JPWO2020095759A1 - Method for producing latex particles for measuring anti-streptolysin O antibody - Google Patents
Method for producing latex particles for measuring anti-streptolysin O antibody Download PDFInfo
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- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F8/00—Chemical modification by after-treatment
- C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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Abstract
少量のストレプトリジンＯを使用することにより、高い感度が得られる抗ストレプトリジンＯ抗体測定用ラテックス粒子の製造方法を提供する。カルボキシル基含量が５０μｅｑ／ｇ〜２５０μｅｑ／ｇ、好ましくは９０μｅｑ／ｇ〜１５０μｅｑ／ｇの範囲で表面に導入されたラテックス粒子に、ストレプトリジンＯを共有結合させる工程を含むストレプトリジンＯ抗体測定用ラテックス粒子の製造方法。Provided is a method for producing latex particles for measuring an anti-streptolysin O antibody, which can obtain high sensitivity by using a small amount of streptolysin O. Latex for measuring streptolysin O antibody, which comprises a step of covalently binding streptolysin O to latex particles introduced into the surface in a carboxyl group content of 50 μeq / g to 250 μeq / g, preferably 90 μeq / g to 150 μeq / g. How to make particles.
Description
本発明は、抗ストレプトリジンＯ抗体測定試薬（以下、「ＡＳＯ試薬」と表記することもある。）に用いられるラテックス粒子の製造方法に関する。より詳しくは、ストレプトリジンＯを使用したラテックス凝集比濁法を測定原理とした抗ストレプトリジンＯ抗体測定用ラテックス粒子の製造方法に関する。ストレプトリジンＯ（以下、「ＳＬＯ」と表記することもある。）は溶血性連鎖球菌（Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｐｙｏｇｅｎｅｓ）が菌体外に生産する溶血活性を有するタンパク質で、溶血性連鎖球菌の感染有無を診断するためのＡＳＯ測定試薬の原料に広く使用されているものである。 The present invention relates to a method for producing latex particles used in an anti-streptolysin O antibody measuring reagent (hereinafter, may be referred to as "ASO reagent"). More specifically, the present invention relates to a method for producing latex particles for measuring an anti-streptolysin O antibody based on a measurement principle of a latex agglutination turbidimetry method using streptolysin O. Streptolysin O (hereinafter, also referred to as "SLO") is a protein having hemolytic activity produced by Streptococcus pyogenes outside the cells, and diagnoses the presence or absence of infection with Streptococcus pyogenes. It is widely used as a raw material for ASO measurement reagents for this purpose.
ラテックス凝集比濁法を測定原理とした測定試薬を製造するためには、測定対象物質と結合する抗体もしくは抗原をラテックス粒子に感作する必要がある。例えば、溶血性連鎖球菌（Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｐｙｏｇｅｎｅｓ）が菌体外に生産するＳＬＯをラテックス粒子に感作すれば、ＳＬＯに対する抗体価（ＡＳＯ価）を測定することが可能であり、ＳＬＯは溶血性連鎖球菌の感染有無を診断するためのＡＳＯ測定試薬の原料として広く使用されている。 In order to produce a measurement reagent based on the latex agglutination turbidimetry method, it is necessary to sensitize the latex particles with an antibody or antigen that binds to the substance to be measured. For example, if SLO produced outside the cells by Streptococcus pyogenes is sensitized to latex particles, the antibody titer (ASO titer) against SLO can be measured, and SLO is hemolytic streptococcus. It is widely used as a raw material for ASO measurement reagents for diagnosing the presence or absence of infection.
従来はラテックス粒子とＳＬＯとの疎水性相互作用による物理的吸着法が広く採られ、得られたラテックス粒子とＳＬＯとの複合体をＢＳＡなどの免疫学的に不活性なタンパク質を溶解した緩衝液に懸濁した試薬の状態で保存するのが一般的であった（特許文献１）。しかし、このようにして調製された試薬では試薬を調製する条件により、担体同士の自己凝集が起こったり、感作したＳＬＯの脱離などが起こったりして試薬の反応性が低下し、本来の測定性能が発揮できない場合があった。そこで、ラテックス粒子とＳＬＯとを共有結合させることにより、ＳＬＯの脱離を防止して試薬の安定性を向上させる方法が提案されている（特許文献２、３）。 Conventionally, a physical adsorption method based on a hydrophobic interaction between latex particles and SLO has been widely adopted, and a buffer solution in which an immunologically inactive protein such as BSA is dissolved in the obtained complex of latex particles and SLO. It was generally stored in the state of a reagent suspended in (Patent Document 1). However, with the reagent prepared in this way, depending on the conditions for preparing the reagent, self-aggregation between carriers may occur, sensitized SLO may be eliminated, and the reactivity of the reagent may decrease, resulting in the original reactivity of the reagent. In some cases, the measurement performance could not be demonstrated. Therefore, a method has been proposed in which the latex particles and the SLO are covalently bonded to prevent the elimination of the SLO and improve the stability of the reagent (Patent Documents 2 and 3).
しかしながら、ＡＳＯ試薬の調製においてはラテックス粒子に感作させるＳＬＯが非常に高価であり、試薬の安定性のみならず、少量のＳＬＯで高い感度が得られるＡＳＯ試薬の調製方法を構築することが望まれていた。ただ、物理吸着法とは異なり、共有結合によるラテックス試薬の調製においては、共有結合の種類、ラテックス粒子の種類、抗体もしくは抗原を感作させる条件、ブロッキング条件、洗浄条件、ラテックス粒子の最終保存緩衝液の種類など試薬性能に与えるファクターが多数存在し、それらの条件を体系的に評価し、最適な条件を見出すことは非常に困難であった。これまでに、感度向上に関わる添加剤を第１試薬に添加する手法も報告されているが、いずれの試薬組成でも十分な効果があるとは限らず、逐一検討する必要があった（特許文献４、５，６）。 However, in the preparation of ASO reagent, SLO that sensitizes latex particles is very expensive, and it is desired to construct a method for preparing ASO reagent that can obtain high sensitivity with a small amount of SLO as well as stability of the reagent. It was rare. However, unlike the physical adsorption method, in the preparation of latex reagents by covalent bond, the type of covalent bond, the type of latex particle, the condition for sensitizing an antibody or antigen, the blocking condition, the washing condition, and the final storage buffer of the latex particle. There are many factors that affect reagent performance, such as the type of liquid, and it was extremely difficult to systematically evaluate these conditions and find the optimum conditions. So far, a method of adding an additive related to sensitivity improvement to the first reagent has been reported, but not all reagent compositions have a sufficient effect, and it is necessary to study them one by one (Patent Documents). 4, 5, 6).
本発明は、かかる現状に鑑み創作されたものであり、その目的は少量のＳＬＯで、高い感度が得られるＡＳＯ試薬に用いられるラテックス粒子の製造方法を提供することにある。 The present invention has been created in view of the present situation, and an object of the present invention is to provide a method for producing latex particles used in an ASO reagent that can obtain high sensitivity with a small amount of SLO.
本発明者は、カルボキシル基が結合した共有結合向けラテックス粒子の粒子サイズとカルボキシル基含量に着目し、ＳＬＯを感作するのに適したラテックス粒子を鋭意探索した結果、カルボキシル基含量がラテックス試薬の感度に大きく影響をすることを見出し、本発明を完成させるに至った。 The present inventor paid attention to the particle size and the carboxyl group content of the covalent bond latex particles to which the carboxyl groups were bonded, and as a result of diligently searching for the latex particles suitable for sensitizing the SLO, the carboxyl group content was the latex reagent. It has been found that it has a great influence on the sensitivity, and the present invention has been completed.
すなわち、本発明は、以下の列挙するものが例示される。
［項１］
カルボキシル基含量が５０μｅｑ／ｇ〜２５０μｅｑ／ｇの範囲で表面に導入されたラテックス粒子に、ストレプトリジンＯを共有結合させる工程を含むストレプトリジンＯ測定用ラテックス粒子の製造方法。
［項２］
カルボキシル基含量が９０μｅｑ／ｇ〜１５０μｅｑ／ｇの範囲で表面に導入されたラテックス粒子を用いる項１に記載のストレプトリジンＯ測定用ラテックス粒子の製造方法。
［項３］
平均粒径が５０ｎｍ〜２５０ｎｍのラテックス粒子を用いる項１または２に記載のストレプトリジンＯ測定用ラテックス粒子の製造方法。
［項４］
平均粒径が９０ｎｍ〜１５０ｎｍのラテックス粒子を用いる項３に記載のストレプトリジンＯ測定用ラテックス粒子の製造方法。
［項５］
ｐＨ５．９からｐＨ６．７の緩衝液中において、ストレプトリジンＯを、カルボキシル基を表面に有するラテックス粒子に共有結合させる工程を含む、項１〜４のいずれかに記載の抗ストレプトリジンＯ抗体測定用ラテックス粒子の製造方法。
［項６］
カルボキシル基含量が９０μｅｑ／ｇ〜１５０μｅｑ／ｇの範囲からなるラテックス粒子を使用する、項５に記載の抗ストレプトリジンＯ抗体測定用ラテックス粒子の製造方法。
［項７］
平均粒径が９５ｎｍから１５０ｎｍの範囲からなるラテックス粒子を使用する、項５に記載の抗ストレプトリジンＯ抗体測定用ラテックス粒子の製造方法。
［項８］
カルボキシル基含量が９０μｅｑ／ｇ〜１５０μｅｑ／ｇの範囲であって、かつ、平均粒径が９５ｎｍから１５０ｎｍの範囲からなるラテックス粒子を使用する、項５に記載の抗ストレプトリジンＯ抗体測定用ラテックス粒子の製造方法。
［項９］
ストレプトリジンＯを、カルボキシル基を表面に有するラテックス粒子に共有結合させる際にリン酸緩衝液、ＭＥＳ緩衝液およびＭＯＰＳ緩衝液からなる群より選択されるいずれかの緩衝液を用いる、項１〜８のいずれかの記載の抗ストレプトリジンＯ抗体測定用ラテックス粒子の製造方法。That is, the present invention exemplifies those listed below.
[Item 1]
A method for producing a latex particle for measuring streptolysin O, which comprises a step of covalently bonding streptolysin O to latex particles introduced into the surface in a carboxyl group content range of 50 μeq / g to 250 μeq / g.
[Item 2]
Item 2. The method for producing latex particles for measuring streptolysin O according to Item 1, wherein latex particles having a carboxyl group content in the range of 90 μeq / g to 150 μeq / g are used.
[Item 3]
Item 3. The method for producing latex particles for measuring streptolysin O according to Item 1 or 2, wherein latex particles having an average particle size of 50 nm to 250 nm are used.
[Item 4]
Item 3. The method for producing latex particles for measuring streptolysin O, which uses latex particles having an average particle size of 90 nm to 150 nm.
[Item 5]
Item 3. Anti-streptolysin O antibody measurement according to any one of Items 1 to 4, which comprises a step of covalently bonding streptolysin O to latex particles having a carboxyl group on the surface in a buffer solution having a pH of 5.9 to 6.7. For manufacturing methods of latex particles.
[Item 6]
Item 5. The method for producing a latex particle for measuring an anti-streptolysin O antibody according to Item 5, which uses latex particles having a carboxyl group content in the range of 90 μeq / g to 150 μeq / g.
[Item 7]
Item 5. The method for producing a latex particle for measuring an anti-streptolysin O antibody, which uses latex particles having an average particle size in the range of 95 nm to 150 nm.
[Item 8]
Item 5. Latex particles for measuring anti-streptolysin O antibody according to Item 5, wherein latex particles having a carboxyl group content in the range of 90 μeq / g to 150 μeq / g and an average particle size in the range of 95 nm to 150 nm are used. Manufacturing method.
[Item 9]
Items 1 to 8 use any buffer selected from the group consisting of phosphate buffer, MES buffer and MOPS buffer when covalently bonding streptolysin O to latex particles having a carboxyl group on the surface. The method for producing latex particles for measuring anti-streptolysin O antibody according to any one of the above.
本発明のＡＳＯ試薬に用いられるラテックス粒子の製造方法は、少量のＳＬＯを用いた場合であっても、感度の高い測定を可能とする点で有用である。 The method for producing latex particles used in the ASO reagent of the present invention is useful in that it enables highly sensitive measurement even when a small amount of SLO is used.
（ＳＬＯ）
本発明に用いるＳＬＯとしては、特に限定されず、例えば、溶血性連鎖球菌（Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｐｙｏｇｅｎｅｓ）が菌体外に生産するＳＬＯや組換え大腸菌などを用いて生産された遺伝子組換えのＳＬＯ、また、一部のアミノ酸配列を欠損させた改変型のＳＬＯ、さらにはＭＢＰなどを融合させた融合型ＳＬＯを使用しても良い。例えば、ＷＯ２０１７／２０４３２５に開示されているような、Ｃ末端領域を欠損させたＳＬＯが、熱安定性に優れたＳＬＯとして、好適に例示される。(SLO)
The SLO used in the present invention is not particularly limited, and for example, an SLO produced by Streptococcus pyogenes outside the cells, a recombinant SLO produced using recombinant Escherichia coli, or the like, or A modified SLO lacking a part of the amino acid sequence, or a fused SLO fused with MBP or the like may be used. For example, an SLO lacking the C-terminal region as disclosed in WO2017 / 204325 is preferably exemplified as an SLO having excellent thermal stability.
（ラテックス粒子）
本発明に用いるラテックス粒子としては、特に限定されないが、例えば、スチレンスルホン酸、メタクリル酸、アクリル酸、アクリル酸エステルなどを共重合させたポリスチレン粒子にカルボキシル基を導入した共有結合法向けのラテックス粒子が挙げられる。また、物理吸着を抑制するため、つまり、親水度を高めるためにポリアクリルアミドを含んだラテックス粒子を使用することもできる。好ましくは、アクリル酸を共重合させたポリスチレン粒子が挙げられ、さらに好ましくはポリアクリルアミドにより親水度が高められたアクリル酸を共重合させたポリスチレン粒子が挙げられる。(Latex particles)
The latex particles used in the present invention are not particularly limited, but for example, latex particles for a covalent bond method in which a carboxyl group is introduced into polystyrene particles obtained by copolymerizing styrene sulfonic acid, methacrylic acid, acrylic acid, acrylic acid ester, or the like. Can be mentioned. In addition, latex particles containing polyacrylamide can also be used in order to suppress physical adsorption, that is, to increase hydrophilicity. Preferred are polystyrene particles copolymerized with acrylic acid, and more preferably polystyrene particles copolymerized with acrylic acid whose hydrophilicity has been increased by polyacrylamide.
カルボキシル基の導入効率は適宜選択されるが、好ましくは５０μｅｑ／ｇ〜３００μｅｑ／ｇ、より好ましくは７０μｅｑ／ｇ〜２５０μｅｑ／ｇ、さらに好ましくは９０μｅｑ／ｇ〜２００μｅｑ／ｇ、特に好ましくは９０μｅｑ／ｇ〜１５０μｅｑ／ｇの割合でカルボキシル基が導入されたラテックス粒子である。本明細書において、カルボキシル基の導入効率は、アルカリ溶液の中和滴定により算出することができる。 The introduction efficiency of the carboxyl group is appropriately selected, but is preferably 50 μeq / g to 300 μeq / g, more preferably 70 μeq / g to 250 μeq / g, still more preferably 90 μeq / g to 200 μeq / g, and particularly preferably 90 μeq / g. Latex particles with a carboxyl group introduced at a rate of ~ 150 μeq / g. In the present specification, the introduction efficiency of a carboxyl group can be calculated by neutralization titration of an alkaline solution.
使用するラテックス粒子の平均粒径は、目的・用途に応じて適宜選択されるが、本発明においては、自動分析機向けであれば、好ましくは５０ｎｍ〜２５０ｎｍ、より好ましくは６０ｎｍ〜２００ｎｍ、さらに好ましくは７０ｎｍ〜１５０ｎｍ、特に好ましくは９５ｎｍから１５０ｎｍの粒径のラテックス粒子であることが好ましい。また、目視判断用の試薬向けであれば、好ましくは１００ｎｍ〜５００ｎｍ、よりこのましくは２００ｎｍ〜４００ｎｍのラテックス粒子であることが好ましい。本明細書において、「平均粒径」は、レーザー回折・散乱法によって求めた粒度分布における積算値５０％での粒径を意味する。 The average particle size of the latex particles used is appropriately selected according to the purpose and application, but in the present invention, it is preferably 50 nm to 250 nm, more preferably 60 nm to 200 nm, and further preferably for an automatic analyzer. Is preferably latex particles having a particle size of 70 nm to 150 nm, particularly preferably 95 nm to 150 nm. Further, for reagents for visual judgment, latex particles having a thickness of 100 nm to 500 nm, more preferably 200 nm to 400 nm are preferable. In the present specification, the "average particle size" means the particle size at an integrated value of 50% in the particle size distribution obtained by the laser diffraction / scattering method.
（ＳＬＯをラテックス粒子へ感作する方法）
ラテックス粒子の表面に有するカルボキシル基にＳＬＯのアミノ基を結合させる方法としては、特に限定されるものではないが、例えば、１−エチル−３−（３−ジメチルアミノプロピル）カルボジイミド塩酸塩（ＥＤＣ）などの水溶性カルボジイミドが使用できる。水溶性カルボジイミドの濃度は、０．１％〜５．０％の条件が好ましく、さらに好ましくは、０．５％〜２．０％の条件で用いるのが好ましい。Ｎ−ヒドロキシスクシンイミドを併用して、不安定なアシルイソ尿素中間体を、アミンと反応しやすいより安定したスクシンイミジルエステルに変換させてもよい。Ｎ−ヒドロキシスクシンイミドの濃度は、０．１％〜６．０％の条件が好ましく、さらに好ましくは０．２％〜２．０％の条件で用いるのが好ましい。(How to sensitize SLO to latex particles)
The method for binding the amino group of SLO to the carboxyl group on the surface of the latex particles is not particularly limited, but for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Water-soluble carbodiimide such as can be used. The concentration of the water-soluble carbodiimide is preferably 0.1% to 5.0%, more preferably 0.5% to 2.0%. N-Hydroxysuccinimide may be used in combination to convert the unstable acylisourea intermediate into a more stable succinimidyl ester that is more reactive with amines. The concentration of N-hydroxysuccinimide is preferably 0.1% to 6.0%, more preferably 0.2% to 2.0%.
ＳＬＯをラテックス粒子へ感作する際に用いる緩衝液としては、ＳＬＯを失活させることがなく、かつ、ラテックス粒子への感作を阻害しないようなイオン濃度やｐＨを有するものであればよく、例えば、リン酸緩衝液、酢酸緩衝液、クエン酸緩衝液、コハク酸緩衝液、リンゴ酸緩衝液、ＭＥＳ緩衝液、ＭＯＰＳ緩衝液、ＰＩＰＥＳ緩衝液、ＨＥＰＥＳ緩衝液などの各種グッド緩衝液等が挙げられる。これらの緩衝液は単独で用いてもよく、２種以上を併用してもよい。ｐＨに関しては適宜決められるが、好ましくはｐＨ４．０〜ｐＨ８．０、さらに好ましくはｐＨ５．０〜ｐＨ７．０の範囲である。温度条件は、４℃〜４０℃が好ましく、さらに好ましくは１０℃〜３０℃の条件である。タンパク濃度は、０．１ｍｇ／ｍｌ〜１００ｍｇ／ｍｌの条件が好ましく、さらに好ましくは１ｍｇ／ｍｌ〜１０ｍｇ／ｍｌの条件が好ましい。反応時間は、１分〜２４時間の反応が好ましく、さらに好ましくは１０分〜５時間の反応が好ましい。 The buffer used when sensitizing SLO to latex particles may be any buffer solution having an ion concentration and pH that does not inactivate SLO and does not inhibit sensitization to latex particles. For example, various good buffers such as phosphate buffer, acetate buffer, citrate buffer, succinic acid buffer, malic acid buffer, MES buffer, MOPS buffer, PIPES buffer, and HEPES buffer can be mentioned. Be done. These buffer solutions may be used alone or in combination of two or more. The pH is appropriately determined, but is preferably in the range of pH 4.0 to pH 8.0, and more preferably pH 5.0 to pH 7.0. The temperature condition is preferably 4 ° C. to 40 ° C., more preferably 10 ° C. to 30 ° C. The protein concentration is preferably 0.1 mg / ml to 100 mg / ml, more preferably 1 mg / ml to 10 mg / ml. The reaction time is preferably 1 minute to 24 hours, more preferably 10 minutes to 5 hours.
ＳＬＯをラテックス粒子へ感作する際のＳＬＯに関しては適宜決められるが、好ましくは０．１〜１０．０（ｍｇ／ｍｌ）、さらに好ましくは０．３〜６．０（ｍｇ／ｍｌ）、特に好ましくは０．７５〜３．０（ｍｇ／ｍｌ）の範囲である。 The SLO for sensitizing the SLO to the latex particles is appropriately determined, but is preferably 0.1 to 10.0 (mg / ml), more preferably 0.3 to 6.0 (mg / ml), particularly. It is preferably in the range of 0.75 to 3.0 (mg / ml).
（ブロッキング）
また、本発明の免疫測定試薬においては、非特異反応を抑制するためや免疫測定試薬自体の安定性を高めるために、特に限定されるものではないが、例えば、アルブミン、カゼイン、ゼラチンなどの蛋白質、これら蛋白質の分解物、アミノ酸、界面活性剤等の１種類もしくは２種類以上を前記不溶性担体に更に担持させても良い。ブロッキング剤の濃度は適宜決められるが、好ましくは０．１％〜５％、さらに好ましくは１％〜３％である。処理温度についても適宜決められるが、好ましくは４℃〜３０℃、さらに好ましくは４℃〜２０℃である。処理時間についても適宜決められるが、好ましくは０．５時間〜２４時間、さらに好ましくは１時間〜１６時間である。(blocking)
Further, the immunoassay reagent of the present invention is not particularly limited in order to suppress a non-specific reaction or enhance the stability of the immunoassay reagent itself, but for example, proteins such as albumin, casein, and gelatin. , One or more kinds of decomposition products of these proteins, amino acids, surfactants and the like may be further supported on the insoluble carrier. The concentration of the blocking agent is appropriately determined, but is preferably 0.1% to 5%, more preferably 1% to 3%. The treatment temperature is also appropriately determined, but is preferably 4 ° C to 30 ° C, more preferably 4 ° C to 20 ° C. The treatment time is also appropriately determined, but is preferably 0.5 hour to 24 hours, more preferably 1 hour to 16 hours.
（洗浄）
ブロッキング反応後の洗浄工程においては、各種界面活性剤を含む緩衝液での洗浄を実施することが望ましい。この場合の緩衝液としては、リン酸緩衝液、ＭＥＳ緩衝液およびＭＯＰＳ緩衝液などが挙げられる。界面活性剤としては、Ｔｗｅｅｎ２０、ＴｒｉｔｏｎＸ−１００、ＣＨＡＰＳ、ＳＤＳ、デオキシコール酸などが挙げられる。使用濃度は適宜決められるが、例えば、０．１％のＴｗｅｅｎ２０を含むリン酸緩衝生理食塩水（ｐＨ７．４）などが好適である。洗浄時間は、１０〜１０００分程度が好ましく、より好ましくは２０〜３００分、特に好ましくは３０〜９０分である。(Washing)
In the washing step after the blocking reaction, it is desirable to carry out washing with a buffer solution containing various surfactants. Examples of the buffer solution in this case include a phosphate buffer solution, a MES buffer solution, and a MOPS buffer solution. Examples of the surfactant include
（測定試薬）
本発明において、ＡＳＯ試薬の好ましい態様としては、２つの試薬から構成されてなり、緩衝溶液を第１試薬、ストレプトリジンＯを感作したラテックス粒子を含むラテックス試液を第２試薬とする構成からなるものが挙げられる。(Measuring reagent)
In the present invention, a preferred embodiment of the ASO reagent is composed of two reagents, the buffer solution being the first reagent, and the latex reagent solution containing the latex particles sensitized to streptolysin O as the second reagent. Things can be mentioned.
本発明の免疫測定法としては、本発明のＡＳＯ試薬を用いるのであれば特に限定されるものではないが、例えば、被測定物質を含む媒体に本発明の免疫測定試薬を加え、被測定物質と本発明の免疫測定試薬に含まれるラテックス粒子上に固定された抗原とが抗原抗体反応により結合して生じた凝集を、光学的に観察または目視により観察する。抗原抗体反応のｐＨの好ましい下限は５、上限は１０であり、より好ましい下限は６、上限は９である。反応の温度の好ましい下限は４℃、上限は５０℃であり、より好ましい下限は２０℃、上限は４０℃である。反応時間は適宜決められるが、第２試薬と混合後、１〜１０分間程度反応させることが望ましい。目視により観察して判定する方法としては、例えば、試料と本発明の免疫測定試薬とを、判定板上にて室温で混合し、１〜５分間揺り動かした後、凝集の有無を判定する方法等が挙げられる。 The immunoassay method of the present invention is not particularly limited as long as the ASO reagent of the present invention is used, but for example, the immunoassay reagent of the present invention is added to a medium containing the substance to be measured to obtain the substance to be measured. Aggregation formed by binding to an antigen immobilized on latex particles contained in the immunoassay reagent of the present invention by an antigen-antibody reaction is observed optically or visually. The preferred lower limit and upper limit of the pH of the antigen-antibody reaction are 5, the more preferable lower limit is 6, and the upper limit is 9. The preferred lower limit of the reaction temperature is 4 ° C. and the upper limit is 50 ° C., the more preferable lower limit is 20 ° C. and the upper limit is 40 ° C. The reaction time is appropriately determined, but it is desirable to react with the second reagent for about 1 to 10 minutes. As a method of visually observing and judging, for example, a method of mixing a sample and the immunoassay reagent of the present invention on a judgment plate at room temperature, shaking for 1 to 5 minutes, and then judging the presence or absence of aggregation, etc. Can be mentioned.
上記媒体としては、被測定物質の種類に応じて適当な各種緩衝液が用いられる。係る緩衝液としては、被測定物質を失活させることがなく、かつ、抗原抗体反応を阻害しないようなイオン濃度やｐＨを有するものであればよく、例えば、リン酸緩衝液、グリシン緩衝液、トリス緩衝液、各種グッド緩衝液等の緩衝液が好適に挙げられる。これらの緩衝液は単独で用いてもよく、２種以上を併用してもよい。 As the medium, various appropriate buffer solutions are used depending on the type of the substance to be measured. The buffer solution may be any buffer solution having an ion concentration and pH that does not inactivate the substance to be measured and does not inhibit the antigen-antibody reaction. For example, a phosphate buffer solution, a glycine buffer solution, and the like. Buffer solutions such as Tris buffer and various Good buffers are preferable. These buffer solutions may be used alone or in combination of two or more.
上記媒体は、反応の感度を高めるために、水溶性添加剤、例えばポリエチレングリコール、カルボキシルメチルセルロース、メチルセルロース、デキストラン、ポリビニルピロリドン、ポリグリコシルエチルメタクリレート、プルラン、デキストラン、エルシナン等の水溶性高分子等を含有してもよい。また、特異性の向上、試薬の安定性向上等の目的から、カゼイン、ゼラチン等のタンパク質又はその分解物、変性物；塩化コリン等の第４級アンモニウム塩、ＥＤＴＡ、ポリアニオン、カオトロピックイオン（Ｃｌ−，Ｉ−，ＳＣＮ−等）、アミノ酸、界面活性剤等を含有してもよい。 The medium contains water-soluble additives such as polyethylene glycol, carboxylmethylcellulose, methylcellulose, dextran, polyvinylpyrrolidone, polyglycosylethylmethacrylate, pullulan, dextran, ersinan and the like in order to increase the sensitivity of the reaction. You may. In addition, for the purpose of improving specificity and stability of reagents, proteins such as casein and gelatin or their decomposition products and denaturants; quaternary ammonium salts such as choline chloride, EDTA, polyanions, chaotropic ions (Cl-) , I-, SCN-, etc.), amino acids, surfactants, etc. may be contained.
好ましい態様の一例を挙げると、特に第１試薬においてポリビニルピロリドンを使用することが望ましい。第１試薬に添加するポリビニルピロリドンの濃度としては、第１試薬溶液の最終濃度として、０．０５％〜０．４％、より好ましくは０．０７％〜０．３６％、さらに好ましくは０．０９％〜０．３３％、特に好ましくは０．１％〜０．３％である。 To give an example of a preferred embodiment, it is particularly desirable to use polyvinylpyrrolidone in the first reagent. The concentration of polyvinylpyrrolidone added to the first reagent is 0.05% to 0.4%, more preferably 0.07% to 0.36%, still more preferably 0., as the final concentration of the first reagent solution. It is 09% to 0.33%, particularly preferably 0.1% to 0.3%.
（ＡＳＯ試薬の評価方法）
ＡＳＯ試薬の性能としては、検出感度、測定範囲、測定精度、再現性、安定性などの項目が挙げられる。本発明によれば、ラテックス粒子のカルボキシル基含量を適宜、選択すれば、測定試薬の感度が向上する。ＡＳＯ試薬における測定レンジの上限としては、６００ＩＵ／ｍｌ、好ましくは８００ＩＵ／ｍｌ、より好ましくは１０００ＩＵ／ｍｌ、特に好ましくは１２００ＩＵ／ｍｌである。尚、ＩＵ／ｍｌとは、世界保健機関（ＷＨＯ）が定めた抗ストレプトリジンＯ抗体の国際単位に基づいたものである。このため、ＡＳＯ試薬の感度評価においては、上記の測定レンジを考慮することが好ましい。(ASO reagent evaluation method)
The performance of the ASO reagent includes items such as detection sensitivity, measurement range, measurement accuracy, reproducibility, and stability. According to the present invention, the sensitivity of the measurement reagent is improved by appropriately selecting the carboxyl group content of the latex particles. The upper limit of the measurement range of the ASO reagent is 600 IU / ml, preferably 800 IU / ml, more preferably 1000 IU / ml, and particularly preferably 1200 IU / ml. IU / ml is based on the international unit of anti-streptolysin O antibody defined by the World Health Organization (WHO). Therefore, it is preferable to consider the above measurement range in the sensitivity evaluation of the ASO reagent.
本発明におけるＡＳＯ試薬は、感度並びに広い範囲での定量性を有するものである。測定レンジの上限としては、６００ＩＵ／ｍｌ、好ましくは８００ＩＵ／ｍｌ、より好ましくは１０００ＩＵ／ｍｌ、特に好ましくは１２００ＩＵ／ｍｌである。ここで、ＩＵ／ｍｌとは、世界保健機関（ＷＨＯ）が定めた抗ストレプトリジンＯ抗体の国際単位に基づいたものである。 The ASO reagent in the present invention has sensitivity and quantification in a wide range. The upper limit of the measurement range is 600 IU / ml, preferably 800 IU / ml, more preferably 1000 IU / ml, and particularly preferably 1200 IU / ml. Here, IU / ml is based on the international unit of anti-streptolysin O antibody defined by the World Health Organization (WHO).
以下に、本発明を実施例により具体的に説明する。なお、本発明は、実施例に限定されるものではない。 Hereinafter, the present invention will be specifically described with reference to Examples. The present invention is not limited to the examples.
［実施例１］ＡＳＯラテックス試薬（第２試薬）の調製
メルク社製カルボキシル基導入ラテックス粒子（製品番号：ＰＳＩ９０−２１）０．１４ｍＬを遠心分離し、上清を抜き取った。次に５０ｍＭ ＭＥＳ（ｐＨ４．５）を１ｍＬ添加し、十分に懸濁した。その後、再び遠心分離を行い、上清を抜き取った。さらに、５０ｍＭ ＭＥＳ（ｐＨ４．５）を０．７ｍＬ添加した。これに４％カルボジイミド（ＥＤＣ）０．３５ｍＬ及び２．４％Ｎ−ヒドロキシスクシンイミド（ＮＨＳ）０．３５ｍＬを加えて、ラテックス粒子の１％濃度とした状態で、２５℃で１５分撹拌し、カルボキシル基を活性化した後、遠心分離を行い、上清を抜き取った。[Example 1] Preparation of ASO Latex Reagent (Second Reagent) 0.14 mL of Merck's carboxyl group-introduced latex particles (Product No .: PSI90-21) were centrifuged, and the supernatant was extracted. Next, 1 mL of 50 mM MES (pH 4.5) was added and sufficiently suspended. Then, centrifugation was performed again and the supernatant was extracted. Further, 0.7 mL of 50 mM MES (pH 4.5) was added. To this, 0.35 mL of 4% carbodiimide (EDC) and 0.35 mL of 2.4% N-hydroxysuccinimide (NHS) were added, and the mixture was stirred at 25 ° C. for 15 minutes at a concentration of 1% of latex particles, and carboxyl. After activating the group, centrifugation was performed and the supernatant was extracted.
これに各種ｐＨ（ｐＨ６．０、ｐＨ６．６、ｐＨ７．５及びｐＨ７．８）の５０ｍＭ カリウムリン酸緩衝液（以下、「ＫＰＢ」と示す。）を０．７ｍＬ添加し、再び、遠心分離を行い、上清を抜き取った。さらに、各ｐＨの５０ｍＭ ＫＰＢをそれぞれ０．７ｍＬ添加し、超音波にて分散させることで、活性化ラテックス粒子とした。ＷＯ／２０１７／２０４３２５の実施例２に記載の方法で調製したＳＬＯを３．０（ｍｇ／ｍＬ）の濃度になるよう、同じく各種ｐＨ（ｐＨ６．０、ｐＨ６．６、ｐＨ７．５及びｐＨ７．８）の５０ｍＭ ＫＰＢで溶解した。次に、活性化ラテックス粒子０．７ｍＬとＳＬＯ溶液０．７ｍＬを同ｐＨの組み合わせで混合し（５０ｍＭ ＫＰＢ（ｐＨ６．０）で懸濁したラテックス粒子は５０ｍＭ ＫＰＢ（ｐＨ６．０）で溶解したＳＬＯと混合）、２５℃にて２時間撹拌混合することで、ＳＬＯをラテックス粒子に共有結合させた。 To this, 0.7 mL of 50 mM potassium phosphate buffer (hereinafter referred to as “KPB”) of various pH (pH 6.0, pH 6.6, pH 7.5 and pH 7.8) was added, and centrifugation was performed again. And the supernatant was extracted. Further, 0.7 mL of 50 mM KPB at each pH was added and dispersed by ultrasonic waves to obtain activated latex particles. Similarly, various pHs (pH 6.0, pH 6.6, pH 7.5 and pH 7.) were prepared so that the SLO prepared by the method described in Example 2 of WO / 2017/20432 had a concentration of 3.0 (mg / mL). It was dissolved in 50 mM KPB of 8). Next, 0.7 mL of activated latex particles and 0.7 mL of SLO solution were mixed in the same pH combination (latex particles suspended at 50 mM KPB (pH 6.0) were dissolved at 50 mM KPB (pH 6.0) SLO. SLO was covalently bonded to the latex particles by stirring and mixing at 25 ° C. for 2 hours.
次に、遠心分離を行い、上清を除去した後、ブロッキンング溶液（２％ＢＳＡを含む１×ＰＢＳ（ｐＨ７．４））で２ｍＬに再懸濁した。再び、遠心分離により、上清を除去し、１．４ｍＬのブロッキング溶液を添加し、超音波にて分散させた後、４℃で一晩、撹拌した。その後、遠心分離を行い、上清を除去し、最終緩衝液（０．５％ＢＳＡ、０．１％Ｔｗｅｅｎ２０及び０．０５％アジ化ナトリウムを含む１×ＰＢＳ（ｐＨ７．４））１．４ｍＬに再懸濁した。続いて、超音波にて分散させた後、４℃で１時間、撹拌した。再び、遠心分離により、上清を除去し、２ｍＬの最終緩衝液を添加し、超音波にて十分に分散させた後、２．６７ｍＬの最終緩衝液を添加して、第２試薬とした。
The supernatant was then removed by centrifugation and then resuspended in 2 mL with blocking solution (1 x PBS (pH 7.4) containing 2% BSA). Again, the supernatant was removed by centrifugation, 1.4 mL of blocking solution was added, sonicated and then stirred at 4 ° C. overnight. Then, centrifugation is performed, the supernatant is removed, and 1.4 mL of the final buffer (1 × PBS (pH 7.4) containing 0.5% BSA, 0.1
［実施例２］ＡＳＯラテックス試薬の感度評価
本発明の免疫学的ラテックス試薬における第１試薬としては、０．５％ＢＳＡを含む１×ＰＢＳ（ｐＨ７．４）を調製した。次に、自動分析装置日立７１８０（日立ハイテクノロジーズ製）を用いて、各ラテックス粒子を用いた場合の感度比較を実施した。まず、第１試薬１２０μｌに各濃度（８００、４００、１００ＩＵ／ｍｌ）のＡＳＯ標準液１．５μｌを添加撹拌し、３７℃で適時保持した後、第２試薬３０μｌを添加撹拌し、この後、３００秒後から６００秒後間の波長５４６ｎｍでの吸光度変化（ΔｍＡｂｓ）を評価した。その結果、ｐＨ７．５やｐＨ７．８の条件下で感作したラテックス粒子を用いた場合は、４００（ＩＵ／ｍＬ）で感度がほぼ定常にとなったが、ｐＨ６．０やｐＨ６．６の条件下で感作したラテックス粒子を用いた場合は、８００（ＩＵ／ｍＬ）まで感度が上昇することを見出した。結果を、表１及び図１に示す。[Example 2] Sensitivity evaluation of ASO latex reagent As the first reagent in the immunological latex reagent of the present invention, 1 × PBS (pH 7.4) containing 0.5% BSA was prepared. Next, a sensitivity comparison was carried out when each latex particle was used using an automatic analyzer Hitachi 7180 (manufactured by Hitachi High Technologies). First, 1.5 μl of ASO standard solution having each concentration (800, 400, 100 IU / ml) is added and stirred to 120 μl of the first reagent, and the mixture is kept at 37 ° C. for an appropriate time, and then 30 μl of the second reagent is added and stirred. The change in absorbance (ΔmAbs) at a wavelength of 546 nm between 300 seconds and 600 seconds was evaluated. As a result, when latex particles sensitized under the conditions of pH 7.5 and pH 7.8 were used, the sensitivity became almost constant at 400 (IU / mL), but the sensitivity was pH 6.0 or pH 6.6. It was found that when latex particles sensitized under the conditions were used, the sensitivity increased to 800 (IU / mL). The results are shown in Table 1 and FIG.
［実施例３］ＡＳＯラテックス試薬（第２試薬）の調製
表２に示した５ロットのメルク社製カルボキシル基導入ラテックス粒子（製品番号：ＰＳＩ９０−２１）０．１４ｍＬをそれぞれ遠心分離し、上清を抜き取った。以後、それぞれのロットは同様の実験操作を行った。次に５０ｍＭ ＭＥＳ（ｐＨ４．５）を１ｍＬ添加し、十分に懸濁した。その後、再び遠心分離を行い、上清を抜き取った。さらに、５０ｍＭ ＭＥＳ（ｐＨ４．５）を０．７ｍＬ添加した。これに４％カルボジイミド（ＥＤＣ）０．３５ｍＬ及び２．４％Ｎ−ヒドロキシスクシンイミド（ＮＨＳ）０．３５ｍＬを加えて、ラテックス粒子の１％濃度とした状態で、２５℃で１５分撹拌し、カルボキシル基を活性化した後、遠心分離を行い、上清を抜き取った。これに５０ｍＭ カリウムリン酸緩衝液（以下、「ＫＰＢ」と示す。）（ｐＨ６．５）を０．７ｍＬ添加し、再び、遠心分離を行い、上清を抜き取った。これに５０ｍＭ ＫＰＢ（ｐＨ６．５）を０．７ｍＬ添加し、超音波にて分散させることで、活性化ラテックス粒子とした。ＷＯ／２０１７／２０４３２５の実施例２に記載の方法で調製したストレプトリジンＯ（ＳＬＯ）を３．０（ｍｇ／ｍＬ）の濃度になるよう５０ｍＭ ＫＰＢ（ｐＨ６．５）で溶解した。次に、活性化ラテックス粒子０．７ｍＬとＳＬＯ溶液０．７ｍＬを混合し、２５℃にて２時間撹拌混合することで、ＳＬＯをラテックス粒子に共有結合させた。[Example 3] Preparation of ASO Latex Reagent (Second Reagent) 0.14 mL of each of the 5 lots of Merck carboxyl group-introduced latex particles (Product No .: PSI90-21) shown in Table 2 were centrifuged, and the supernatant was obtained. Was pulled out. After that, each lot performed the same experimental operation. Next, 1 mL of 50 mM MES (pH 4.5) was added and sufficiently suspended. Then, centrifugation was performed again and the supernatant was extracted. Further, 0.7 mL of 50 mM MES (pH 4.5) was added. To this, 0.35 mL of 4% carbodiimide (EDC) and 0.35 mL of 2.4% N-hydroxysuccinimide (NHS) were added, and the mixture was stirred at 25 ° C. for 15 minutes at a concentration of 1% of latex particles, and carboxyl. After activating the group, centrifugation was performed and the supernatant was extracted. 0.7 mL of 50 mM potassium phosphate buffer (hereinafter referred to as “KPB”) (pH 6.5) was added thereto, and centrifugation was performed again to remove the supernatant. 0.7 mL of 50 mM KPB (pH 6.5) was added thereto and dispersed by ultrasonic waves to obtain activated latex particles. Streptolysin O (SLO) prepared by the method described in Example 2 of WO / 2017/20432 was dissolved in 50 mM KPB (pH 6.5) to a concentration of 3.0 (mg / mL). Next, 0.7 mL of activated latex particles and 0.7 mL of SLO solution were mixed and mixed by stirring at 25 ° C. for 2 hours to covalently bond SLO to the latex particles.
次に、遠心分離を行い、上清を除去した後、ブロッキンング溶液（２％ＢＳＡを含む１×ＰＢＳ（ｐＨ７．４））で２ｍＬに再懸濁した。再び、遠心分離により、上清を除去し、１．４ｍＬのブロッキング溶液を添加し、超音波にて分散させた後、４℃で一晩、撹拌した。その後、遠心分離を行い、上清を除去し、最終緩衝液（０．５％ＢＳＡ、０．１％Ｔｗｅｅｎ２０及び０．０５％アジ化ナトリウムを含む１×ＰＢＳ（ｐＨ７．４））１．４ｍＬに再懸濁した。続いて、超音波にて分散させた後、４℃で１時間、撹拌した。再び、遠心分離により、上清を除去し、２ｍＬの最終緩衝液を添加し、超音波にて十分に分散させた後、２．６７ｍＬの最終緩衝液を添加して、第２試薬とした。
The supernatant was then removed by centrifugation and then resuspended in 2 mL with blocking solution (1 x PBS (pH 7.4) containing 2% BSA). Again, the supernatant was removed by centrifugation, 1.4 mL of blocking solution was added, sonicated and then stirred at 4 ° C. overnight. Then, centrifugation is performed, the supernatant is removed, and 1.4 mL of the final buffer (1 × PBS (pH 7.4) containing 0.5% BSA, 0.1
［実施例４］ＡＳＯラテックス試薬の感度評価
本発明の免疫学的ラテックス試薬における第１試薬としては、０．５％ＢＳＡを含む１×ＰＢＳ（ｐＨ７．４）を調製した。次に、自動分析装置日立７１８０（日立ハイテクノロジーズ製）を用いて、各ラテックス粒子を用いた場合の感度比較を実施した。まず、第１試薬１２０μｌに各濃度（４００、１００ＩＵ／ｍｌ）のＡＳＯ標準液１．５μｌを添加撹拌し、３７℃で適時保持した後、第２試薬６０μｌを添加撹拌し、この後、３００秒後から６００秒後間の波長５４６ｎｍでの吸光度変化（ΔｍＡｂｓ）を評価した。その結果、メルク社製カルボキシル基導入ラテックス粒子（製品番号：ＰＳＩ９０−２１）のＬｏｔ Ｎｏ．８００／７９（１３３ｎｍ、ＣＯＯＨ＝１０９μｅｑ／ｇ）、Ｌｏｔ Ｎｏ．３９３０／９（１０６ｎｍ、ＣＯＯＨ＝１０７μｅｑ／ｇ）及びＬｏｔ Ｎｏ．３７７８／１７（１１３ｎｍ、ＣＯＯＨ＝１３５μｅｑ／ｇ）を用いて、ＡＳＯ試薬を調製した場合は、図２に示すように高感度な試薬を調製することができた。一方、Ｌｏｔ Ｎｏ．２３７４／２２（１２８ｎｍ、ＣＯＯＨ＝１７２μｅｑ／ｇ）及びＬｏｔ Ｎｏ．２５４２／７（１０４ｎｍ、ＣＯＯＨ＝１６８μｅｑ／ｇ）を用いて、ＡＳＯ試薬を調製した場合は感度が悪いことが明らかとなった。以上の結果より、ＳＬＯのアミノ基を共有結合によりラテックス粒子に感作する際には、ラテックス粒子のカルボキシル基含量が感度に影響をすることを明らかにした。[Example 4] Sensitivity evaluation of ASO latex reagent As the first reagent in the immunological latex reagent of the present invention, 1 × PBS (pH 7.4) containing 0.5% BSA was prepared. Next, using an automatic analyzer Hitachi 7180 (manufactured by Hitachi High-Technologies Corporation), a sensitivity comparison was carried out when each latex particle was used. First, 1.5 μl of ASO standard solution of each concentration (400, 100 IU / ml) is added and stirred to 120 μl of the first reagent, and after holding at 37 ° C. for a timely period, 60 μl of the second reagent is added and stirred, and then 300 seconds. The change in absorbance (ΔmAbs) at a wavelength of 546 nm after 600 seconds was evaluated. As a result, the Lot No. of the carboxyl group-introduced latex particles (product number: PSI90-21) manufactured by Merck & Co., Inc. 800/79 (133 nm, COOH = 109 μeq / g), Lot No. 3930/9 (106 nm, COOH = 107 μeq / g) and Lot No. When the ASO reagent was prepared using 3778/17 (113 nm, COOH = 135 μeq / g), a highly sensitive reagent could be prepared as shown in FIG. On the other hand, Lot No. 2374/22 (128 nm, COOH = 172 μeq / g) and Lot No. It was revealed that the sensitivity was poor when the ASO reagent was prepared using 2542/7 (104 nm, COOH = 168 μeq / g). From the above results, it was clarified that when the amino group of SLO is covalently bonded to the latex particles, the carboxyl group content of the latex particles affects the sensitivity.
[実施例５] ＡＳＯラテックス試薬（第２試薬）の調製におけるＳＬＯ濃度の影響
実施例３に記載の方法でラテックス試薬（第２試薬）を調製した。尚、その際、ラテックス粒子には表２中のＬｏｔ Ｎｏ．８００／７９（１３３ｎｍ、ＣＯＯＨ＝１０９μｅｑ／ｇ）を使用し、ストレプトリジンＯ（ＳＬＯ）を１．５（ｍｇ／ｍＬ）、２．０（ｍｇ／ｍＬ）、３．０（ｍｇ／ｍＬ）、４．０（ｍｇ／ｍＬ）、５．０（ｍｇ／ｍＬ）及び６．０（ｍｇ／ｍＬ）の濃度になるよう５０ｍＭ ＫＰＢ（ｐＨ６．５）でそれぞれ溶解したものを調製し、ＳＬＯ濃度の影響を検討した。実施例３に記載の通り、ＳＬＯをラテックス粒子に共有結合させた際は、活性化ラテックス粒子０．７ｍＬとＳＬＯ溶液０．７ｍＬを混合したため、感作時のＳＬＯ濃度はそれぞれ０．７５（ｍｇ／ｍＬ）、１．０（ｍｇ／ｍＬ）、１．５（ｍｇ／ｍＬ）、２．０（ｍｇ／ｍＬ）、２．５（ｍｇ／ｍＬ）及び３．０（ｍｇ／ｍＬ）となった。その結果、終濃度１．０（ｍｇ／ｍＬ）で感作した際、感度は最も高くなりことが明らかとなった。一方で、終濃度１．０（ｍｇ／ｍＬ）より高濃度のＳＬＯを感作すると、感度は下がるものの、８００ＩＵ／ｍｌまでの希釈直線性は高まることが明らかとなった。結果を図３に示した。[Example 5] Effect of SLO concentration on preparation of ASO latex reagent (second reagent) Latex reagent (second reagent) was prepared by the method described in Example 3. At that time, the latex particles were labeled with Lot No. in Table 2. Using 800/79 (133 nm, COOH = 109 μeq / g), streptidine O (SLO) was 1.5 (mg / mL), 2.0 (mg / mL), 3.0 (mg / mL), Prepare each dissolved in 50 mM KPB (pH 6.5) so as to have concentrations of 4.0 (mg / mL), 5.0 (mg / mL) and 6.0 (mg / mL), and prepare the SLO concentration. The impact was examined. As described in Example 3, when SLO was co-bonded to latex particles, 0.7 mL of activated latex particles and 0.7 mL of SLO solution were mixed, so that the SLO concentration at the time of sensitization was 0.75 (mg). / ML), 1.0 (mg / mL), 1.5 (mg / mL), 2.0 (mg / mL), 2.5 (mg / mL) and 3.0 (mg / mL) rice field. As a result, it was clarified that the sensitivity was the highest when sensitized at a final concentration of 1.0 (mg / mL). On the other hand, it was revealed that when SLO having a concentration higher than the final concentration of 1.0 (mg / mL) was sensitized, the sensitivity decreased, but the dilution linearity up to 800 IU / ml increased. The results are shown in FIG.
[実施例６] ＡＳＯラテックス試薬（第２試薬）の調製におけるＳＬＯ濃度及びラテックス粒子のロット間差の影響
実施例５に記載のＳＬＯ濃度がＡＳＯラテックス試薬の感度に与える影響に関して、ラテックス粒子のロット間差が出ないことを確認するため、表２に示されているＬｏｔ Ｎｏ．８００／７９（１３３ｎｍ、ＣＯＯＨ＝１０９μｅｑ／ｇ）に加えて、表２中のＬｏｔ Ｎｏ．３７７８／１７（１１３ｎｍ、ＣＯＯＨ＝１３５μｅｑ／ｇ）及び新規ロットとして、同メルク社製カルボキシル基導入ラテックス粒子（製品番号：ＰＳＩ９０−２１）のＬｏｔ Ｎｏ．３７７８／１７（１１３ｎｍ、ＣＯＯＨ＝１３５μｅｑ／ｇ）を用いて、実施例５と同様の実験を行った。その結果、カルボキシル基含量が同等のラテックス粒子の場合は実施例５の結果と同等の結果が得られることを明らかにした。結果を図４に示す。[Example 6] Effect of difference between lots of SLO concentration and latex particles in preparation of ASO latex reagent (second reagent) With respect to the effect of the SLO concentration described in Example 5 on the sensitivity of ASO latex reagent, a lot of latex particles. In order to confirm that there is no difference, the Lot Nos. shown in Table 2 are shown. In addition to 800/79 (133 nm, COOH = 109 μeq / g), Lot No. in Table 2 3778/17 (113 nm, COOH = 135 μeq / g) and as a new lot, Lot No. of carboxyl group-introduced latex particles (product number: PSI90-21) manufactured by Merck & Co., Inc. The same experiment as in Example 5 was performed using 3778/17 (113 nm, COOH = 135 μeq / g). As a result, it was clarified that in the case of latex particles having the same carboxyl group content, the same result as the result of Example 5 can be obtained. The results are shown in FIG.
本発明は、測定感度に優れた診断薬に用いられるラテックス粒子を提供するものとして、特に医療分野において有用な技術を提供するものである。
The present invention provides a technique particularly useful in the medical field as a latex particle used for a diagnostic agent having excellent measurement sensitivity.
Claims (9)
Claims 1 to 2, which use any buffer selected from the group consisting of phosphate buffer, MES buffer and MOPS buffer when covalently bonding streptolysin O to latex particles having a carboxyl group on the surface. 8. The method for producing latex particles for measuring anti-streptolysin O antibody according to any one of 8.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4329151A (en) * | 1979-08-17 | 1982-05-11 | Icl Scientific | Stable diagnostic reagent and method for qualitative determinations of streptococci infections |
| JPH10239316A (en) * | 1997-02-24 | 1998-09-11 | Sekisui Chem Co Ltd | Carrier for immunodiagnosis |
| WO2001092885A1 (en) * | 2000-05-30 | 2001-12-06 | Iatron Laboratories, Inc. | Immunological latex turbidimetry method and reagent therefor |
| JP2002082117A (en) * | 2000-09-08 | 2002-03-22 | Sekisui Chem Co Ltd | Test kit |
| JP2003344410A (en) * | 2002-05-23 | 2003-12-03 | Sekisui Chem Co Ltd | Immuno-measurement reagent and immuno-measurement method |
| WO2018043687A2 (en) * | 2016-08-31 | 2018-03-08 | 積水化学工業株式会社 | Fluorescent particles for diagnostic agent and immunoassay reagent using same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4329151A (en) * | 1979-08-17 | 1982-05-11 | Icl Scientific | Stable diagnostic reagent and method for qualitative determinations of streptococci infections |
| JPH10239316A (en) * | 1997-02-24 | 1998-09-11 | Sekisui Chem Co Ltd | Carrier for immunodiagnosis |
| WO2001092885A1 (en) * | 2000-05-30 | 2001-12-06 | Iatron Laboratories, Inc. | Immunological latex turbidimetry method and reagent therefor |
| JP2002082117A (en) * | 2000-09-08 | 2002-03-22 | Sekisui Chem Co Ltd | Test kit |
| JP2003344410A (en) * | 2002-05-23 | 2003-12-03 | Sekisui Chem Co Ltd | Immuno-measurement reagent and immuno-measurement method |
| WO2018043687A2 (en) * | 2016-08-31 | 2018-03-08 | 積水化学工業株式会社 | Fluorescent particles for diagnostic agent and immunoassay reagent using same |
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