JP2013127472A - Measured value lowering inhibitor for immunity measuring method, and immunity measuring method using the same - Google Patents
Measured value lowering inhibitor for immunity measuring method, and immunity measuring method using the same Download PDFInfo
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
本発明は、妨害物質による測定値低下を抑制するための免疫測定法用測定値低下抑制剤並びにそれを用いた免疫測定法及び免疫測定法用試薬に関する。 The present invention relates to a measurement value decrease inhibitor for immunoassay for suppressing a decrease in measurement value due to an interfering substance, an immunoassay using the same, and a reagent for immunoassay.
臨床検査において、生体試料を被検試料とする検査項目は多数存在する。その中の免疫学的測定法においては、目的物質の免疫反応に影響を与える妨害物質が生体試料中に存在することが知られている。この影響とは測定値の正確性に影響するものであり、いくつかの報告もなされている。 In clinical tests, there are many test items that use biological samples as test samples. In the immunoassay among them, it is known that a disturbing substance that affects the immune response of the target substance exists in the biological sample. This effect affects the accuracy of measured values, and several reports have been made.
これら妨害物質による影響の確認及び低減の最も一般的な方法として、被検試料を前希釈することにより妨害物質の濃度を低下させる方法がある。しかしこの方法の場合、希釈操作により妨害物質と共に目的物質濃度も低下するため、試料中の低濃度物質を検出する測定系では利用が困難となる。更に、希釈操作が加わることにより測定時間も延長し、迅速性に欠けるものとなる。一方で、妨害物質が既知の場合は妨害活性を抑制又は抑止する機能を持つ化合物の添加や加温などの前処理方法がとられるが、既知の特定物質だけに有効であり、測定項目によっては未知の物質が測定に悪影響を与える可能性も考えられる。 As the most common method for confirming and reducing the influence of these interfering substances, there is a method of reducing the concentration of interfering substances by prediluting a test sample. However, in this method, the concentration of the target substance as well as the interfering substance is reduced by the dilution operation, so that it is difficult to use in a measurement system that detects a low concentration substance in a sample. Further, the addition of the dilution operation extends the measurement time and lacks rapidity. On the other hand, when the interfering substance is known, pretreatment methods such as addition of a compound having a function to suppress or suppress interfering activity or heating are taken, but it is effective only for known specific substances, and depending on the measurement item It is possible that unknown substances may adversely affect the measurement.
また、特開平９−３０４３８４号公報では免疫測定法における偽陽性回避のため、スルホン酸基やその塩を含有する共役ジエン系重合体を用いることが記載されているが、特殊な試料中に存在する偽陽性物質よりも、全ての生体試料中に存在しうる妨害物質の回避が正確性向上に必要である。 JP-A-9-304384 describes that a conjugated diene polymer containing a sulfonic acid group or a salt thereof is used to avoid false positives in an immunoassay, but it exists in a special sample. In order to improve accuracy, it is necessary to avoid interfering substances that may be present in all biological samples rather than false positive substances.
本発明の目的は、被検試料中の妨害物質による影響を抑制して免疫測定法の正確性を向上させることができる、免疫測定法用測定値低下抑制剤並びにそれを用いた、妨害物質による測定値低下が抑制された免疫測定法及び免疫測定法用試薬を提供することである。 The object of the present invention is to suppress the influence of interfering substances in a test sample to improve the accuracy of immunoassay, and to prevent the measurement value from being decreased for immunoassay, and to the interfering substance using the same. It is to provide an immunoassay and an immunoassay reagent in which a decrease in measured value is suppressed.
本願発明者らは、鋭意研究の結果、イオン性官能基を有する疎水性環式モノマーが重合されたポリマーであって、分子量が１０００ないし１０万であるイオン性界面活性剤を免疫測定法を行なう反応溶液に共存させることにより、被検試料中の妨害物質による影響を抑制して免疫測定法の正確性を向上させることができることを見出し、本発明を完成した。 As a result of diligent research, the inventors of the present application perform an immunoassay method for an ionic surfactant having a molecular weight of 1,000 to 100,000, which is a polymer obtained by polymerizing a hydrophobic cyclic monomer having an ionic functional group. It has been found that the coexistence in the reaction solution can improve the accuracy of the immunoassay by suppressing the influence of the interfering substance in the test sample.
すなわち、本発明は、イオン性官能基を有する疎水性環式モノマーが重合されたポリマーであって、分子量が１０００ないし１０万であるイオン性界面活性剤から成る、妨害物質による測定値低下を抑制するための免疫測定法用測定値低下抑制剤を提供する。また、本発明は、上記本発明の測定値低下抑制剤の存在下において行なう免疫測定法を提供する。さらに、本発明は、緩衝液と、感作粒子又は抗血清とを少なくとも含む免疫測定法用試薬において、上記本発明の測定値低下抑制剤を含むことを特徴とする、免疫測定法用試薬を提供する。 That is, the present invention is a polymer in which a hydrophobic cyclic monomer having an ionic functional group is polymerized, and comprises an ionic surfactant having a molecular weight of 1,000 to 100,000, and suppresses a decrease in measured value due to an interfering substance. The present invention provides a measurement value decrease inhibitor for immunoassay. The present invention also provides an immunoassay method performed in the presence of the measured value decrease inhibitor of the present invention. Furthermore, the present invention provides an immunoassay reagent characterized in that the immunoassay reagent contains at least a buffer solution and a sensitizing particle or antiserum, which comprises the measured value decrease inhibitor of the present invention. provide.
本発明の免疫測定法の測定値低下抑制剤の存在下において免疫測定法を行なうことにより、被検試料中の妨害物質の影響が低減され、測定値低下抑制剤の非存在下において行なう場合よりも免疫測定法の正確性が向上する。 By performing the immunoassay in the presence of the measurement value lowering inhibitor of the immunoassay of the present invention, the influence of the interfering substance in the test sample is reduced, and compared with the case of performing in the absence of the measurement value lowering inhibitor. Even the accuracy of the immunoassay improves.
免疫測定法のうち、免疫凝集法は、抗原抗体反応により生じる、反応液の濁度や吸光度のような光学的性質の変化に基づいて、被検試料中の抗原又は抗体を検出又は定量する方法であり、免疫比濁法及び免疫比ろう法（ネフェロメトリー）が包含される。測定の感度を高めるため、通常、被検試料中の目的抗原又は目的抗体と抗原抗体反応する抗体又は抗原が、ラテックス粒子のような粒子上に不動化されており（感作粒子）、抗原抗体反応により該感作粒子が凝集することに起因する光学的性質の変化に基づいて被検物質の検出又は定量が行なわれる（ラテックス粒子を用いる場合は特にラテックス凝集法とも呼ばれる）。もっとも、感作粒子を用いることなく抗血清もしばしば用いられる。 Among immunoassays, immunoagglutination is a method for detecting or quantifying an antigen or antibody in a test sample based on a change in optical properties such as turbidity or absorbance of a reaction solution caused by an antigen-antibody reaction. And immunoturbidimetric methods and immunotrophic methods (nepherometry) are included. In order to increase the sensitivity of the measurement, the target antigen or antibody or antigen that reacts with the target antibody in the test sample is usually immobilized on particles such as latex particles (sensitized particles). The test substance is detected or quantified based on a change in optical properties caused by the aggregation of the sensitized particles by the reaction (in particular, when latex particles are used, it is also called a latex agglutination method). However, antisera are often used without using sensitizing particles.
本発明の免疫測定法の測定値低下抑制剤は、イオン性官能基を有する疎水性環式モノマーが重合されたポリマーであって、分子量が１０００ないし１０万であるイオン性界面活性剤から成る。 The measured value decrease inhibitor of the immunoassay method of the present invention is a polymer obtained by polymerizing a hydrophobic cyclic monomer having an ionic functional group, and comprises an ionic surfactant having a molecular weight of 1,000 to 100,000.
イオン性官能基としては、スルホン基若しくはその塩、カルボン酸若しくはその塩又はアミン（水溶液中でイオン化される第４級アミン等）が好ましく、特にスルホン基又はその塩が好ましい。このように、イオン性界面活性剤は、陰イオン界面活性剤でも陽イオン界面活性剤でもよい。 As the ionic functional group, a sulfone group or a salt thereof, a carboxylic acid or a salt thereof, or an amine (such as a quaternary amine ionized in an aqueous solution) is preferable, and a sulfone group or a salt thereof is particularly preferable. Thus, the ionic surfactant may be an anionic surfactant or a cationic surfactant.
また、疎水性環としては、芳香環やシクロアルキル環が挙げられ、酸素原子、窒素原子又はイオウ原子等を含む複素環であってもよいし、それらが縮合した縮合環であってもよい。疎水性環としては芳香環が好ましく、芳香環としては、ベンゼン環、ナフタレン環、アントラセン環等が例示され、好ましくはベンゼン環又はナフタレン環、さらに好ましくはベンゼン環である。 In addition, examples of the hydrophobic ring include an aromatic ring and a cycloalkyl ring, which may be a heterocyclic ring containing an oxygen atom, a nitrogen atom, a sulfur atom, or the like, or a condensed ring in which they are condensed. As the hydrophobic ring, an aromatic ring is preferable, and examples of the aromatic ring include a benzene ring, a naphthalene ring, and an anthracene ring, and a benzene ring or a naphthalene ring is preferable, and a benzene ring is more preferable.
測定値低下抑制剤として用いられる好ましいポリマーとして、ポリアネト−ルスルホン酸ナトリウム塩、ポリスチレンスルホン酸ナトリウム塩、ナフタレンスルホン酸ホルマリン縮合物のナトリウム塩、芳香族スルホン酸ホルマリン縮合物のナトリウム塩（具体的には、ディスロ−ル（商品名、日本乳化剤社製）、デモ−ル（商品名、花王社製）、ポリティＰＳ−１９００（ライオン）、ポリティＮ−１００Ｋ（商品名、ライオン社製）等）を挙げることができる。 Preferred polymers used as measured value decrease inhibitors include polyanetolsulfonic acid sodium salt, polystyrenesulfonic acid sodium salt, sodium salt of naphthalenesulfonic acid formalin condensate, sodium salt of aromatic sulfonic acid formalin condensate (specifically, , Disroll (trade name, manufactured by Nippon Emulsifier Co., Ltd.), Demol (trade name, manufactured by Kao Corporation), Politi PS-1900 (Lion), Politi N-100K (trade name, manufactured by Lion Corporation) and the like. be able to.
また、測定値低下抑制剤として用いられる好ましいポリマーとして、繰返し単位が、下記一般式[I]で表されるものを挙げることができる。 Moreover, as a preferable polymer used as a measured value decrease inhibitor, a polymer represented by the following general formula [I] can be exemplified.
（ただし、Arは疎水性環、Ｘはイオン性官能基、R1ないしR3は互いに独立に水素原子又はアルキル基を示し、nは０ないし１０の整数を示し、Arを構成する各炭素原子に結合している水素原子は、本発明の効果を阻害しない置換基で置換されていてもよい）。 (Wherein Ar represents a hydrophobic ring, X represents an ionic functional group, R 1 to R 3 each independently represent a hydrogen atom or an alkyl group, n represents an integer of 0 to 10, and each carbon atom constituting Ar The hydrogen atom bonded to may be substituted with a substituent that does not inhibit the effect of the present invention).
上記一般式[I]において、Ｘ及びArは、それぞれ上記したイオン性官能基及び疎水性環である。R1ないしR3がアルキル基の場合、低級（炭素数１〜４、以下同じ）アルキル基が好ましい。また、ｎは０ないし３が好ましい。また、Arを構成する各炭素原子に結合している水素原子は、本発明の効果を阻害しない置換基で置換されていてもよく、このような置換基の例として、低級アルキル基及び低級アルコキシル基を挙げることができる。 In the above general formula [I], X and Ar are the above-described ionic functional group and hydrophobic ring, respectively. When R 1 to R 3 are alkyl groups, lower (1 to 4 carbon atoms, the same shall apply hereinafter) alkyl groups are preferred. N is preferably 0 to 3. In addition, a hydrogen atom bonded to each carbon atom constituting Ar may be substituted with a substituent that does not inhibit the effect of the present invention. Examples of such a substituent include a lower alkyl group and a lower alkoxyl. The group can be mentioned.
一般式[I]で表される繰返し単位のうち、下記一般式[II]で表されるものが好ましい。 Of the repeating units represented by the general formula [I], those represented by the following general formula [II] are preferred.
式[II]中、Ｍは、水溶液中で１価の陽イオンとなる原子又は基、好ましくはナトリウムやカリウムのようなアルカリ金属、R1ないしR3は、互いに独立に水素原子又は低級アルコキシル基、R4ないしR6は、互いに独立に水素原子又は低級アルキル基を示す。 In the formula [II], M is an atom or group that becomes a monovalent cation in an aqueous solution, preferably an alkali metal such as sodium or potassium, and R 1 to R 3 are independently a hydrogen atom or a lower alkoxyl group. R 4 to R 6 each independently represent a hydrogen atom or a lower alkyl group.
上記一般式[I]で表される繰返し単位のうち、特に好ましいものとして、下記式[III]で表されるアネトールスルホン酸塩、下記式[IV]で表されるスチレンスルホン酸塩及び下記式[V]で示されるナフタレン酸スルホン酸ホルマリン縮合物塩を挙げることができる。 Of the repeating units represented by the above general formula [I], particularly preferred are anethole sulfonates represented by the following formula [III], styrene sulfonates represented by the following formula [IV], and the following formulas: Mention may be made of the naphthalene sulfonic acid formalin condensate salt represented by [V].
ただし、式[III]、[IV]及び[V]中、Ｍは式[I]と同義であり、好ましくはナトリウムやカリウムのようなアルカリ金属である。 However, in the formulas [III], [IV] and [V], M has the same meaning as in the formula [I], and is preferably an alkali metal such as sodium or potassium.
上記ポリマーの分子量は、１０００〜１０万であり、好ましくは、１０００〜６万である。 The molecular weight of the polymer is 1000 to 100,000, preferably 1000 to 60,000.
上記繰返し単位は、１種のみでも２種以上を組み合わせて採用することもできる。測定値低下抑制剤として用いられるポリマーは、上記繰返し単位のみから成ることが好ましいが、本発明の効果に悪影響を与えない範囲で他の単位を含んでいてもよい。通常、このような他の単位のポリマー中の含量は、２０モル％以下、好ましくは１０モル％以下、さらに好ましくは５モル％以下である。 The above repeating units can be used alone or in combination of two or more. The polymer used as the measured value decrease inhibitor is preferably composed of only the above repeating units, but may contain other units as long as the effects of the present invention are not adversely affected. Usually, the content of such other units in the polymer is 20 mol% or less, preferably 10 mol% or less, more preferably 5 mol% or less.
本発明の免疫測定法は、上記した本発明の測定値低下抑制剤の存在下において免疫測定法を行なうものである。測定値低下抑制剤の使用量は、特に限定されないが、通常、反応溶液中の最終濃度が0.01%ないし５％（重量／容量）であり、好ましくは0.1〜１％程度である。もっとも、使用する高分子イオン性界面活性剤の種類、被検試料と緩衝液および抗血清液または、感作ラテックス粒子の混合比などにより効果が得られる最適な濃度を選択することが好ましい。 The immunoassay method of the present invention is an immunoassay method in the presence of the above-described measured value decrease inhibitor of the present invention. The amount of the measured value decrease inhibitor used is not particularly limited, but the final concentration in the reaction solution is usually 0.01% to 5% (weight / volume), preferably about 0.1 to 1%. However, it is preferable to select an optimum concentration that is effective depending on the type of the polymeric ionic surfactant to be used, the test sample, the buffer solution and the antiserum solution, or the mixing ratio of the sensitized latex particles.
本発明の免疫測定法に付される被検試料は、特に限定されないが、妨害物質の影響を抑制するという本発明の効果を大いに発揮できる生体試料が好ましく、特に、血液、血清、血漿、尿、便、唾液、組織液、髄液、ぬぐい液等の体液等又はその希釈物が好ましく、さらには血液、血清及び血漿又はこれらの希釈物が好ましい。 The test sample to be subjected to the immunoassay method of the present invention is not particularly limited, but a biological sample capable of greatly exerting the effect of the present invention to suppress the influence of interfering substances is preferable, and in particular, blood, serum, plasma, urine Body fluids such as stool, saliva, tissue fluid, cerebrospinal fluid, and wiping fluid or the like, or dilutions thereof, and blood, serum and plasma, or dilutions thereof are preferred.
免疫測定法が免疫凝集法である場合に用いられる感作粒子に感作される抗体又は抗原は特に限定されるものではなく、従来と同様、例えば、C-反応性蛋白（CRP）、リウマトイド因子（RF）、フェリチン（FER）及びミオグロビン（Mb）並びにこれらに対する抗体を例示することができるが、もちろんこれらに限定されるものではない。 The antibody or antigen sensitized to the sensitized particles used when the immunoassay is an immunoagglutination method is not particularly limited. For example, C-reactive protein (CRP), rheumatoid factor is the same as in the past. (RF), ferritin (FER) and myoglobin (Mb) and antibodies against them can be exemplified, but of course not limited thereto.
上記測定値低下抑制剤の存在下において免疫測定法を行なうことを除き、本発明の免疫測定法は従来と同様に行なうことができる。すなわち、免疫測定法が免疫凝集法である場合、反応液中の感作粒子の濃度は、特に限定されないが、通常、０．０１〜０．５％程度であり、反応は、通常、１℃〜５６℃、好ましくは３７℃で１分間〜１０分間程度行なわれる。もっとも、反応条件はこれらに限定されるものではない。また、反応媒体としては、通常、グリシン緩衝液等の各種緩衝液が用いられる。反応溶液の濁度又は吸光度を反応の開始前と開始後一定時間後測定し、又は、反応の開始前及び開始後経時的に測定し、濁度若しくは吸光度の変化の大きさ（エンドポイント法）又は変化の速度（レート法）に基づき、被検物質の検出又は定量を行なう。なお、免疫凝集法は、手動により行なうこともできるし、自動分析装置を用いて行うこともできる。 The immunoassay method of the present invention can be performed in the same manner as in the prior art except that the immunoassay method is performed in the presence of the above-described measurement value decrease inhibitor. That is, when the immunoassay is an immunoagglutination method, the concentration of the sensitizing particles in the reaction solution is not particularly limited, but is usually about 0.01 to 0.5%, and the reaction is usually performed at 1 ° C. It is carried out at ˜56 ° C., preferably 37 ° C. for about 1 to 10 minutes. However, the reaction conditions are not limited to these. As the reaction medium, various buffer solutions such as glycine buffer solution are usually used. Measure the turbidity or absorbance of the reaction solution before the start of the reaction and after a certain period of time after the start of the reaction, or measure it before and after the start of the reaction over time, and the magnitude of the change in turbidity or absorbance (endpoint method) Alternatively, the test substance is detected or quantified based on the rate of change (rate method). The immunoaggregation method can be performed manually or using an automatic analyzer.
免疫測定法が免疫凝集法である場合、本発明の方法では、測定値低下抑制剤と、感作粒子又は抗血清を同時に被検試料と混合しても良いが、被検試料を測定値低下抑制剤と接触させる第１工程と、次いで、第１工程後の被検試料を感作粒子又は抗血清と抗原抗体反応させる第２工程とを含むことが、本発明の効果を最大限に得る上で好ましい。この場合、第１工程、第２工程とも、特に限定されないが、反応時間はそれぞれ、通常１分間〜１０分間、好ましくは１分間〜５分間、反応温度は通常、１℃〜５６℃、好ましくは３７℃で行なわれる。また、２段階で反応を行なう場合、測定値低下抑制剤の濃度は、第１工程の反応液中、好ましくは0.01%ないし５％（重量／容量）であり、さらに好ましくは０．０５〜１％程度である。 When the immunoassay is an immunoagglutination method, in the method of the present invention, the measurement value decrease inhibitor and the sensitized particles or antiserum may be mixed with the test sample at the same time. Including the first step of contacting with the inhibitor and then the second step of reacting the test sample after the first step with the sensitized particles or the antiserum with the antigen-antibody results in the maximum effect of the present invention. Preferred above. In this case, the first step and the second step are not particularly limited, but the reaction time is usually 1 minute to 10 minutes, preferably 1 minute to 5 minutes, and the reaction temperature is usually 1 ° C. to 56 ° C., preferably Performed at 37 ° C. When the reaction is carried out in two stages, the concentration of the measured value reduction inhibitor is preferably 0.01% to 5% (weight / volume) in the reaction solution of the first step, more preferably 0.05 to 1. %.
本発明の免疫測定法は、免疫凝集法に限定されるものではなく、サンドイッチELISA等のサンドイッチ法、イムノクロマト法、競合法等も含まれる。これらの免疫測定法の場合にも、本発明の測定値低下抑制剤を免疫反応の媒体中に含ませてそれぞれの免疫測定法を行なえばよい。その際の測定値低下抑制剤の濃度は上記と同様でよい。 The immunoassay method of the present invention is not limited to the immunoaggregation method, and includes a sandwich method such as a sandwich ELISA, an immunochromatography method, a competitive method, and the like. Also in the case of these immunoassays, each immunoassay may be performed by including the measured value decrease inhibitor of the present invention in an immune reaction medium. The concentration of the measured value decrease inhibitor at that time may be the same as described above.
本発明は、また、緩衝液と、感作粒子と、上記測定値低下抑制剤を含む免疫測定法用試薬をも提供する。上記のように、免疫凝集法を２段階で反応を行なう場合には、免疫測定法用試薬は、緩衝液を少なくとも含み、被検試料と先に混合される第１試薬と、緩衝液及び感作粒子を少なくとも含み、被検試料と第１試薬の混合物に添加される第２試薬とから成る２液系試薬であることが操作性及び試薬の安定性の観点から好ましく、この場合、前記測定値低下抑制剤は、前記第１試薬中に含まれる。この場合、第１試薬中の測定値低下抑制剤の濃度は、特に限定されないが、通常、０．０１〜５％(w/v)、好ましくは、０．０１〜１％(w/v)程度である。なお、これらの試薬の他に、希釈液を用いて被検試料を希釈してもよい。 The present invention also provides an immunoassay reagent containing a buffer solution, sensitized particles, and the above-described measurement value decrease inhibitor. As described above, when the immunoagglutination method is performed in two steps, the immunoassay reagent contains at least a buffer solution, the first reagent previously mixed with the test sample, the buffer solution and the sensitivity. From the viewpoints of operability and reagent stability, it is preferable that the reagent is a two-component reagent comprising at least a particle-forming particle and comprising a second reagent added to the mixture of the test sample and the first reagent. The value decrease inhibitor is contained in the first reagent. In this case, the concentration of the measured value decrease inhibitor in the first reagent is not particularly limited, but is usually 0.01 to 5% (w / v), preferably 0.01 to 1% (w / v). Degree. In addition to these reagents, the test sample may be diluted with a diluent.
以下、本発明を実施例に基づきより具体的に説明する。もっとも、本発明は下記実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
試薬
以下の組成からなるミオグロビン測定用ラテックス比濁用試薬を調製した。
第1試薬
170ｍM グリシン緩衝液 ｐH7.0
50ｍM EDTA
100ｍM 塩化ナトリウム
0.3％ ポリアネト−ルスルホン酸ナトリウム塩（分子量４万）
第２試薬
Ｍｂ-ラテックス「生研」/ラテックス浮遊液（デンカ生研社製）
A latex turbidimetric reagent for measuring myoglobin having the following composition was prepared.
First reagent
170 mM glycine buffer, pH 7.0
50 mM EDTA
100 mM sodium chloride
0.3% Polyanetolsulfonic acid sodium salt (molecular weight 40,000)
Second Reagent Mb-Latex “Seiken” / Latex suspension (Denka Seiken)
試薬
以下の組成からなるミオグロビン測定用ラテックス比濁用試薬を調製した。
第1試薬
170ｍM グリシン緩衝液 ｐH7.0
50ｍM EDTA
100ｍM 塩化ナトリウム
0.3％ ポリスチレンp-スルホン酸ナトリウム塩（分子量１万４千）
第２試薬
Ｍｂ-ラテックス「生研」/ラテックス浮遊液（デンカ生研社製）
Reagent A latex turbidimetric reagent for measuring myoglobin having the following composition was prepared.
First reagent
170 mM glycine buffer, pH 7.0
50 mM EDTA
100 mM sodium chloride
0.3% Polystyrene p-sulfonic acid sodium salt (Molecular weight 14,000)
Second Reagent Mb-Latex “Seiken” / Latex suspension (Denka Seiken)
（比較例１）
試薬
比較用試薬として第1試薬中にポリアネト−ルスルホン酸ナトリウム塩及びポリスチレンp-スルホン酸ナトリウム塩を含まない、以下の組成からなるミオグロビン測定用ラテックス比濁用試薬を調製した。
第1試薬
170ｍM グリシン緩衝液 ｐH7.0
50ｍM EDTA
100ｍM 塩化ナトリウム
第２試薬
Ｍｂ-ラテックス「生研」/ラテックス浮遊液（デンカ生研社製）
(Comparative Example 1)
Reagents As a comparative reagent, a latex turbidimetric reagent for measuring myoglobin having the following composition was prepared, which did not contain polyanetolsulfonic acid sodium salt and polystyrene p-sulfonic acid sodium salt in the first reagent.
First reagent
170 mM glycine buffer, pH 7.0
50 mM EDTA
100mM sodium chloride second reagent Mb-latex "Seiken" / latex suspension (manufactured by Denka Seiken)
なお、実施例１、実施例２及び比較例１の第1試薬、第2試薬で用いる緩衝液はリン酸緩衝液、グッド緩衝液等を使用することができ、ｐHについても緩衝液の好適なｐHを使用できる。 The buffer used in the first reagent and the second reagent of Example 1, Example 2 and Comparative Example 1 can be a phosphate buffer, a Good buffer, etc., and a suitable buffer for pH. pH can be used.
測定例
被検試料 無作為に抽出した臨床検体1例（血清）
測定方法 試料調製 被検試料を、生理食塩液を希釈液として希釈濃度1/10（１０倍希釈）〜10/10（希釈せず）の段階に希釈する。
測定方法 東芝TBA-30R型自動分析装置による測定
Measurement example Test sample Randomly extracted clinical sample (serum)
Measurement method Sample preparation A test sample is diluted in a dilution concentration of 1/10 (10-fold dilution) to 10/10 (not diluted) using physiological saline as a diluent.
Measurement method Measurement with Toshiba TBA-30R automatic analyzer
自動分析装置による測定
実施例１、実施例２及び比較例１の各試薬を用いて測定を行った。前述の調製した試料20μＬに第1試薬200μＬを添加し、37℃で攪拌混合し、5分放置後、第2試薬100μＬを添加し、更に37℃で攪拌混合した後、約2分間の凝集反応を570nmの吸光度変化量として測定した。またあらかじめ既知濃度の試料を同一条件で測定し、濃度と吸光度変化量の関係を表す検量線を作成しておいた。第一試薬中の測定値低下抑制剤の有無により、求めたそれぞれの測定値(ng/mL)を比較した。
Measurement using an automatic analyzer Measurement was performed using the reagents of Example 1, Example 2, and Comparative Example 1. Add 200 μL of the first reagent to 20 μL of the sample prepared above, stir and mix at 37 ° C., leave it for 5 minutes, add 100 μL of the second reagent, stir and mix at 37 ° C., and then agglutinate for about 2 minutes. Was measured as the amount of change in absorbance at 570 nm. A sample having a known concentration was measured in advance under the same conditions, and a calibration curve representing the relationship between the concentration and the amount of change in absorbance was prepared. The obtained measured values (ng / mL) were compared depending on the presence or absence of the measured value decrease inhibitor in the first reagent.
結果を下記表１に示す。表１中、「理論値」は、各例における共存する妨害物質が引き起こす測定値低下作用を無視し得る希釈濃度1/10（１０倍希釈）の測定値を基準とし、これに希釈濃度の分子を乗じた値（希釈濃度10/10なら１０倍、2/10なら２倍）を意味する。また、差（％）は、（理論値−測定値）／理論値 ×１００（％）を意味し、差（％）が小さいほど、理論値との乖離が小さいことを示す。 The results are shown in Table 1 below. In Table 1, “Theoretical value” is based on the measurement value of dilution concentration 1/10 (diluted 10 times) that can ignore the measurement value reduction effect caused by coexisting interfering substances in each example. Means the value multiplied by 10 (10 times for a dilution concentration of 10/10 and 2 for 2/10). The difference (%) means (theoretical value−measured value) / theoretical value × 100 (%), and the smaller the difference (%), the smaller the deviation from the theoretical value.
表１
表１に示されるように、測定値低下抑制剤を含まない比較例１では、無希釈の試料（希釈濃度10/10）で、理論値と測定値の差が３３％にも達するのに対し、本発明の実施例１及び２では、無希釈の場合でも差は５％又は４％であった。これらの結果は、本発明の測定値低下抑制剤の添加により理論値と測定値の差が小さくなることを示しており、妨害物質の影響が低減されることによって測定値の正確性が大幅に向上していることがわかる。 As shown in Table 1, in Comparative Example 1 that does not contain a measured value decrease inhibitor, the difference between the theoretical value and the measured value reaches 33% in the undiluted sample (diluted concentration 10/10). In Examples 1 and 2 of the present invention, the difference was 5% or 4% even without dilution. These results show that the difference between the theoretical value and the measured value is reduced by the addition of the measured value lowering inhibitor of the present invention, and the accuracy of the measured value is greatly improved by reducing the influence of interfering substances. It can be seen that it has improved.
すなわち、本発明は、スルホン基又はその塩を有する芳香族モノマーが重合されたポリマーであって、分子量が１０００ないし１０万であるイオン性界面活性剤から成る、被検試料中の妨害物質による測定値低下を抑制するための免疫測定法用測定値低下抑制剤の存在下において、希釈した被検試料について行なう免疫測定法であって、前記ポリマーは、その繰返し単位が下記式[III]で表されるアネトールスルホン酸塩又は下記式[V]で表されるナフタレンスルホン酸ホルマリン縮合物塩である、免疫測定法。
を提供する。
That is, the present invention is a measurement by an interfering substance in a test sample, which is a polymer obtained by polymerizing an aromatic monomer having a sulfone group or a salt thereof, and which is composed of an ionic surfactant having a molecular weight of 1,000 to 100,000. An immunoassay method for a diluted test sample in the presence of a measurement value decrease inhibitor for immunoassay for suppressing a decrease in value , wherein the polymer has a repeating unit represented by the following formula [III]: An immunoassay that is an anethole sulfonate salt or a naphthalene sulfonic acid formalin condensate salt represented by the following formula [V] .
I will provide a.
測定値低下抑制剤として用いられる好ましいポリマーとして、ポリアネト−ルスルホン酸ナトリウム塩、ナフタレンスルホン酸ホルマリン縮合物のナトリウム塩、芳香族スルホン酸ホルマリン縮合物のナトリウム塩（具体的には、ディスロ−ル（商品名、日本乳化剤社製）、デモ−ル（商品名、花王社製）、ポリティＰＳ−１９００（ライオン）、ポリティＮ−１００Ｋ（商品名、ライオン社製）等）を挙げることができる。 Preferable polymers used as a measured value decrease inhibitor include polyanetol sulfonic acid sodium salt, sodium salt of naphthalene sulfonic acid formalin condensate, sodium salt of aromatic sulfonic acid formalin condensate (specifically, distrol (product Name, manufactured by Nippon Emulsifier Co., Ltd.), demol (trade name, manufactured by Kao Corporation), Politi PS-1900 (Lion), Politi N-100K (trade name, manufactured by Lion Corporation) and the like.
本発明の測定値低下抑制剤として用いられるポリマーの繰り返し単位は、下記式[III]で表されるアネトールスルホン酸塩又は下記式[V]で示されるナフタレン酸スルホン酸ホルマリン縮合物塩である。 Repeating units of the polymer to be used as a measure lowering inhibitor of the present invention is a naphthalene acid acid formalin condensate salts represented by anethole sulfonate or formula represented by the following formula [III] [V].
ただし、式[III]及び[V]中、Ｍは水溶液中で１価の陽イオンとなる原子又は基、好ましくはナトリウムやカリウムのようなアルカリ金属である。
However, in the formulas [III] and [V], M is an atom or group that becomes a monovalent cation in an aqueous solution , preferably an alkali metal such as sodium or potassium.
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| WO1991010747A1 (en) * | 1990-01-16 | 1991-07-25 | Prutech Research Development Partnership Ii | Combination assay for antibody or antigen |
| WO1992008978A1 (en) * | 1990-10-31 | 1992-05-29 | Prutech Research And Development Partnership Ii | Immunoassay for the determination of anti-hiv antibodies in human samples |
| JP2006524319A (en) * | 2003-04-14 | 2006-10-26 | カリパー・ライフ・サイエンシズ・インク． | Reduced mobility shift assay interference |
-
2013
- 2013-02-12 JP JP2013023979A patent/JP5694401B2/en not_active Expired - Lifetime
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0326964A (en) * | 1989-06-14 | 1991-02-05 | Hoechst Ag | Method of stabilizing biological active material stabilized on solid phase |
| WO1991010747A1 (en) * | 1990-01-16 | 1991-07-25 | Prutech Research Development Partnership Ii | Combination assay for antibody or antigen |
| JPH05504400A (en) * | 1990-01-16 | 1993-07-08 | プルテック・リサーチ・ディベロプメント・パートナーシップ Ii | Antibody or antigen combination assays |
| WO1992008978A1 (en) * | 1990-10-31 | 1992-05-29 | Prutech Research And Development Partnership Ii | Immunoassay for the determination of anti-hiv antibodies in human samples |
| JP2006524319A (en) * | 2003-04-14 | 2006-10-26 | カリパー・ライフ・サイエンシズ・インク． | Reduced mobility shift assay interference |
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