原因につながることが確認された。。
【００５９】
【表４】

【００６０】
５．遊離サイロキシン測定系への影響−２−
遊離サイロキシンの免疫学的な測定に与える各種抗菌剤の影響を調査するために、以下のような実験を行った。利用した抗菌剤と使用濃度（検体中の濃度）は次のとおりである。ｐ−ヒドロキシ安息香酸エチル（表中にＥｔｈｙｌｐａｒａｂｅｎで示した）、あるいはｐ−ヒドロキシ安息香酸メチル（表中にＭｅｔｈｙｌｐａｒａｂｅｎで示した、いずれも和光純薬工業製）とキノロン剤の組み合わせは、試薬用の防腐剤として過去に提案された［ ２］ものである。
抗菌剤の組み合わせを変更した他は４と同じ操作により遊離サイロキシン測定系への影響を観察した。結果は表５に示すとおりである。
公知の抗菌剤の組み合わせであるｐ−ヒドロキシ安息香酸エチル（あるいはメチル）を利用した場合に測定値が小さくなることが確認された。本発明による抗菌剤であるＬＦＬＸが４ではるかに高い濃度で用いているのにもかかわらず測定値に影響を与えないのに対して、ｐ−ヒドロキシ安息香酸エチル等を組み合わせると大きな誤差の原因となることが確認された。４と同じように、これらの薬剤が遊離サイロキシン−ＴＢＧ結合サイロキシンとの平衡関係に影響を与えたものと推測された。

【００６１】
【表５】

【００６２】
６．酵素学的な測定系への影響
酵素学的な測定系への影響を観察するため、コレステロールとトリグリセライドの酵素学的な反応による測定結果に与える各種抗菌剤の影響を調査した。コレステロールの測定にはコレステザイム−Ｖ５５５’栄研’（栄研化学製、登録商標）を、またトリグリセライドの測定にはトリグリザイム−６００’栄研’（栄研化学製、登録商標）を用いた。本実施例では以下の濃度の抗菌剤を添加したヒト血清検体を試料として用意した。対照として各抗菌剤を溶解した溶媒のみを添加した血清をそれぞれ測定した。測定操作は試薬キットの指示書にしたがい、各検体を２重測定してその平均値を求めた。
【００６３】

【００６４】
結果は表６に示すとおりである。表５における測定値は、試料によって最終的な希釈率が異なる場合が有るので容量補正を施した後の値である。また表中に「測定値／対照」の数値を％で示した。本発明による抗菌剤の添加によって、酵素学的な反応による測定結果は影響を受けていないことが確認できた。
なお各薬剤の溶解に用いた溶媒、すなわちＮａＯＨ（０．０５Ｎと０．００５Ｎ）、精製水、ジメチルスルホキシド（０．００５％）＋ＮａＯＨ（０．０５Ｎ）、およびジメチルスルホキシド（０．０００５％）＋ＮａＯＨ（０．００５Ｎ）のみを添加したものについても測定値の変化を観察したが、これらの溶媒添加による影響は確認できなかった。
【００６５】
【表６】

【００６６】
引用文献のリスト
［ １］ 特公平７−７８００５
［ ２］ 特開平１−２８３２２３
［ ３］ 特開平７−６３７５２
［ ４］ 特開平８−４３３８５
［ ５］ ＵＳＰ ３７９９９３０ ；１９７４年
［ ６］ Ａｎｔｉｍｉｃｒｏｂｉａｌ Ａｇｅｎｔｓ ＆ Ｃｅｍｏｔｈｅｒａｐｙ． １８（１）：３７−４０，１９８０ Ｊｕｌ
［ ７］ ＵＳＰ ４４９９０９１ ；１９８５
［ ８］ ＲＥＶＩＥＷＳ ＯＦ ＩＮＦＥＣＴＩＯＵ ＤＩＳＥＡＳＥＳ，１９８９，Ｖ１１，Ｓ５，ＰＳ９１２
［ ９］ 特開昭６３−１０７９９０
［１０］ 「ニューキノロン剤の正しい使い方」 −医薬ジャーナル社 藤井良知 島田 馨編ｐ２９０
［１１］ ｓｔｒｕｃｔｕｒｅ−ａｃｔｉｖｉｔｙ ｒｅｌａｔｉｏｎｓｈｉｐ ｉｎ ｑｕｉｎｏｌｏｎｅ ａｎｔｉｂａｃｔｅｒｉａｌｓ：Ｄｅｓｉｇｎ，ｓｙｎｔｈｅｓｉｓ ａｎｄ ｂｉｏｌｏｇｉｃａｌ ａｃｔｉｖｉｔｉｅｓ ｏｆ ｎｏｖｅｌ ｉｓｏｔｈｉａｚｏｌｏｑｕｉｎｏｌｏｎｅｓ．Ｄｒｕｇｓ Ｅｘｐｔｌ．Ｃｌｉｎ．Ｒｅｓ．ＸＩＶ（６）．３７９−３８３ １９８８．
［１２］ Ａｎｔｉｍｉｃｒｏｂ． Ａｇｅｎｔｓ Ｃｈｅｍｏｔｈｅｒ．，３３，１１６７−１１７３（１９８９）
［１３］ 特開昭６３−１０４９７４
［１４］ ５−Ａｍｉｎｏ−７−（３−ａｍｉｎｏ−１−ｐｙｒｒｏｌｉｄｉｎｙｌ）−１−ｃｙｃｌｏｐｒｏｐｙｌ−６，８−ｄｉｆｌｕｏｒｏ−１，４−ｄｉｈｙｄｒｏ−４−ｏｘｏ−３−ｑｕｉｎｏｌｉｎｅｃａｒｂｏｘｙｌｉｃ ａｃｉｄ（ＰＤ １２４８１６）：ｓｙｎｔｈｅｓｉｓ ａｎｄ ｂｉｏｌｏｇｉｃａｌ ｅｖａｌｕａｔｉｏｎ ｏｆ ａ ｎｅｗ ｃｌａｓｓ ｏｆ ｑｕｉｎｏｌｏｎｅ ａｎｔｉｂａｃｔｅｒｉａｌｓ．Ｄｒｕｇｓ Ｅｘｐ Ｃｌｉｎ Ｒｅｓ １４：４５３，１９８８
［１５］ Ｄｉａｇｎｏｓｔｉｃ Ｍｉｃｒｏｂｉｏｌｏｇｙ ＆ Ｉｎｆｅｃｔｉｏｕｓ Ｄｉｓｅａｓｅ． １４（３）：２４５−５８，１９９１ Ｍａｙ−Ｊｕｎ
［１６］ 特開昭５９−６７２６９
［１７］ 「ニューキノロン」三橋 進編 学会出版センター ｐ５７
［１８］ Ｌ．Ａ．Ｍｉｔｓｃｈｅｒ ｅｔ ａｌ．：ｉｎｔｅｒｎａｔｉｏｎａｌ ｔｅｌｅｓｙｍｐａｓｉｕｍ ｏｎ Ｑｕｉｎｏｌｏｎｅｓ Ｐ．Ｂ．Ｆｅｒｎａｎｄｅｓ．Ｊ．Ｒ．Ｐｒｏｕｓ Ｓｃｉｅｎｓｅ，Ｓ．Ａ．，ｐｐ３−２０（１９８９）
［１９］ 特開平１−１００１６６
［２０］ Ａｎｔｉｍｉｃｒｏｂ．Ａｇｅｎｔｓ Ｃｈｅｍｏｔｈｅｒ．３１（６）：８５４−８５９，１９８７
［２１］ 医薬ジャーナル ＶＯＬ．３１，Ｓ−１，Ｐ１３５−１４３，１９９５
［２２］ Ａｎｔｉｂｉｏｔｉｃｓ ｉｎ Ｌａｂｏｒａｔｏｒｙ Ｍｅｄｉｃｉｎｅ ２ｎｄ ｅｄ． Ｃ．１３ ｐ．４７７−５１４（１９８６）[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a biological component measuring reagent used in fields such as clinical examinations. The reagent for measuring biological components is composed of enzymes necessary for analysis, immunologically active components such as antigens and antibodies, or proteins such as serum proteins containing these components. The standard substances necessary for various types of analysis are composed of serum whose substance concentration has been tested and those obtained by adding various components to the serum so that the concentration is constant.
[0002]
[Prior art]
Many of the reagents used in fields such as clinical tests contain proteins such as antibodies and enzymes, or biological components such as sugars and vitamins. For example, reagents for immunological analysis include antigens and antibodies. When an enzyme label is used, an enzyme and a compound serving as a substrate thereof are also included. In addition, in the case of a reagent for enzymatic analysis, an enzyme and an enzyme substrate are present in the reagent. Further, in the analysis, a standard substance is required for preparing a calibration curve and confirming the analysis accuracy. The standard substance is constituted by adding serum to which the content of the compound to be analyzed is assayed or a certain amount of the analyte to be added to the serum. These biological components are utilized as microbial nutrients and cause microbial growth.
Propagation of microorganisms in the reagent is a problem that must be avoided because it not only degrades the quality of the reagent itself by decomposing components but also causes contamination of the analysis environment. However, it is not easy to completely protect reagents and reference materials containing high concentrations of biological components from the growth of microorganisms.
[0003]
Many compounds (hereinafter referred to as antibacterial agents) that inhibit the growth of bacteria and fungi are known, but the following precautions are necessary to add them to the reagent. First, care must be taken not to interfere with the reactivity expected of the reagent. A reagent is for performing an analysis using some kind of reaction, whether it is immunological or enzymatic. Therefore, antimicrobial agents that inhibit this reaction should not be used.
[0004]
For example, azide that has been added to reagents as an antibacterial agent for a long time hardly affects the immunological reaction, but depending on the concentration used, it is known to have a significant effect on the enzyme reaction, especially the activity of peroxidase. It has been. Since peroxidase is an enzyme that is widely used for the measurement of hydrogen peroxide produced by a labeling enzyme or oxidase for binding analysis, an antibacterial agent that inhibits its activity is often inconvenient when added to a reagent. In addition to this, azide is known to act as an inhibitory action on ascorbate oxidase. Ascorbate oxidase is an enzyme that is used as an enzyme that oxidizes and removes ascorbic acid, which is an interfering substance in the color development system of hydrogen peroxide by peroxidase, and antibacterial agents that act inhibitoryly on this enzyme should avoid combinations .
[0005]
There are also attempts to use various antibiotics instead of azides. For example, a mixed solution of penicillin G-streptomycin-fungizone (hereinafter abbreviated as PSF, commercially available as Penicillin-Streptomycin-Fungizone-Mixture from Wako Pure Chemical Industries, Ltd.) was used as an antibacterial agent for the reagent. However, the antibacterial activity of PSF may not always be sufficiently maintained in an environment in which bacteria are likely to grow, such as a standard product based on serum. In addition, PSF has no antibacterial activity against mycoplasma which is often brought into the experimental environment in the state of being infected with animal blood. Furthermore, according to the knowledge of the present inventors, it became clear that some reactions are affected in the presence of PSF. Specifically, for example, in the immunological measurement of free thyroxine, it was observed that the measured value may increase in the presence of PSF.
[0006]
In order to avoid such problems, the presently used antibacterial agents for reagents currently use various compounds according to the usage of the reagents. However, it is not easy to select an antibacterial agent suitable for each of a wide range of reagents composed of very diverse components.
On the other hand, some of the reagents currently on the market cannot be limited to specific analytes. This is the case, for example, for multi-item standards that test many components simultaneously. Since the multi-item standard is used as a standard in the analysis of many items, there is a high possibility that a completely different reaction system is applied to each item. Specifically, substance A may be a standard for RIA (radioimmunoassay), but substance B may also be used as a standard for ELISA (enzyme immunoassay). In such a case, an antibacterial agent that is not a problem in RIA may interfere with the enzyme reaction in ELISA. Therefore, an antibacterial agent that can be applied to as wide a range of reagents as possible is required.
[0007]
In addition, the antimicrobial agent added to the reagent must not denature the components in the reagent during storage. There may be various activities and components having various structures such as immunologically active substances, enzymatically active substances, and chemically active substances in the reagent. Those that may denature the activity of these compounds during storage are not available.
[0008]
Further, there may be disadvantages depending on the properties of the compound itself added as an antibacterial agent. For example, thimerosal, which was often used in place of azide, has become obsolete due to environmental health problems because it contains organic mercury in its structure.
In addition, it is known that sodium azide, which is a typical azide, reacts with metals such as copper and lead to produce explosive metal azides. In the waste liquid at the terminal inspection facility, the concentration of sodium azide is low, so the danger is small, but it is a problem that cannot be ignored in manufacturing facilities that may always come into contact with a relatively high concentration of sodium azide. is there.
[0009]
Finally, the most important thing for antibacterial agents is to maintain sufficient antibacterial activity under the environment in which they are used. Since reagents provide various reactions, the environment such as pH, salt concentration, and solvent fluctuates greatly. Therefore, it is required for the reagent antibacterial agent to maintain sufficient antibacterial activity under various conditions. In the background as described above, several combinations useful as reagent antibacterial agents have been reported.
[0010]
As one of the new antibacterial agents, 2-methyl-4-isothiazolin-3-one-hydrochloride, 2-hydroxypyridine-N-oxide, chloroacetamide, {N, N-methylene-bis [(N-1- Hydroxymethyl) -2,5-dioxo-4-imidazolidinyl]}-urea and 5-bromo-5-nitro-1,3-dioxane have been proposed [1]. A combination of a specific arylfluoroquinolone and a p-hydroxybenzoic acid derivative is also known [2]. In addition, there have been reports of attempts to combine sulfamethoxazole and trimethoprim [3], or to apply microsides [4].
These reports have been proposed for the purpose of solving the above problems. However, for the reasons mentioned above, it is preferable to select a wide range of antibacterial agents in order to support various reaction systems, and the continuous use of specific antibacterial agents is the emergence of resistant bacteria. There is a possibility to lead to.
Furthermore, the present inventors have observed that the combination of these antibacterial agents utilizing a p-hydroxybenzoic acid derivative [2] has a significant influence on the reaction system in some immunological measurement systems. It was confirmed that there is a case. That is, when a combination of an arylfluoroquinolone and a p-hydroxybenzoic acid derivative is added as an antibacterial agent to the immunoassay standard for free-type thyroxine, compared with the case where the binding rate between the label and the antibody is not added. May decrease to 1/3 or less. Such antibacterial agents cannot be used at least as reagents or standards for specific measurement items.
[0011]
[Problems to be solved by the invention]
The present invention provides a new antibacterial agent that can be applied to a wide range of reagents, exhibits a sufficient antibacterial activity against various microorganisms under various environments, and hardly causes the appearance of resistant bacteria. It is an issue. It is an object of the present invention to effectively prevent the growth of microorganisms in the reagent and protect the active ingredients in the reagent from the growth of microorganisms without adversely affecting the original performance of the reagent.
[0012]
[Means for Solving the Problems]
An object of the present invention is to add at least one drug selected from quinolone antibiotics having the following structure:And does not contain a p-hydroxybenzoic acid derivativeIt is solved by the reagent for measuring biological components. The following quinolone antibiotics may be salts.
[0018]
Compound1
[Chemical 7]

[0023]
Compound2
[Chemical 8]

[0024]
DETAILED DESCRIPTION OF THE INVENTION
The quinolone antibiotics used in the present invention are compounds having the above-mentioned structure and the following general names, respectively. Many of the quinolone antibiotics used in the present invention are so-called fluoroquinolone antibiotics containing fluorine in the structure. Although these quinolone antibiotics were originally developed for the treatment of various infectious diseases, the present inventors had many problems as described above when these antibacterial agents were added to the reagents. As a result, the present invention was completed. Hereinafter, each antibacterial agent will be described.
[0030]
Compound1: Sparfloxacin
Sparfloxacin (5-amino-1-cyclopropyl-6, 8-difluoro-7- (cis-3, 5-dimethyl-1-piperazinyl) -4 (1H) -oxoquinoline-3-carboxylic acid) [ 12] is a tricyclic quinolone synthetic antibacterial agent having an amino group at the 5-position of the oxoquinoline skeleton, fluorine at the 6- and 8-positions, and a 3,5-dimethylpiperazinyl group at the 7-position. It has antibacterial activity against a wide range of microorganisms such as pneumococci, Staphylococcus aureus including MRSA, gram positive bacteria, gram negative bacteria, mycoplasma, chlamydia, tuberculosis and the like, and its action is bactericidal. It is said that the antibacterial activity belongs to the strongest class among quinolones.
[0035]
Compound2: Lomefloxacin
Lomefloxacin ((±) -1-ethyl-6, 8-difluoro-1, 4-dihydro-7- (3-methyl-1-piperazinyl) -4-oxo-3-quinolinecarboxylic acid) [20] It is a synthetic quinolone antibacterial agent belonging to tricyclic quinolone. It exhibits a broad antibacterial spectrum against gram positive bacteria, negative bacteria, and anaerobic bacteria, and its action is bactericidal. On the acidic side, the antibacterial activity tends to decrease. It is mainly distributed as hydrochloride.
[0036]
The compounds as described above are easily available as long as they are commercially available. Even those not commercially available can be synthesized based on a method for producing a compound having a similar structure.
[0037]
These antibacterial agents may be added alone, or several types may be mixed and added. If several types are mixed and used, an effect that resistant bacteria hardly appear can be expected. Whether used alone or mixed, the preferred use concentration of these quinolone antibiotics in the reagent is 0.0005% W / V (5 μg / ml) or more. When the concentration is lower than this concentration, the antibacterial activity may be insufficient particularly in a reagent based on serum that is easy to propagate microorganisms. In addition, in order to ensure sufficient antibacterial activity against a wider range of microorganisms and to realize an environment in which resistant bacteria hardly appear, 0.001-0.01% W / V (10-100 μg / ml) Use within a range. Even if the antibacterial agent of the present invention is used at such a very high concentration, it is unlikely to affect the immune reaction or the enzyme reaction. For example, in the case of lomefloxacin, it has been confirmed that the reaction does not affect the reaction up to 0.05% W / V (500 μg / ml) in RIA, but it may act to inhibit some enzyme reactions. Too high concentrations may be available only in limited reaction systems.
Basically, the quinolone antibacterial agent used in the present invention hardly affects various reaction systems even when used at a high concentration. Therefore, it does not mean that it should not be used at high concentrations. However, an unnecessarily high concentration is not preferable because it leads to an increase in raw material costs. Also, even if it is used at a concentration exceeding the solubility, an effect according to the amount added cannot be expected, so it is efficient to use at this level of concentration.
[0038]
In the present invention, the reagent to which the antibacterial agent can be applied is a reagent containing a component to be protected from the growth of microorganisms. Specific examples include those containing enzymes, those containing immunologically active substances such as antigens and antibodies, and those containing serum proteins.
Reagents containing immunologically active substances such as antigens and antibodies include triiodothyronine, thyroxine, thyroid stimulating hormone, ovarian stimulating hormone, luteinizing hormone, chorionic gonadotropin, various hormones such as insulin, interferon, interleukin, macrophage Reagents including antibodies for analyzing lymphokines such as migration inhibitory factor and granulocyte colony stimulating factor, virus particles and fragments thereof, bacteria, toxins, tumor markers such as CEA and AFP can be shown. Alternatively, a reagent containing an antigen for measuring an antibody against virus particles, bacteria, toxins or the like falls into the category of a reagent containing this immunologically active component. These immunologically active components may be bound to labeling components such as radioisotopes, enzymes, coenzymes, enzyme substrates, luminescent substances, or fluorescent substances. Further, it may be in a state of being bound to a solid phase such as latex particles, metal colloid particles, an inner wall of a test tube or beads.
Furthermore, the reagents in the present invention are included, including standard substances that have been tested in advance for the contents of these components to be measured. The standard substance is often based on serum in which microorganisms easily propagate, and the addition of an antibacterial agent is particularly important. Serum-based reagents are high breeding sites for microorganisms because of their high protein concentration and the abundance of other nutrients necessary for the growth of microorganisms. For this reason, the effect of this type of antibacterial agent is significant.
[0039]
Among reagents containing immunologically active substances, triiodothyronine and thyroxine are one of the measurement items that can be expected to have a special effect according to the present invention. These thyroid hormones are known to be bound to serum proteins such as TBG (Thyroxine Binding Globulin) and albumin in blood and to exist in a free state. Both are in a delicate equilibrium state in the living body, and among these, those in a free state have physiological activity as hormones. This measurement of free hormone is useful information for diagnosis of endocrine function. A hormone in the free state will not be a clinically meaningful measurement unless it is measured so as not to break the equilibrium with the bound one. The inventors have confirmed that some antibiotics affect this equilibrium state, making the measured free fraction larger than it actually is. The antibacterial agent proposed by the present invention has a small influence on the measurement value in such a measurement system, and is intended to protect a reagent for immunological measurement of a hormone in a free state and a standard substance from the growth of microorganisms. Useful.
[0040]
The present inventors have found that an antibacterial agent having a binding rate with serum protein of 50% or less is useful in such special measurement items. Therefore, the present invention not only provides a use as a reagent preservative for quinolone antibiotics having the specific structure as described above, but also a free ligand of an antibacterial agent having a serum protein binding rate of 50% or less. It provides a new use as an antibacterial agent for measuring reagents. A typical antibacterial agent having a serum protein binding rate of 50% or less is lomefloxacin, and the binding rate to human serum protein is 24.4%. Examples of the antibacterial agent having a binding rate of 50% or less in the present invention include the following antibacterial agents [21] [22]. Antibacterial agents having a binding rate with these serum proteins of 50% or less are excellent compounds that hardly affect the measured value of the free ligand.

The binding rate of a drug with serum protein is important information for grasping pharmacokinetics. Therefore, as long as it is a compound intended to be administered to humans as an antibacterial agent, at least the binding rate to human serum proteins is known. When used as an antibacterial agent for a reagent, it is possible that non-human animal blood may be used as a sample, or that non-human serum may be included as a medium for a measurement reagent component or standard reagent. In such a case, it is also possible to obtain the expected blood protein binding rate rather than the human serum protein binding rate. Methods for determining serum protein binding rates are known [22].
[0041]
It should be noted that the serum protein binding rate that can be determined by such a known method does not matter which of the various proteins contained in the serum binds, and simply how a certain compound binds to the serum protein. I'm just asking what to do. Therefore, it is difficult to estimate the influence of a specific free ligand on the measured value from the general numerical value indicating such pharmacokinetics. The inventors of the present invention have compared the serum protein binding rate of antibacterial agents that are less likely to affect the newly found free ligand measurement values with those of known antibacterial agents, and measured values with a specific serum protein binding rate as a boundary. The present invention was completed by confirming that the influence on the temperature could be ignored and suppressed.
[0042]
On the other hand, an enzyme-containing reagent is a reagent for measuring various enzyme substrates such as glucose, cholesterol, triglyceride, and creatinine by an enzyme reaction, and an enzyme such as amylase, LDH, γGTP, GOT, and GPT. Examples include reagents containing substrates and conjugated enzymes. As with the immunologically active substance, a standard substance whose content has been tested in advance is required for the enzyme and the substrate, so that an antibacterial agent must be added.
[0043]
One microbiological analysis technique is an analysis method called a drug sensitivity test. This is an analytical technique for culturing an isolated microorganism in the presence of an antibacterial agent and determining the sensitivity to the antibacterial agent based on the culture results. In this analysis technique, a combination of a reagent component such as a nutrient for culture and a growth indicator and an antibacterial agent is used. However, since these reagent components are components consumed by microorganisms and cannot be said to be components that should be protected from the growth of microorganisms, they are clearly distinguished from the reagents in the present invention.
[0044]
Since the reagent provided by the present invention has excellent antibacterial activity, the addition of antibacterial components that have been conventionally used can be omitted. For example, known antibacterial agents such as sodium azide, which may produce explosive metal azides in the manufacturing process, and thimerosal which contain organic mercury and which should be avoided, no longer need to be added, taking advantage of the present invention Should not be added.
Also, an antibacterial agent that may interfere with the reaction of the reagent is unnecessary. For example, PSF, which greatly interferes with the measurement of free thyroxine, no longer needs to be added by the reagent of the present invention.
[0045]
The specific quinolone preparation added as an antibacterial agent in the present invention is a drug that is mainly effective against bacteria and mycoplasma. Therefore, antibacterial activity may be insufficient for filamentous fungi. In such a case, addition of amphotericin B (hereinafter abbreviated as AMPH-B) is effective. AMPH-B, also called fungizone, is an antifungal compound contained in PSF, but it is preferable since it does not interfere with the measurement of free thyroxine when combined with a specific quinolone preparation according to the present invention. Can be shown as a combination. When AMPH-B is added at a concentration of about 0.5 to 30 μg / ml, sufficient antifungal activity can be expected.
Nystatin, flucytosine, miconazole, itraconazole, and the like can be shown as compounds that can be expected to have similar antifungal activity in addition to AMPH-B. Of these compounds having antifungal activity, flucytosine hardly binds to serum protein and is advantageous when added to the reagent for measuring free ligand provided by the present invention.
[0046]
[Action]
The specific quinolone antibacterial agent used in the present invention protects the reagent components that are easily decomposed by microorganisms from the propagation of microorganisms, without adversely affecting the reactivity originally expected for the reagents. Moreover, it provides sufficient antibacterial activity even in serum-based reagents that are very prone to microbial growth. These antibacterial agents do not denature various active ingredients contained in reagents such as immunologically active ingredients and enzymatically active ingredients during storage.
[0047]
Quinolone antibiotics are compounds that exhibit antibacterial activity by inhibiting an enzyme involved in nucleic acid synthesis called DNA gyrase. This enzyme is not used in currently used clinical laboratory reagents and is not an analysis target, so it can be used as an antibacterial agent that does not affect the reaction system.
However, any compound that exhibits antibacterial activity by the same mechanism is not applicable as an antibacterial agent for reagents. The physiological activity of a drug is very complex and cannot be grasped in one aspect. In fact, it goes without saying that even the same quinolone drugs are being developed in consideration of characteristics other than the original antibacterial activity such as side effects, antibacterial performance, and solubility. Such consideration is a matter of course to be taken into consideration when using as an antibacterial agent for reagents. Of course, the judgment criteria for treatment and the matters to be considered when using them as antibacterial agents for reagents are completely different. For example, safety is the most important point to consider when using it for treatment. However, in the reagent, the priority should be given to not affecting the reaction of the reagent rather than safety. The present inventors have found usefulness of various quinolone drugs developed for treatment as antibacterial agents for reagents.
[0048]
【The invention's effect】
The present invention provides a reagent comprising a new antimicrobial agent. The antibacterial agent in the present invention effectively suppresses the growth of microorganisms in the reagent. In addition, various reagents having a wide variety of reactivity ranging from immunological reagents to enzymatic reagents do not significantly affect the original reactivity. Furthermore, it is a highly safe antibacterial agent that is unlikely to be a problem in terms of production and environmental hygiene. Some specific examples of the advantages expected from the quinolone agent of the present invention are shown below.
First, peroxidase whose enzyme activity is inhibited by azide is not affected by the antibacterial agent of the present invention. Does not produce explosive compounds in the manufacturing process like sodium azide. Free thyroxine whose measurement value is affected by PSF is hardly affected by the measurement value in the presence of the quinolone agent of the present invention.
[0049]
In addition, the specific quinolone antibacterial agent used in the present invention hardly affects the reactivity even when used at a higher concentration than various antibacterial agents used so far, and can be used at a high concentration. It is effective in preventing the appearance of resistant bacteria. In particular, in a measurement system based on an immunological reaction of a hormone in a free state that is likely to cause an error due to an antibacterial agent, the antibacterial agent according to the present invention can be said to be an excellent antibacterial agent that hardly affects the measured value. Hereinafter, the present invention will be described more specifically with reference to examples.
[0050]
【Example】
1. Antibacterial effects of quinolone antibacterial agents in human serum
Antibacterial effect in human serum using lomefloxacin hydrochloride (Hokuriku Pharmaceutical, trade name “Bareon”, hereinafter referred to as LFX) and sparfloxacin (Dainippon Pharmaceutical, trade name “SPARA”, hereinafter referred to as SPFX) Was investigated. LFLX was once dissolved in 1N NaOH, and SPFX was dissolved in distilled water as it was to obtain the desired concentration. As comparative controls, streptomycin sulfate (manufactured by Wako Pure Chemical Industries, Ltd., hereinafter referred to as SM) and penicillin G potassium (manufactured by Wako Pure Chemical Industries, Ltd., hereinafter referred to as PCG) were diluted with distilled water to a target concentration and used.
[0051]
50 μl of various antibacterial agent solutions and 50 μl of microbial turbid solution were added to 200 μl of human pooled serum so that the following concentrations (final concentrations) were obtained. The microorganisms used in the experiment are the following three species. After measuring the number of each bacterium, an equal number of bacteria was mixed so as to obtain a total of 1,000,000 cells / ml to obtain a turbid liquid. Six kinds of test materials (pool serum) to which an antibacterial agent was added were stored in a 37 ° C. incubator for 1 week. For each test material after storage, the absorbance at 650 nm was measured, and when the absorbance of the same pooled serum in a sterile state was 0, it was determined that the absorbance was 0.1 or more, and the effect of the antibacterial agent was confirmed. . The results are shown in Table 1.

[0052]
[Table 1]

[0053]
From Table 1, serum turbidity is observed in 24 hours when no antibacterial agent is added (condition 1), and when sodium azide (condition 2), SM (condition 3), and PCG (condition 4) are added, respectively. And the growth of bacteria was confirmed. On the other hand, when the antibacterial agent according to the present invention was added (conditions 5 and 6), no serum white turbidity was observed even after standing at 37 ° C. for 6 days, and it was confirmed that no bacteria were growing at this point. It was.
[0054]
2. Effect of this antibacterial agent on various measurement systems
The effect of each antibacterial agent on RIA was investigated. IgE was selected as the measurement item, and the antibacterial agents used in the experiments and the concentrations used were as follows.

In this example, an antibacterial agent was added to the labeled antibody and the specimen. 20 μl of serum specimen (or standard substance) to which antibacterial agent was added at this concentration, anti-human IgE antibody-immobilized beads, and each antibacterial agent were also added at this concentration to the test tube¹²⁵100 μl of I-labeled anti-human IgE antibody (300,000 cpm) was mixed, and after incubation for 2 hours at 25 ° C., unreacted labeled antibody in the test tube was washed with physiological saline. Finally bound to the immobilized beads¹²⁵The radioactivity of the I-labeled anti-human IgE antibody was measured with a gamma counter. The results are shown in Table 2. This value is obtained by taking a double measurement and taking an average value of the data. In the case of RIA, it was confirmed that these antibacterial agents did not affect the measurement system.
[0055]
[Table 2]

[0056]
3. Chemiluminescence analysis system
In this example, an antibacterial agent was added to the specimen. After reacting with 60 μl of serum sample and 200 μl of dilution buffer for 7 minutes in a reaction cup on which the anti-CEA antibody was immobilized, 40 μl of POD-labeled anti-human CEA monoclonal antibody was added and incubated at 25 ° C. for 7 minutes to remove the reaction solution. Wash the reaction cup 5 times with dilution buffer.₂O₂And 100 μl each of luminescence reagent were added, and the luminescence value (integrated value for 3 seconds) was measured. H₂O₂Was diluted with 0.1 M phosphate buffer (pH 8.0) to 30% W / V. As the luminescent reagent, luminol 220 mg and p-iodophenol 11 mg dissolved in the same phosphate buffer were used. The measurement results are shown in Table 3. This measurement value is obtained by taking a double measurement and taking an average value of the data.
In the chemiluminescence analysis system, it was confirmed that the measurement value was not affected as long as the antibacterial agent according to the present invention was added to the specimen. On the other hand, a decrease in the measured value was observed with sodium azide used so far. It is considered that the enzyme activity is affected while the POD-labeled antibody is in contact with the specimen, and the measured value is lowered despite the enzyme reaction being performed after the specimen is removed.
[0057]
[Table 3]

[0058]
4). Effect on free thyroxine measurement system
In order to investigate the effect of various antibacterial agents on the immunological measurement of free thyroxine, the following experiment was conducted. The antibacterial agents used and the concentrations used (concentrations in the specimen) are as follows.

The reaction is 20 μl of a sample containing the antibacterial agent at the above concentration,¹²⁵50 μl of I-labeled HCG-conjugated thyroxine solution, 50 μl of anti-thyroxine / rabbit serum-containing antiserum, and 1 anti-rabbit IgG antibody-bound bead were mixed and reacted in a 37 ° C. water bath for 2 hours. After the reaction, the unreacted reagent was washed, and the count of labeled antigen bound to the beads was measured with a γ counter.
HCG-binding thyroxine has an immunological binding activity with an anti-thyroxine antibody, but cannot bind to TBG or the like, which is a binding partner in vivo, due to modification by HCG. By using such a special labeled antigen, free thyroxine can be measured without disturbing the equilibrium between free thyroxine and thyroxine in a state of being bound to TBG or the like. Each sample was subjected to 10-fold measurement, and the average value of the measured values is shown in Table 4.
When SM or PCG was added, the measured value increased, and it was confirmed that an error may occur due to the addition of the antibacterial agent. Such a large error does not occur in LFRX, which is the antibacterial agent of the present invention, and it is clear that the influence on the measurement system is small. Such a phenomenon occurs between a thyroxine in which an antibacterial agent such as PCG is bound to TBG or albumin (not a measurement target in this example) and a thyroxine existing in a free state (a measurement target in this example). This is presumed to have been caused by the subtle equilibrium relationship that was established. PCG is a compound [22] with a serum protein binding rate of 53% (ultracentrifugation) to 56% (dialysis equilibrium method), whereas LFRX is 24.4% and the binding rate is 50%. It was confirmed that it would lead to an error of a size that cannot be ignored if exceeded. .
[0059]
[Table 4]

[0060]
5. Effect on free thyroxine measurement system-2-
In order to investigate the effect of various antibacterial agents on the immunological measurement of free thyroxine, the following experiment was conducted. The antibacterial agents used and the concentrations used (concentrations in the specimen) are as follows. The combination of ethyl p-hydroxybenzoate (indicated in Ethylparaben in the table) or methyl p-hydroxybenzoate (indicated in Methylparaben in the table, both manufactured by Wako Pure Chemical Industries, Ltd.) and quinolone It has been proposed in the past as a preservative [2].
The effect on the free thyroxine measurement system was observed by the same operation as 4 except that the combination of antibacterial agents was changed. The results are as shown in Table 5.
It was confirmed that the measured value was small when ethyl p-hydroxybenzoate (or methyl), which is a combination of known antibacterial agents, was used. Although the antibacterial agent LFX according to the present invention is used at a much higher concentration of 4 and does not affect the measured value, the combination of ethyl p-hydroxybenzoate and the like causes a large error. It was confirmed that Similar to 4, it was speculated that these drugs affected the equilibrium relationship with free thyroxine-TBG-bound thyroxine.

[0061]
[Table 5]

[0062]
6). Influence on the enzymatic measurement system
In order to observe the influence on the enzymatic measurement system, the influence of various antibacterial agents on the measurement result by the enzymatic reaction of cholesterol and triglyceride was investigated. Cholestezyme-V555 'Eiken' (registered trademark) was used for the measurement of cholesterol, and Triglyzyme-600'Eiken '(registered trademark) of Eiken Chemical was used for the measurement of triglyceride. In this example, a human serum specimen to which an antibacterial agent having the following concentration was added was prepared as a sample. As controls, sera to which only a solvent in which each antibacterial agent was dissolved were added. The measurement operation was carried out according to the reagent kit instructions, and each sample was measured in duplicate to determine the average value.
[0063]

[0064]
The results are as shown in Table 6. The measured values in Table 5 are values after performing volume correction since the final dilution rate may vary depending on the sample. In the table, the value of “measured value / control” is shown in%. It was confirmed that the measurement result by the enzymatic reaction was not affected by the addition of the antibacterial agent according to the present invention.
The solvent used for dissolving each drug, that is, NaOH (0.05N and 0.005N), purified water, dimethyl sulfoxide (0.005%) + NaOH (0.05N), and dimethyl sulfoxide (0.0005%) + NaOH Although the change of the measured value was also observed for the sample to which only (0.005N) was added, the effect of the addition of these solvents could not be confirmed.
[0065]
[Table 6]

[0066]
List of cited references
[1] Japanese Patent Publication No. 7-78005
[2] JP-A-1-283223
[3] JP-A-7-63752
[4] JP-A-8-43385
[5] USP 3799930; 1974
[6] Antimicrobial Agents & Chemotherapy. 18 (1): 37-40, 1980 Jul
[7] USP 4490991; 1985
[8] REVIEWS OF INFECTIOU DISEASES, 1989, V11, S5, PS912
[9] JP 63-107990 A
[10] “Proper use of new quinolone”-Pharmaceutical Journal, Yoshitoshi Fujii, Satoshi Shimada, p290
[11] structure-activity relationship in quinolone antibacterials: Design, synthesis and biologics of novelisiozoquinolones. Drugs Exptl. Clin. Res. XIV (6). 379-383 1988.
[12] Antimicrob. Agents Chemother. 33, 1167-1173 (1989)
[13] JP 63-104974 A
[14] 5-Amino-7- (3-amino-1-pyrrolidinyl) -1-cyclopropyl-6,8-difluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid (PD 124816): synthesis and bioevaluation of a new class of quinolone antibacterials. Drugs Exp Clin Res 14: 453, 1988
[15] Diagnostic Microbiology & Infectious Disease. 14 (3): 245-58, 1991 May-Jun
[16] JP 59-67269 A
[17] "New quinolone" Susumu Mitsuhashi Society of Sciences Publishing Center p57
[18] L.M. A. Mitscher et al. : International telesympasium on Quinones P. B. Fernandes. J. et al. R. Prous Science, S.M. A. , Pp3-20 (1989)
[19] JP-A-1-100166
[20] Antimicrob. Agents Chemother. 31 (6): 854-859, 1987
[21] Medicinal Journal VOL. 31, S-1, P135-143, 1995
[22] Antibiotics in Laboratory Medicine 2nd ed. C. 13 p. 477-514 (1986)

Claims (17)

A biological component measuring reagent characterized by adding at least one drug selected from quinolone antibiotics having the following structure and not containing a p-hydroxybenzoic acid derivative

The reagent according to claim 1, wherein the biological component measuring reagent is not intended for the propagation of microorganisms. The reagent according to claim 1, wherein the quinolone antibiotic is added at 0.001-0.01% W / V. The reagent of claim 1 further comprising an antifungal agent. The reagent of claim 4, wherein the antifungal agent is selected from the group consisting of amphotericin B, nystatin, flucytosine, miconazole, and itraconazole. The reagent according to claim 1, wherein the biological component measurement reagent comprises a component selected from the group consisting of an enzyme, an antibody, an antigen, and a serum protein. The reagent according to claim 1, wherein the biological component measuring reagent is an enzymatic analysis reagent or an immunological analysis reagent. The reagent according to claim 1, wherein the biological component measuring reagent is a standard substance for enzymatic analysis or a standard substance for immunological analysis. The reagent according to claim 6, 7 or 8, wherein the biological component measuring reagent is for in vitro diagnosis. The reagent according to claim 8, wherein the biological component measurement reagent is based on serum. The reagent according to claim 1, wherein the biological component measuring reagent is in a liquid state. The reagent according to claim 1, wherein the biological component measurement reagent is in a dry state. The reagent according to claim 1, which contains neither azide nor thimerosal. The reagent according to claim 1, wherein the biological component measuring reagent is a reagent for immunological analysis of thyroxine and / or triiodothyronine in a free state not bound to serum protein, or a standard substance for immunological analysis. The reagent according to claim 1, which does not contain any of antibiotics selected from penicillin G and streptomycin. In a biological component measurement reagent that does not contain a p-hydroxybenzoic acid derivative, a component to be protected from the growth of microorganisms contained in the reagent is protected by at least one drug selected from quinolone antibiotics having the following structure: how to

does not include the p- hydroxybenzoic acid derivatives immunological reagent for thyroxine and / or triiodothyronine, and adding at least one agent selected from the quinolone antibiotic having the following structure, serum proteins For inhibiting the growth of microorganisms without affecting the immunoassay of thyroxine and / or triiodothyronine in a free state that is not bound to