JP2024044817A - Method for inhibiting sedimentation of specific binding substance-immobilized carrier particles - Google Patents
Method for inhibiting sedimentation of specific binding substance-immobilized carrier particles Download PDFInfo
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Abstract
【課題】測定対象物質に特異的に結合する物質を固定化した担体粒子を緩衝液中で保存した際の、該担体粒子の沈降を抑制できる方法を提供する。【解決手段】本発明の担体粒子の沈降抑制方法は、測定対象物質に特異的に結合する物質を固定化した担体粒子を、グッド緩衝剤及びアルカリ金属ハロゲン化物からなる群から選ばれる少なくとも１種を含有する緩衝液中で保存するものである。【選択図】なし[Problem] To provide a method capable of suppressing the precipitation of carrier particles onto which a substance that specifically binds to a substance to be measured is immobilized when the carrier particles are stored in a buffer solution. [Solution] The method for suppressing the precipitation of carrier particles of the present invention comprises storing carrier particles onto which a substance that specifically binds to a substance to be measured is immobilized in a buffer solution containing at least one selected from the group consisting of Good's buffer and an alkali metal halide. [Selected Figure] None
Description
本発明は、測定対象物質を測定する際に用いる、測定対象物質に対して特異的に結合する物質を固定化した担体粒子の沈降抑制方法に関するものである。
本発明は、特に、化学、生命科学、分析化学及び臨床検査等の分野において有用なものである。
The present invention relates to a method for suppressing sedimentation of carrier particles on which a substance that specifically binds to a substance to be measured is immobilized, which is used when measuring a substance to be measured.
The present invention is particularly useful in fields such as chemistry, life science, analytical chemistry, and clinical testing.
抗原と抗体、糖とレクチン、ヌクレオチド鎖とそれに相補的なヌクレオチド鎖、リガンドとレセプター等の特異的な親和性を有する物質間の反応を利用した試料中に含まれる測定対象物質の測定試薬及び測定方法は種々のものが知られている。 There are various known measurement reagents and methods for measuring a substance contained in a sample that utilize reactions between substances with specific affinity, such as antigens and antibodies, sugars and lectins, nucleotide chains and complementary nucleotide chains, and ligands and receptors.
これは、試料中に含まれる測定対象物質と、この測定対象物質に対して特異的に結合する物質（測定対象物質に対する特異的結合物質）との結合の有無、又は結合の量を測ることにより、試料中に含まれる測定対象物質の有無の測定〔定性測定〕、又はその含有量（濃度）の測定〔定量測定〕を行うものである。 This is done by measuring the presence or absence of binding, or the amount of binding, between the analyte contained in the sample and a substance that specifically binds to the analyte (substance that specifically binds to the analyte). , to measure the presence or absence of a substance to be measured contained in a sample (qualitative measurement), or to measure its content (concentration) (quantitative measurement).
特に、測定対象物質に対する特異的結合物質を担体に固定化したものを使用して測定を行う方法、又は測定試薬が繁用されている。 In particular, a method or measurement reagent that uses a carrier on which a substance that specifically binds to the substance to be measured is immobilized is frequently used.
例えば、抗原と抗体の間の抗原抗体反応（免疫反応、免疫学的反応）を利用した免疫学的測定試薬（免疫学的測定方法）においては、ラテックス粒子を担体として使用するラテックス免疫比濁法；ラテックス粒子、有機高分子粒子、無機粒子、金属粒子、若しくは赤血球などを担体として使用する間接凝集反応測定法；マイクロタイタープレート、ビーズ、粒子、試験管、若しくは容器などを担体として使用する酵素免疫測定法、発光免疫測定法などの標識免疫測定法；又は、セルロース、若しくは不織布などを担体として使用するイムノクロマトグラフィー法等が繁用されている。 For example, in immunological measurement reagents (immunological measurement methods) that utilize the antigen-antibody reaction (immune reaction, immunological reaction) between an antigen and an antibody, latex immunoturbidimetry using latex particles as a carrier is used. ; Indirect agglutination measurement method using latex particles, organic polymer particles, inorganic particles, metal particles, or red blood cells as carriers; Enzyme immunoassay using microtiter plates, beads, particles, test tubes, containers, etc. as carriers Measurement methods, labeled immunoassay methods such as luminescence immunoassay; or immunochromatography methods using cellulose, nonwoven fabric, etc. as carriers are frequently used.
これらの中でも、ラテックス粒子を担体として使用するラテックス免疫比濁法は、操作が簡便であり測定時間も短いため、病院や診療所等の医療機関の臨床検査室等で繁用されている。 Among these, the latex immunoturbidimetric method, which uses latex particles as a carrier, is widely used in clinical laboratories of medical institutions such as hospitals and clinics because it is easy to operate and has a short measurement time.
しかしながら、このラテックス免疫比濁法は、担体として使用するラテックス粒子が、その保存中に沈降を生じやすいため、ラテックス粒子が沈降した場合には、測定時に転倒混和等によって、ラテックス粒子を均一にする必要があり、操作が煩雑となり、試料中の測定対象物質の測定に誤差が生じ易いという問題点がある。 However, in this latex immunoturbidimetric method, the latex particles used as a carrier tend to sediment during storage, so if the latex particles settle, it is necessary to make the latex particles uniform by inverting and mixing during measurement. There are problems in that it is necessary, the operation is complicated, and errors are likely to occur in the measurement of the substance to be measured in the sample.
そこで、ラテックス粒子等の沈降を抑制するため、ラテックス粒子等の分散液中に、ポリアニオンおよびその塩、デキストラン、シクロデキストリン、ポリエチレングリコール、およびグリセロールからなる群より選択される少なくとも１種の化合物を共
存させる方法（例えば、特許文献１参照。）が提案されているが、必ずしも完全とは言えなかった。
In order to suppress the settling of latex particles and the like, a method has been proposed in which at least one compound selected from the group consisting of polyanions and their salts, dextran, cyclodextrin, polyethylene glycol, and glycerol is allowed to coexist in a dispersion of latex particles and the like (see, for example, Patent Document 1). However, this method is not necessarily complete.
従って、本発明の課題は、測定対象物質に特異的に結合する物質を固定化した担体粒子を緩衝液中で保存した際の、該担体粒子の沈降を抑制できる方法を提供することである。 Therefore, an object of the present invention is to provide a method that can suppress sedimentation of carrier particles on which a substance that specifically binds to a substance to be measured is stored in a buffer solution.
本発明者は、上記課題の解決を目指して鋭意検討を行った結果、測定対象物質に特異的に結合する物質を固定化した担体粒子を緩衝液中で保存する際に、緩衝液にグッド緩衝剤及びアルカリ金属ハロゲン化物からなる群から選ばれる少なくとも１種を含有させることにより、担体粒子の沈降を抑制できることを見出し、本発明を完成するに至った。 As a result of intensive studies aimed at solving the above problems, the present inventor discovered that when storing carrier particles on which a substance that specifically binds to a substance to be measured is stored in a buffer solution, Good buffer is added to the buffer solution. The present inventors have discovered that sedimentation of carrier particles can be suppressed by containing at least one member selected from the group consisting of agents and alkali metal halides, and have completed the present invention.
すなわち、本発明は、以下の発明を提供する。
（１） 測定対象物質に特異的に結合する物質を固定化した担体粒子を、グッド緩衝剤及びアルカリ金属ハロゲン化物からなる群から選ばれる少なくとも１種を含有する緩衝液中で保存することを特徴とする、担体粒子の沈降抑制方法。
（２） 測定対象物質に特異的に結合する物質が抗ＨＭＧＢ１抗体である、前記（１）に記載の担体粒子の沈降抑制方法。
（３） 担体粒子がラテックス粒子である、前記（１）又は（２）に記載の担体粒子の沈降抑制方法。
（４） アルカリ金属ハロゲン化物が、塩化ナトリウム、塩化カリウム、又は臭化ナトリウムである、前記（１）又は（２）に記載の担体粒子の沈降抑制方法。
（５） アルカリ金属ハロゲン化物が、塩化ナトリウム、塩化カリウム、又は臭化ナトリウムである、前記（３）に記載の担体粒子の沈降抑制方法。
（６） グッド緩衝剤及びアルカリ金属ハロゲン化物からなる群から選ばれる少なくとも１種を含有する緩衝液中で保存された、抗ＨＭＧＢ１抗体を固定化した担体粒子を用いることを特徴とする、試料中のＨＭＧＢ１の測定方法。
（７） 担体粒子がラテックス粒子である、前記（６）に記載の試料中のＨＭＧＢ１の測定方法。
（８） アルカリ金属ハロゲン化物が、塩化ナトリウム、塩化カリウム、又は臭化ナトリウムである、前記（６）又は（７）に記載の試料中のＨＭＧＢ１の測定方法。
That is, the present invention provides the following inventions.
(1) A method for inhibiting the sedimentation of carrier particles, characterized in that carrier particles having immobilized thereon a substance that specifically binds to a substance to be measured are stored in a buffer solution containing at least one selected from the group consisting of Good's buffer and an alkali metal halide.
(2) The method for inhibiting precipitation of carrier particles according to (1) above, wherein the substance that specifically binds to the substance to be measured is an anti-HMGB1 antibody.
(3) The method for inhibiting precipitation of carrier particles according to (1) or (2) above, wherein the carrier particles are latex particles.
(4) The method for inhibiting precipitation of carrier particles according to (1) or (2) above, wherein the alkali metal halide is sodium chloride, potassium chloride, or sodium bromide.
(5) The method for inhibiting precipitation of carrier particles according to (3) above, wherein the alkali metal halide is sodium chloride, potassium chloride, or sodium bromide.
(6) A method for measuring HMGB1 in a sample, characterized by using carrier particles having anti-HMGB1 antibodies immobilized thereon, the carrier particles being stored in a buffer solution containing at least one selected from the group consisting of Good's buffer and an alkali metal halide.
(7) The method for measuring HMGB1 in a sample according to (6) above, wherein the carrier particles are latex particles.
(8) The method for measuring HMGB1 in a sample according to (6) or (7) above, wherein the alkali metal halide is sodium chloride, potassium chloride, or sodium bromide.
本発明は、測定対象物質に特異的に結合する物質を固定化した担体粒子の沈降を抑制することができるものである。
そして、これにより、担体粒子が沈降することなく、長期間保存することができるため、例えば、本発明により沈降が抑制された担体粒子をラテックス比濁法等の測定に用いることにより、誤差を含まない、安定した性能が得られる測定を行うことができるものである。
The present invention is capable of suppressing sedimentation of carrier particles on which a substance that specifically binds to a substance to be measured is immobilized.
As a result, the carrier particles can be stored for a long period of time without sedimentation, so that, for example, by using the carrier particles whose sedimentation has been suppressed according to the present invention in measurements such as latex turbidimetry, errors can be eliminated. It is possible to perform measurements that provide stable performance.
以下、本発明を詳細に説明するが、以下の実施の形態は、本発明を説明するための例示であり、本発明はこの実施の形態に限定されるものではない。また、本発明は、その要旨を逸脱しない限り、様々な形態で実施することができる。 The present invention will be described in detail below, but the following embodiments are illustrative for explaining the present invention, and the present invention is not limited to these embodiments. Moreover, the present invention can be implemented in various forms without departing from the gist thereof.
１．測定対象物質に特異的に結合する物質を固定化した担体粒子の沈降抑制方法
本発明の担体粒子の沈降抑制方法は、測定対象物質に特異的に結合する物質を固定化した担体粒子を、グッド緩衝剤及びアルカリ金属ハロゲン化物からなる群から選ばれる少なくとも１種を含有する緩衝液中で保存するものである。
1. Method for inhibiting sedimentation of carrier particles having immobilized thereon a substance that specifically binds to a substance to be measured The method for inhibiting sedimentation of carrier particles of the present invention comprises storing carrier particles having immobilized thereon a substance that specifically binds to a substance to be measured in a buffer solution containing at least one selected from the group consisting of Good's buffer and an alkali metal halide.
（１）測定対象物質に特異的に結合する物質
本発明において、測定対象物質に特異的に結合する物質とは、測定対象物質に特異的に結合することができる物質であれば特に限定はない。
(1) Substance that specifically binds to the substance to be measured In the present invention, the substance that specifically binds to the substance to be measured is not particularly limited as long as it is a substance that can specifically bind to the substance to be measured. .
この測定対象物質に特異的に結合する物質としては、例えば、測定対象物質に結合することができる抗体（抗測定対象物質抗体）、アプタマー（核酸アプタマー若しくはペプチドアプタマー）、アフィボディー、糖若しくはレクチン、ヌクレオチド鎖、又はレセプター等を挙げることができる。 Examples of the substance that specifically binds to the analyte are antibodies that can bind to the analyte (anti-analyte antibody), aptamers (nucleic acid aptamers or peptide aptamers), affibodies, sugars or lectins, Examples include nucleotide chains and receptors.
なお、測定対象物質に結合することができる抗体（抗測定対象物質抗体）としては、例えば、測定対象物質に結合することができるモノクローナル抗体、ポリクローナル抗体、抗血清、抗体の断片〔Ｆａｂ及びＦ（ａｂ’）２など〕、又は一本鎖抗体（ｓｃＦｖ）等を挙げることができる。 In addition, examples of antibodies capable of binding to the analyte (anti-analyte antibody) include monoclonal antibodies, polyclonal antibodies, antiserum, antibody fragments [Fab and F( ab')2], single chain antibodies (scFv), and the like.
また、本発明においては、測定対象物質に特異的に結合する物質が、特に抗ＨＭＧＢ１抗体であることが好ましい。 Furthermore, in the present invention, it is particularly preferable that the substance that specifically binds to the substance to be measured is an anti-HMGB1 antibody.
（２）担体粒子
本発明において、担体粒子は、前記の測定対象物質に特異的に結合する物質を固定化することができるものであれば特に限定されない。
(2) Carrier Particles In the present invention, the carrier particles are not particularly limited as long as they are capable of immobilizing a substance that specifically binds to the above-mentioned target substance to be measured.
この担体粒子の材質は、特に限定はなく、例えば、ポリスチレン、スチレン－スチレンスルホン酸塩共重合体、アクリロニトリル－ブタジエン－スチレン共重合体、塩化ビニル－アクリル酸エステル共重合体、酢酸ビニル－アクリル酸共重合体、ポリアクロレイン、スチレン－メタクリル酸共重合体、スチレン－グリシジル（メタ）アクリル酸共重合体、スチレン－ブタジエン共重合体、メタクリル酸重合体、アクリル酸重合体、ゼラチン、シリカ、アルミナ、カーボンブラック、金属化合物、金属、セラミックス又は磁性体等を挙げることができる。 The material of the carrier particles is not particularly limited, and examples include polystyrene, styrene-styrene sulfonate copolymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylic acid ester copolymer, vinyl acetate-acrylic acid. Copolymer, polyacrolein, styrene-methacrylic acid copolymer, styrene-glycidyl (meth)acrylic acid copolymer, styrene-butadiene copolymer, methacrylic acid polymer, acrylic acid polymer, gelatin, silica, alumina, Examples include carbon black, metal compounds, metals, ceramics, and magnetic materials.
そして、この担体粒子としては、例えば、ラテックス粒子、金属コロイド粒子、リポソーム、マイクロカプセル、又は赤血球等の粒子等を挙げることができる。
また、本発明においては、担体粒子が、特にラテックス粒子であることが好ましい。
Examples of the carrier particles include latex particles, metal colloid particles, liposomes, microcapsules, and particles such as red blood cells.
Further, in the present invention, the carrier particles are preferably latex particles.
本発明において、測定対象物質に特異的に結合する物質を担体粒子に固定化することは、物理的吸着法、化学的結合法又はこれらの併用等の公知の方法により行うことができる。
物理的吸着法による場合は、公知の方法に従い、測定対象物質に特異的に結合する物質と、担体粒子とを、緩衝液等の溶液中で混合し接触させたり、或いは緩衝液等に溶解した測定対象物質に特異的に結合する物質を、担体粒子に接触させること等により行うことができる。
In the present invention, a substance that specifically binds to a substance to be measured can be immobilized on carrier particles by a known method such as a physical adsorption method, a chemical bonding method, or a combination thereof.
When using the physical adsorption method, a substance that specifically binds to the substance to be measured and carrier particles are mixed in a solution such as a buffer solution and brought into contact with each other, or dissolved in a buffer solution or the like, according to a known method. This can be carried out by, for example, bringing a substance that specifically binds to the substance to be measured into contact with carrier particles.
また、化学的結合法により行う場合は、日本臨床病理学会編「臨床病理臨時増刊特集第５３号 臨床検査のためのイムノアッセイ－技術と応用－」，臨床病理刊行会，１９８３年発行；日本生化学会編「新生化学実験講座１ タンパク質ＩＶ」，東京化学同人，１９９１年発行等に記載の公知の方法に従い、測定対象物質に特異的に結合する物質と、担体粒子とを、グルタルアルデヒド、カルボジイミド、イミドエステル又はマレイミド等の二価性の架橋試薬と混合、接触させ、測定対象物質に特異的に結合する物質と、担体粒子の、それぞれのアミノ基、カルボキシル基、チオール基、アルデヒド基又は水酸基等と前記の二価性の架橋試薬とを反応させること等により行うことができる。 In addition, when using a chemical binding method, it can be performed according to known methods described in "Clinical Pathology Special Issue No. 53: Immunoassays for Clinical Tests - Techniques and Applications" edited by the Japanese Society of Clinical Pathology, published by Clinical Pathology Publishing Society, 1983; "New Biochemical Experiment Course 1: Protein IV" edited by the Japanese Biochemical Society, published by Tokyo Kagaku Dojin, 1991, etc., by mixing and contacting a substance that specifically binds to the substance to be measured and carrier particles with a bivalent cross-linking reagent such as glutaraldehyde, carbodiimide, imide ester, or maleimide, and reacting the amino groups, carboxyl groups, thiol groups, aldehyde groups, hydroxyl groups, etc. of the substance that specifically binds to the substance to be measured and the carrier particles with the bivalent cross-linking reagent.
更に、測定対象物質に特異的に結合する物質を固定化した担体粒子の非特異的反応等を抑制するために処理を行う必要があれば、測定対象物質に特異的に結合する物質を固定化した担体粒子の表面に、ウシ血清アルブミン（ＢＳＡ）、カゼイン、ゼラチン、卵白アルブミン若しくはその塩などのタンパク質、界面活性剤又は脱脂粉乳等を接触させ被覆させること等の公知の方法により処理して、担体粒子のブロッキング処理（マスキング処理）を行ってもよい。 Furthermore, if it is necessary to perform treatment to suppress non-specific reactions of carrier particles on which a substance that specifically binds to the substance to be measured is immobilized, the substance that specifically binds to the substance to be measured is immobilized. The surface of the prepared carrier particles is treated by a known method such as contacting and coating a protein such as bovine serum albumin (BSA), casein, gelatin, ovalbumin or a salt thereof, a surfactant, or skim milk powder, etc., The carrier particles may be subjected to blocking treatment (masking treatment).
（３）グッド緩衝剤及びアルカリ金属ハロゲン化物
本発明において、グッド緩衝剤については、担体粒子を緩衝液中で保存した際の、該担体粒子の沈降を抑制させる作用を有するものであれば特に限定されないが、例えば、Ｎ－（２－アセトアミド）－２－アミノエタンスルホン酸（ＡＣＥＳ）、Ｎ－（２－アセトアミド）イミノジ酢酸（ＡＤＡ）、Ｎ，Ｎ‐ビス（２‐ヒドロキシエチル）‐２‐アミノエタンスルホン酸（ＢＥＳ）、Ｎ，Ｎ－ビス（２－ヒドロキシエチル）グリシン（Ｂｉｃｉｎｅ）、２，２－ビス（ヒドロキシエチル）－（イミノトリス）－（ヒドロキシメチル）メタン（Ｂｉｓ－Ｔｒｉｓ）、１，３－ビストリスヒドロキシメチルメチルアミノプロパン（Ｂｉｓ－Ｔｒｉｓプロパン）、Ｎ－シクロヘキシル－２－ヒドロキシ－３－アミノプロパンスルホン酸（ＣＡＰＳＯ）、Ｎ－シクロヘキシル－２－ヒドロキシ－３－アミノエタンスルホン酸（ＣＡＰＳ）、Ｎ－シクロヘキシル－２－アミノエタンスルホン酸（ＣＨＥＳ）、３－［Ｎ，Ｎ－ビス（ヒドロキシエチル）アミノ］－２－ヒドロキシプロパンスルホン酸（ＤＩＰＳＯ）、２－〔４－（２－ヒドロキシエチル）－１－ピペラジニル〕エタンスルホン酸（ＨＥＰＥＳ）、２－ヒドロキシ－３－［４－（２－ヒドロキシエチル）－１－ピペラジニル］－プロパンスルホン酸モノハイドレート（ＨＥＰＰＳＯ）、２－モルホリノエタンスルホン酸（ＭＥＳ）、３－モルホリノプロパンスルホン酸（ＭＯＰＳ）、２－ヒドロキシ－３－モルホリノプロパンスルホン酸（ＭＯＰＳＯ）、ピペラジン－１，４－ビス（２－エタンスルホン酸）（ＰＩＰＥＳ）、ピペラジン－１，４－ビス（２－ヒドロキシ－３－プロパンスルホン酸）デハイドレート（ＰＯＰＳＯ）、Ｎ－トリス（ヒドロキシメチル）メチル－３－アミノプロパンスルホン酸（ＴＡＰＳ）、Ｎ－トリス（ヒドロキシメチル）メチル－２－ヒドロキシ－３－アミノプロパンスルホン酸（ＴＡＰＳＯ）、Ｎ－トリス（ヒドロキシメチル）メチル－２－アミノエタンスルホン酸（ＴＥＳ）、若しくはＮ－［トリス（ヒドロキシメチル）メチル］グリシン（Ｔｒｉｃｉｎｅ）、又はこれらの塩等を挙げることができる。
(3) Good's buffer and alkali metal halide In the present invention, the Good's buffer is particularly limited as long as it has the effect of suppressing sedimentation of carrier particles when the carrier particles are stored in a buffer solution. However, for example, N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), N-(2-acetamido)iminodiacetic acid (ADA), N,N-bis(2-hydroxyethyl)-2- Aminoethanesulfonic acid (BES), N,N-bis(2-hydroxyethyl)glycine (Bicine), 2,2-bis(hydroxyethyl)-(iminotris)-(hydroxymethyl)methane (Bis-Tris), 1 , 3-Bis-Trishydroxymethylaminopropane (Bis-Trispropane), N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (CAPSO), N-cyclohexyl-2-hydroxy-3-aminoethanesulfonic acid ( CAPS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 3-[N,N-bis(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), 2-[4-(2- Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]-propanesulfonic acid monohydrate (HEPPSO), 2-morpholinoethane Sulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), piperazine- 1,4-bis(2-hydroxy-3-propanesulfonic acid) dehydrate (POPSO), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), N-tris(hydroxymethyl)methyl- 2-hydroxy-3-aminopropanesulfonic acid (TAPSO), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), or N-[tris(hydroxymethyl)methyl]glycine (Tricine), or These salts can be mentioned.
また、本発明において、アルカリ金属ハロゲン化物については、担体粒子を緩衝液中で保存した際の、該担体粒子の沈降を抑制させる作用を有するものであれば特に限定されないが、特に、塩化ナトリウム、塩化カリウム、又は臭化ナトリウムを使用することが好ましい。 In the present invention, the alkali metal halide is not particularly limited as long as it has the effect of suppressing the precipitation of the carrier particles when the carrier particles are stored in a buffer solution, but it is particularly preferable to use sodium chloride, potassium chloride, or sodium bromide.
なお、このグッド緩衝剤及びアルカリ金属ハロゲン化物は、１種類のものだけを用いてもよいし、又は複数種類のものを同時に用いてもよい。 The Good's buffer and alkali metal halide may be used alone or in combination.
また、緩衝液に含有させるグッド緩衝剤及びアルカリ金属ハロゲン化物の濃度については、担体粒子を緩衝液中で保存した際の、該担体粒子の沈降を抑制させる作用を発揮する濃度であれば特に限定されず、その種類に応じて適宜設定される。 In addition, the concentration of Good's buffer and alkali metal halide to be contained in the buffer solution is particularly limited as long as it has the effect of suppressing sedimentation of the carrier particles when the carrier particles are stored in the buffer solution. It is set appropriately depending on the type.
例えば、グッド緩衝剤を使用する場合には、０．２５ｍｏｌ／Ｌ以上であればよく、０．２５～１ｍｏｌ／Ｌの範囲にあれば好ましく、０．５～１ｍｏｌ／Ｌの範囲であることがより好ましく、０．７５～１ｍｏｌ／Ｌの範囲が特に好ましい。
また、アルカリ金属ハロゲン化物を使用する場合には、０．５ｍｏｌ／Ｌ以上であればよく、０．５～３ｍｏｌ／Ｌの範囲にあれば好ましく、０．７５～２ｍｏｌ／Ｌの範囲であることがより好ましく、１～２ｍｏｌ／Ｌの範囲が特に好ましい。
For example, when using a Good buffer, it is sufficient if it is 0.25 mol/L or more, preferably in the range of 0.25 to 1 mol/L, and preferably in the range of 0.5 to 1 mol/L. More preferably, the range is from 0.75 to 1 mol/L.
In addition, when using an alkali metal halide, it is sufficient if it is 0.5 mol/L or more, preferably in the range of 0.5 to 3 mol/L, and preferably in the range of 0.75 to 2 mol/L. is more preferable, and a range of 1 to 2 mol/L is particularly preferable.
なお、本発明において、担体粒子を保存するための緩衝液は、特に限定されないが、例えば、水、生理食塩水又はトリス（ヒドロキシメチル）アミノメタン緩衝液〔Ｔｒｉｓ緩衝液〕、リン酸緩衝液若しくはリン酸緩衝生理食塩水などの各種緩衝液等の水系溶媒を用いることができる。 In the present invention, the buffer solution for storing the carrier particles is not particularly limited, but may be, for example, water, physiological saline, or an aqueous solvent such as various buffer solutions such as tris(hydroxymethyl)aminomethane buffer solution [Tris buffer solution], phosphate buffer solution, or phosphate buffered physiological saline solution.
２．試料中のＨＭＧＢ１の測定方法
本発明の試料中のＨＭＧＢ１の測定方法は、「試料中のＨＭＧＢ１」と「抗ＨＭＧＢ１抗体を固定化した担体粒子」とを接触させ、ＨＭＧＢ１を介して結合した「抗ＨＭＧＢ１抗体を固定化した担体粒子」の凝集物を測定することにより、試料中のＨＭＧＢ１の濃度を測定するものである。
2. Method for measuring HMGB1 in a sample The method for measuring HMGB1 in a sample of the present invention involves contacting "HMGB1 in a sample" with "carrier particles having immobilized anti-HMGB1 antibodies" and measuring the aggregates of "carrier particles having immobilized anti-HMGB1 antibodies" bound via HMGB1, thereby measuring the concentration of HMGB1 in the sample.
すなわち、「試料中のＨＭＧＢ１」と「抗ＨＭＧＢ１抗体を固定化した担体粒子」とを接触させ、ＨＭＧＢ１を介して結合した「抗ＨＭＧＢ１抗体を固定化した担体粒子」の免疫複合体凝集物の生成を、その透過光や散乱光を光学的方法により測るか、又は目視的に測るものである。つまり、「抗原抗体反応による複合体」の凝集物の生成を測るものである（凝集反応法）。 That is, "HMGB1 in the sample" and "carrier particles on which anti-HMGB1 antibody is immobilized" are brought into contact, and an immune complex aggregate of "carrier particles on which anti-HMGB1 antibody is immobilized" bound via HMGB1 is generated. The transmitted light and scattered light are measured by an optical method or visually. In other words, it measures the formation of aggregates of "complexes resulting from antigen-antibody reactions" (agglutination reaction method).
なお、本発明は、「試料中のＨＭＧＢ１」を「グッド緩衝剤及びアルカリ金属ハロゲン化物からなる群から選ばれる少なくとも１種を含有する緩衝液中で保存された、抗ＨＭＧＢ１抗体を固定化した担体粒子」と接触させることを特徴とするものである。 The present invention is characterized in that "HMGB1 in a sample" is contacted with "carrier particles having anti-HMGB1 antibodies immobilized thereon, the carrier particles being stored in a buffer solution containing at least one selected from the group consisting of Good's buffer and an alkali metal halide."
この抗ＨＭＧＢ１抗体を固定化した担体粒子の凝集物の測定方法としては、例えば、ラテックス比濁法、ラテックス凝集反応法又は粒子凝集反応法等を挙げることができる。 Examples of methods for measuring aggregates of carrier particles on which anti-HMGB1 antibodies are immobilized include latex turbidimetry, latex agglutination method, and particle agglutination method.
抗ＨＭＧＢ１抗体を固定化した担体粒子の凝集物の測定を、ラテックス比濁法、ラテックス凝集反応法又は粒子凝集反応法等の免疫複合体凝集物の生成を、その透過光や散乱光を光学的方法により測るか、又は目視的に測る測定法により実施する場合には、溶媒としてリン酸緩衝液、グリシン緩衝液、トリス緩衝液又はグッド緩衝液等を用いることができる。 Measurement of aggregates of carrier particles on which anti-HMGB1 antibodies are immobilized can be carried out using latex turbidimetry, latex agglutination reaction method, particle agglutination reaction method, etc., and the generation of immune complex aggregates can be performed by optically measuring the transmitted light and scattered light. When the measurement is carried out by a measuring method or a visual measuring method, a phosphate buffer, a glycine buffer, a Tris buffer, a Good's buffer, or the like can be used as a solvent.
なお、ラテックス比濁法を測定原理とする場合、担体として用いるラテックス粒子の粒径については、特に制限はないものの、ラテックス粒子が測定対象物質（ＨＭＧＢ１）を介して結合し、凝集物を生成する程度、及びこの生成した凝集物の測定の容易さ等の理由より、ラテックス粒子の粒径は、その平均粒径が、０．０４～１μｍであることが好ましい。 In addition, when using latex turbidimetry as the measurement principle, there is no particular restriction on the particle size of the latex particles used as a carrier, but the latex particles may bond through the substance to be measured (HMGB1) to form aggregates. For reasons such as ease of measurement of the produced aggregates and the like, the average particle size of the latex particles is preferably 0.04 to 1 μm.
また、ラテックス比濁法を測定原理とする場合、抗ＨＭＧＢ１抗体を固定化したラテックス粒子を含ませる濃度については、試料中のＨＭＧＢ１の濃度、本発明における抗体のラテックス粒子表面上での分布密度、ラテックス粒子の粒径、試料と測定試薬の混合比率等の各種条件により最適な濃度は異なるので一概にいうことはできない。 In addition, when using latex turbidimetry as the measurement principle, the concentration of latex particles immobilized with anti-HMGB1 antibody is determined by the concentration of HMGB1 in the sample, the distribution density of the antibody on the latex particle surface of the present invention, The optimal concentration cannot be generalized because it varies depending on various conditions such as the particle size of the latex particles and the mixing ratio of the sample and the measurement reagent.
しかし、通常は、試料と測定試薬が混合され、ラテックス粒子に固定化された「抗ＨＭＧＢ１抗体」と試料中に含まれていた「ＨＭＧＢ１」との抗原抗体反応が行われる測定反応時に、「抗ＨＭＧＢ１抗体を固定化したラテックス粒子」の濃度が、「試料中のＨＭＧＢ１」と「抗ＨＭＧＢ１抗体を固定化したラテックス粒子」との接触時において０．００５～１％（ｗ／ｖ）となるようにするのが一般的であり、この場合、「試料中のＨＭＧＢ１」と「抗ＨＭＧＢ１抗体を固定化したラテックス粒子」との接触時においてこのような濃度になるような濃度の「抗ＨＭＧＢ１抗体を固定化したラテックス粒子」を試料中のＨＭＧＢ１の測定試薬に含ませる。 However, typically, during the measurement reaction in which the sample and the measurement reagent are mixed and an antigen-antibody reaction takes place between the "anti-HMGB1 antibody" immobilized on the latex particles and the "HMGB1" contained in the sample, the concentration of the "latex particles with immobilized anti-HMGB1 antibody" is adjusted to 0.005 to 1% (w/v) when the "HMGB1 in the sample" comes into contact with the "latex particles with immobilized anti-HMGB1 antibody." In this case, the measurement reagent for HMGB1 in the sample contains a concentration of "latex particles with immobilized anti-HMGB1 antibody" that will result in such a concentration when the "HMGB1 in the sample" comes into contact with the "latex particles with immobilized anti-HMGB1 antibody."
また、ラテックス凝集反応法又は粒子凝集反応法等の間接凝集反応法を測定原理とする場合、担体として用いる粒子の粒径については、特に制限はないものの、その平均粒子径が０．０１～１００μｍの範囲内にあることが好ましく、０．３～１０μｍの範囲内にあることがより好ましい。そして、これらの粒子の比重は、１～１０の範囲内にあることが好ましく、１～２の範囲内にあることがより好ましい。 When the measurement principle is an indirect agglutination method such as a latex agglutination method or a particle agglutination method, there are no particular restrictions on the particle size of the particles used as the carrier, but the average particle size is preferably in the range of 0.01 to 100 μm, and more preferably in the range of 0.3 to 10 μm. The specific gravity of these particles is preferably in the range of 1 to 10, and more preferably in the range of 1 to 2.
なお、ラテックス凝集反応法又は粒子凝集反応法等の間接凝集反応法を測定原理とする場合の測定に使用する容器としては、例えば、ガラス、ポリスチレン、ポリ塩化ビニル又はポリメタクリレートなどからなる、試験管、マイクロプレート（マイクロタイタープレート）又はトレイ等を挙げることができる。これらの容器の溶液収容部分（マイクロプレートのウェル等）の底面は、Ｕ型、Ｖ型又はＵＶ型等の底面中央から周辺にかけて傾斜を持つ形状であることが好ましい。 In addition, when the measuring principle is indirect aggregation reaction method such as latex agglutination reaction method or particle agglutination reaction method, the container used for the measurement is, for example, a test tube made of glass, polystyrene, polyvinyl chloride, or polymethacrylate. , a microplate (microtiter plate), or a tray. The bottom surface of the solution-accommodating portion of these containers (wells of microplates, etc.) is preferably U-shaped, V-shaped, or UV-shaped, with a slope from the center of the bottom surface to the periphery.
測定の操作法は公知の方法等により行うことができるが、例えば、光学的方法により測定する場合には、試料と「抗ＨＭＧＢ１抗体を固定化した担体粒子」を反応させ、エンドポイント法又はレート法により、透過光や散乱光を測定する。
また、目視的に測定する場合には、プレートやマイクロプレート等の前記容器中で、試料と「抗ＨＭＧＢ１抗体を固定化した担体粒子」を反応させ、凝集の状態を目視的に判定する。なお、この目視的に測定する代わりにマイクロプレートリーダー等の機器を用いて測定を行ってもよい。
The measurement can be performed by known methods, etc., but for example, when measuring by an optical method, the sample is reacted with "carrier particles having immobilized anti-HMGB1 antibodies" and the transmitted light or scattered light is measured by an endpoint method or a rate method.
In addition, in the case of visual measurement, the sample is reacted with "anti-HMGB1 antibody-immobilized carrier particles" in the above-mentioned container such as a plate or microplate, and the state of agglutination is visually determined. Note that instead of this visual measurement, the measurement may be performed using an instrument such as a microplate reader.
なお、本発明の試料中のＨＭＧＢ１の測定方法における試料としては、血液、血清、血漿、尿、髄液、唾液、汗、涙、腹水もしくは羊水などの体液；大便；血管もしくは肝臓などの臓器；組織；細胞；又は大便、臓器、組織もしくは細胞などの抽出液等、ＨＭＧＢ１が含まれる可能性のある生体試料であれば対象となる。 In addition, samples in the method for measuring HMGB1 in a sample of the present invention include body fluids such as blood, serum, plasma, urine, spinal fluid, saliva, sweat, tears, ascites or amniotic fluid; stool; organs such as blood vessels or liver; Any biological sample that may contain HMGB1, such as tissue; cells; or extracts of stool, organs, tissues, or cells, is eligible.
以下、本発明を実施例により具体的に説明するが、本発明はこれらの実施例により限定されるものではない。 EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention is not limited to these Examples.
〔実施例１〕（抗ＨＭＧＢ１抗体（２Ｈ６）の調製）
抗ＨＭＧＢ１抗体（２Ｈ６）として、受託番号ＮＩＴＥ Ｐ－０２８４３として寄託されたハイブリドーマにより産生されたモノクローナル抗体を用いた。
なお、受託番号ＮＩＴＥ Ｐ－０２８４３で特定されるハイブリドーマは、２０１８年１２月２０日付けで、独立行政法人製品評価技術基盤機構の特許微生物寄託センター［ＮＰＭＤ］（日本国千葉県木更津市かずさ鎌足二丁目５番８号）に寄託されている。
Example 1 Preparation of anti-HMGB1 antibody (2H6)
As the anti-HMGB1 antibody (2H6), a monoclonal antibody produced by the hybridoma deposited under accession number NITE P-02843 was used.
The hybridoma identified by the accession number NITE P-02843 was deposited at the National Institute of Technology and Evaluation's Patent Microorganism Deposit Center [NPMD] (5-8 Kazusa Kamatari 2-chome, Kisarazu City, Chiba Prefecture, Japan) on December 20, 2018.
〔実施例２〕（抗ＨＭＧＢ１抗体（２Ｄ４）の調製）
抗ＨＭＧＢ１抗体（２Ｄ４）として、受託番号ＮＩＴＥ Ｐ－０２８４２として寄託されたハイブリドーマにより産生されたモノクローナル抗体を用いた。
なお、受託番号ＮＩＴＥ Ｐ－０２８４２で特定されるハイブリドーマは、２０１８年１２月２０日付けで、独立行政法人製品評価技術基盤機構の特許微生物寄託センター［ＮＰＭＤ］（日本国千葉県木更津市かずさ鎌足二丁目５番８号）に寄託されている。
[Example 2] (Preparation of anti-HMGB1 antibody (2D4))
As the anti-HMGB1 antibody (2D4), a monoclonal antibody produced by a hybridoma deposited under accession number NITE P-02842 was used.
The hybridoma identified by the accession number NITE P-02842 was deposited at the National Institute of Technology and Evaluation's Patent Microorganism Deposit Center [NPMD] (5-8 Kazusa Kamatari 2-chome, Kisarazu City, Chiba Prefecture, Japan) on December 20, 2018.
〔実施例３〕（抗ＨＭＧＢ１，２モノクローナル抗体の調製）
ＨＭＧＢ１及びＨＭＧＢ２に結合するモノクローナル抗体（抗ＨＭＧＢ１，２モノクローナル抗体）として、受託番号ＮＩＴＥ Ｐ－０２８４４として寄託されたハイブリドーマにより産生されたモノクローナル抗体を用いた。
なお、受託番号ＮＩＴＥ Ｐ－０２８４４で特定されるハイブリドーマは、２０１８年１２月２０日付けで、独立行政法人製品評価技術基盤機構の特許微生物寄託センター［ＮＰＭＤ］（日本国千葉県木更津市かずさ鎌足二丁目５番８号）に寄託されている。
[Example 3] (Preparation of anti-HMGB1,2 monoclonal antibodies)
As the monoclonal antibody that binds to HMGB1 and HMGB2 (anti-HMGB1,2 monoclonal antibody), a monoclonal antibody produced by a hybridoma deposited under accession number NITE P-02844 was used.
The hybridoma identified by the accession number NITE P-02844 was deposited at the National Institute of Technology and Evaluation's Patent Microorganism Deposit Center [NPMD] (5-8 Kazusa Kamatari 2-chome, Kisarazu City, Chiba Prefecture, Japan) on December 20, 2018.
〔実施例４〕（グッド緩衝剤又はアルカリ金属ハロゲン化物の効果の確認）
グッド緩衝剤又はアルカリ金属ハロゲン化物を緩衝液に含有させた場合の担体粒子の沈降抑制効果を確認した。
[Example 4] (Confirmation of the effect of Good buffer or alkali metal halide)
The effect of suppressing sedimentation of carrier particles when Good's buffer or an alkali metal halide was contained in the buffer was confirmed.
〔１〕試料中のＨＭＧＢ１の測定試薬
１．第１試薬
２００ｍＭの緩衝液を含む溶液（ｐＨ８．０）を第１試薬とした。
[1] Reagents for measuring HMGB1 in a sample 1. First reagent A solution containing 200 mM buffer (pH 8.0) was used as the first reagent.
２．第２試薬
（１）前記実施例１で調製した抗体（２Ｈ６）と前記実施例２で調製した抗体（２Ｄ４）を等量混合して、抗ＨＭＧＢ１モノクローナル抗体の混合物を調製した。これを、「抗ＨＭＧＢ１モノクローナル抗体混合物」と名付けた。
2. Second Reagent (1) Equal amounts of the antibody (2H6) prepared in Example 1 and the antibody (2D4) prepared in Example 2 were mixed to prepare a mixture of anti-HMGB1 monoclonal antibodies. This was named "anti-HMGB1 monoclonal antibody mixture."
（２）前記（１）の抗ＨＭＧＢ１モノクローナル抗体混合物を、緩衝液で１～２ｍｇ／ｍＬになるよう希釈し、この抗体液１ｍＬとラテックス粒子（藤倉化成社［日本国］）１ｍＬを混合して接触させ、２～８℃で１６時間静置し、抗ＨＭＧＢ１モノクローナル抗体混合物をラテックス粒子に固定化した。 (2) Dilute the anti-HMGB1 monoclonal antibody mixture from (1) above with a buffer solution to 1 to 2 mg/mL, and mix 1 mL of this antibody solution with 1 mL of latex particles (Fujikura Kasei Co., Ltd. [Japan]). The anti-HMGB1 monoclonal antibody mixture was immobilized on the latex particles by contacting and standing at 2 to 8°C for 16 hours.
（３） 抗ＨＭＧＢ１モノクローナル抗体混合物を固定化したラテックス粒子に、５％ウシ血清アルブミン（ＢＳＡ）を含むトリス緩衝液（ｐＨ７．４）の６．０ｍＬを添加混合し、ブロッキング処理を行い、ブロッキング処理を行った抗ＨＭＧＢ１モノクローナル抗体混合物を固定化したラテックス粒子を得た。 (3) 6.0 mL of Tris buffer (pH 7.4) containing 5% bovine serum albumin (BSA) was added to and mixed with the latex particles onto which the anti-HMGB1 monoclonal antibody mixture was immobilized, and a blocking treatment was performed to obtain latex particles onto which the blocked anti-HMGB1 monoclonal antibody mixture was immobilized.
（４）前記実施例３で調製した抗ＨＭＧＢ１，２モノクローナル抗体を、緩衝液で１～２ｍｇ／ｍＬになるよう希釈し、この抗体液１ｍＬとラテックス粒子（藤倉化成社［日本国］）１ｍＬを混合して接触させ、２～８℃で１６時間静置し、抗ＨＭＧＢ１，２モノクローナル抗体をラテックス粒子に固定化した。 (4) Dilute the anti-HMGB1,2 monoclonal antibody prepared in Example 3 above with a buffer solution to a concentration of 1 to 2 mg/mL, and add 1 mL of this antibody solution and 1 mL of latex particles (Fujikura Kasei Co., Ltd. [Japan]). The anti-HMGB1,2 monoclonal antibody was immobilized on the latex particles by mixing and contacting and standing at 2 to 8°C for 16 hours.
（５）抗ＨＭＧＢ１，２モノクローナル抗体を固定化したラテックス粒子に、５％ウシ血清アルブミン（ＢＳＡ）を含むトリス緩衝液（ｐＨ７．４）の６．０ｍＬを添加混合し、ブロッキング処理を行い、ブロッキング処理を行った抗ＨＭＧＢ１，２モノクローナル抗体を固定化したラテックス粒子を得た。 (5) 6.0 mL of Tris buffer (pH 7.4) containing 5% bovine serum albumin (BSA) was added to and mixed with the latex particles with anti-HMGB1,2 monoclonal antibodies to perform a blocking treatment, thereby obtaining latex particles with anti-HMGB1,2 monoclonal antibodies that had been subjected to a blocking treatment.
（６）前記（３）で調製したブロッキング処理を行った抗ＨＭＧＢ１モノクローナル抗体混合物を固定化したラテックス粒子と、前記（４）で調製したブロッキング処理を行った抗ＨＭＧＢ１，２モノクローナル抗体を固定化したラテックス粒子とを、それぞれ等量混合して、抗ＨＭＧＢ１モノクローナル抗体を固定化したラテックス粒子の混合物を調製した。これを、「抗ＨＭＧＢ１モノクローナル抗体固定化ラテックス粒子混合物」と名付けた。 (6) Equal amounts of latex particles immobilized with the anti-HMGB1 monoclonal antibody mixture prepared in (3) above and latex particles immobilized with the anti-HMGB1,2 monoclonal antibodies prepared in (4) above were mixed to prepare a mixture of latex particles immobilized with anti-HMGB1 monoclonal antibodies. This was named "anti-HMGB1 monoclonal antibody immobilized latex particle mixture."
（７）前記（６）の「抗ＨＭＧＢ１モノクローナル抗体固定化ラテックス粒子混合物」に、表１に記載したグッド緩衝剤又はアルカリ金属ハロゲン化物をそれぞれ表１に記載した濃度になるように添加して、グッド緩衝剤又はアルカリ金属ハロゲン化物をそれぞれ含有する（又は含有しない）１０種類の第２試薬を調製した。 (7) The Good's buffer or alkali metal halide shown in Table 1 was added to the "anti-HMGB1 monoclonal antibody-immobilized latex particle mixture" in (6) above to the concentrations shown in Table 1, to prepare 10 types of second reagents each containing (or not containing) a Good's buffer or an alkali metal halide.
〔２〕試料
生理食塩水（０．９％塩化ナトリウム水溶液）を試料として用いた。
[2] Sample Physiological saline (0.9% sodium chloride aqueous solution) was used as the sample.
〔３〕第２試薬の保存
前記〔１〕の２で調製した１０種類の第２試薬を試験管に５ｍＬずつ分注し、冷蔵（２～８℃）で１３カ月保存した。
[3] Storage of second reagents 5 mL of the 10 types of second reagents prepared in 2 of [1] above were dispensed into test tubes and stored under refrigeration (2 to 8°C) for 13 months.
〔４〕試料の測定
試料中のＨＭＧＢ１測定試薬の第１試薬として前記〔１〕の１の第１試薬を使用し、第２試薬として前記〔３〕の第２試薬を使用して、前記〔２〕の試料の測定を行った。
[4] Measurement of sample The sample in [2] was measured using the first reagent in [1] above as the first reagent for measuring HMGB1 in the sample, and the second reagent in [3] above as the second reagent.
測定は、日立７１８０形自動分析装置（日立ハイテク社［日本国］）を使用して行った。
前記〔２〕の試料に前記〔１〕の１の第１試薬を添加混合し、その５分後に前記〔３〕の第２試薬を添加混合した。
具体的には、前記〔２〕の試料の２４μＬを使用し、第１試薬として前記〔１〕の１の第１試薬の１２０μＬを使用し、第２試薬として前記〔３〕の第２試薬の４０μＬを使用して測定を行った。
The measurements were carried out using a Hitachi 7180 model automatic analyzer (Hitachi High-Tech Corporation, Japan).
The first reagent in item 1 of item 1 of item 1 of item 2 ...
Specifically, the measurement was performed using 24 μL of the sample described in [2] above, 120 μL of the first reagent described in [1] 1 above as the first reagent, and 40 μL of the second reagent described in [3] above as the second reagent.
なお、反応温度は３７℃、測定波長８００ｎｍにおいて、測定タイムコースの１６ポイント目と１７ポイント目（第２試薬添加直後）の吸光度を測定し、その差（吸光度差）を求め、これをラテックス濃度とした。なお、当該吸光度差（ラテックス濃度）は、前記〔３〕の第２試薬を添加混合する際に、転倒混和しなかった場合（転倒混和なし）と、測定直前に転倒混和した場合（転倒混和あり）のそれぞれについて求めた。 The reaction temperature was 37°C, and the measurement wavelength was 800 nm. The absorbance was measured at the 16th and 17th points (immediately after the addition of the second reagent) of the measurement time course, and the difference (absorbance difference) was calculated and used as the latex concentration. The absorbance difference (latex concentration) was calculated for the cases where the second reagent in [3] above was not mixed by inversion (no inversion mixing) and where it was mixed by inversion immediately before measurement (mixing by inversion).
〔５〕測定結果
試料の測定結果を表１に示した。また、表１に示した値は、試料を５回測定した際の吸光度差（ラテックス濃度）の平均値を示している。なお、表１に示した沈降抑制率は、転倒混和なしの吸光度差（単位：Ａｂｓ．）を転倒混和ありの吸光度差（単位：Ａｂｓ．）で除した値（単位：％）を示している。
[5] Measurement results The measurement results of the samples are shown in Table 1. The values shown in Table 1 indicate the average value of the absorbance difference (latex concentration) when the sample is measured five times. The sedimentation inhibition rate shown in Table 1 indicates the value (unit: %) obtained by dividing the absorbance difference (unit: Abs.) without inversion mixing by the absorbance difference (unit: Abs.) with inversion mixing.
表１から明らかなように、第２試薬にグッド緩衝剤又はアルカリ金属ハロゲン化物を含有させていない場合（無添加）は、転倒混和なしの場合の吸光度が極めて低く、沈降抑制率も５％となっている。これは、第２試薬の保存中に試薬中の抗体固定化ラテックス粒子が沈降してしまい、第１試薬に添加混合される抗体固定化ラテックス粒子の量が減少することで、吸光度が低くなったことに起因するものと推察される。
これに対して、第２試薬にグッド緩衝剤又はアルカリ金属ハロゲン化物を含有させた場合は、いずれの場合も、無添加の場合に比べて、転倒混和なしと転倒混和ありの吸光度差にほとんど変化がなく、沈降抑制率が改善されていることが分かる。
As is clear from Table 1, when the second reagent does not contain a Good's buffer or an alkali metal halide (no addition), the absorbance without mixing by inversion is extremely low and the sedimentation inhibition rate is also 5%. This is presumably due to the fact that the antibody-immobilized latex particles in the second reagent settle during storage, reducing the amount of antibody-immobilized latex particles added and mixed into the first reagent, thereby lowering the absorbance.
In contrast, when the second reagent contained Good's buffer or an alkali metal halide, in either case, there was almost no change in the absorbance difference between the cases with and without inversion mixing, compared to the case where no additive was added, indicating that the sedimentation inhibition rate was improved.
このように、抗体固定化ラテックス粒子を緩衝液中で保存する際に、緩衝液にグッド緩衝剤又はアルカリ金属ハロゲン化物を含有させることによって、抗体固定化ラテックス粒子の沈降を長期間にわたって抑制できることが確認された。したがって、測定時に転倒混和等によって、ラテックス粒子を均一にする必要がなく、誤差を含まない、安定した性能が得られる測定を行えることが確かめられた。 In this way, it was confirmed that when storing antibody-immobilized latex particles in a buffer solution, the sedimentation of the antibody-immobilized latex particles can be suppressed for a long period of time by adding a Good's buffer or an alkali metal halide to the buffer solution. Therefore, it was confirmed that it is not necessary to homogenize the latex particles by inversion mixing or the like during measurement, and that measurements can be performed that are free of errors and provide stable performance.
〔実施例５〕（グッド緩衝剤又はアルカリ金属ハロゲン化物の効果の確認）
グッド緩衝剤又はアルカリ金属ハロゲン化物を緩衝液に含有させた場合の担体粒子の沈降抑制効果を確認した。
Example 5 (Confirmation of the effect of Good's buffer or alkali metal halide)
The effect of suppressing the sedimentation of carrier particles when a Good's buffer or an alkali metal halide was contained in a buffer solution was confirmed.
〔１〕試料中のＨＭＧＢ１の測定試薬
１．第１試薬
２００ｍＭの緩衝液を含む溶液（ｐＨ８．０）を第１試薬とした。
[1] Reagent for measuring HMGB1 in sample 1. First Reagent A solution containing 200 mM buffer (pH 8.0) was used as the first reagent.
２．第２試薬
前記実施例４の〔１〕の（１）～（６）の記載の通りに操作を行い、抗ＨＭＧＢ１モノクローナル抗体を固定化したラテックス粒子の混合物を調製した。これを、「抗ＨＭＧＢ１モノクローナル抗体固定化ラテックス粒子混合物」と名付けた。
この「抗ＨＭＧＢ１モノクローナル抗体固定化ラテックス粒子混合物」に、表２に記載したグッド緩衝剤又はアルカリ金属ハロゲン化物をそれぞれ表２に記載した濃度になるように添加して、グッド緩衝剤又はアルカリ金属ハロゲン化物をそれぞれ含有する（又は含有しない）２３種類の第２試薬を調製した。
2. Second Reagent The operations were performed as described in (1) to (6) of [1] of Example 4 to prepare a mixture of latex particles on which anti-HMGB1 monoclonal antibody was immobilized. This was named "anti-HMGB1 monoclonal antibody-immobilized latex particle mixture."
Good's buffer or alkali metal halide listed in Table 2 was added to this "anti-HMGB1 monoclonal antibody-immobilized latex particle mixture" to the concentrations listed in Table 2, and Good's buffer or alkali metal halide was Twenty-three types of second reagents each containing (or not containing) a compound were prepared.
〔２〕試料
生理食塩水（０．９％塩化ナトリウム水溶液）を試料として用いた。
[2] Sample Physiological saline (0.9% sodium chloride aqueous solution) was used as a sample.
〔３〕第２試薬の保存
前記〔１〕の２で調製した２３種類の第２試薬を試験管に５ｍＬずつ分注し、冷蔵（２～８℃）で１カ月保存した。
[3] Storage of the second reagent The 23 types of second reagent prepared in [1] 2 above were dispensed into test tubes in 5 mL portions and stored in a refrigerator (2 to 8° C.) for one month.
〔４〕試料の測定
試料中のＨＭＧＢ１測定試薬の第１試薬として前記〔１〕の１の第１試薬を使用し、第２試薬として前記〔３〕の第２試薬を使用して、前記実施例４の〔４〕と同様にして前記〔２〕の試料の測定を行った。
[4] Measurement of sample The first reagent of 1 above [1] is used as the first reagent for measuring HMGB1 in the sample, and the second reagent of [3] above is used as the second reagent. The sample [2] was measured in the same manner as in Example 4 [4].
〔５〕測定結果
試料の測定結果を表２に示した。また、表２に示した値は、試料を５回測定した際の吸光度差（ラテックス濃度）の平均値を示している。なお、表２に示した沈降抑制率は、転倒混和なしの吸光度差（単位：Ａｂｓ．）を転倒混和ありの吸光度差（単位：Ａｂｓ．）で除した値（単位：％）を示している。
[5] Measurement results The measurement results of the samples are shown in Table 2. The values shown in Table 2 indicate the average value of the absorbance difference (latex concentration) when the sample is measured five times. The sedimentation inhibition rate shown in Table 2 indicates the value (unit: %) obtained by dividing the absorbance difference (unit: Abs.) without inversion mixing by the absorbance difference (unit: Abs.) with inversion mixing.
表２から明らかなように、第２試薬にグッド緩衝剤又はアルカリ金属ハロゲン化物を含有させていない場合（無添加）は、転倒混和なしの場合の吸光度が極めて低く、沈降抑制率も１８％となっている。これは、第２試薬の保存中に試薬中の抗体固定化ラテックス粒子が沈降してしまい、第１試薬に添加混合される抗体固定化ラテックス粒子の量が減少することで、吸光度が低くなったことに起因するものと推察される。
これに対して、第２試薬にグッド緩衝剤又はアルカリ金属ハロゲン化物を含有させた場合は、グッド緩衝剤又はアルカリ金属ハロゲン化物の濃度がいずれの場合も、無添加の場合に比べて、転倒混和なしと転倒混和ありの吸光度差の変化が少なく、沈降抑制率が改善されていることが分かる。
As is clear from Table 2, when the second reagent does not contain Good's buffer or alkali metal halide (no addition), the absorbance is extremely low without mixing by overturning, and the sedimentation inhibition rate is 18%. It has become. This is because the antibody-immobilized latex particles in the reagent precipitated during storage of the second reagent, reducing the amount of antibody-immobilized latex particles added to and mixed with the first reagent, resulting in lower absorbance. It is assumed that this is due to this.
On the other hand, when the second reagent contains Good's buffer or alkali metal halide, no matter the concentration of Good's buffer or alkali metal halide, mixing by overturning is more difficult than when no Good's buffer or alkali metal halide is added. It can be seen that there is little change in the difference in absorbance between without and with overturning mixing, indicating that the sedimentation suppression rate has been improved.
このように、抗体固定化ラテックス粒子を緩衝液中で保存する際に、緩衝液にグッド緩衝剤又はアルカリ金属ハロゲン化物を含有させることによって、抗体固定化ラテックス粒子の沈降を長期間にわたって抑制できることが確認された。したがって、測定時に転倒混和等によって、ラテックス粒子を均一にする必要がなく、誤差を含まない、安定した性能が得られる測定を行えることが確かめられた。 In this way, when storing antibody-immobilized latex particles in a buffer solution, it is possible to suppress sedimentation of antibody-immobilized latex particles for a long period of time by including Good's buffer or an alkali metal halide in the buffer solution. confirmed. Therefore, it was confirmed that it is not necessary to make the latex particles uniform by inverting and mixing during measurement, and that it is possible to perform measurements that do not include errors and provide stable performance.
これらのことより、本発明の測定対象物質に特異的に結合する物質を固定化した担体粒子の沈降抑制方法では、担体粒子が沈降することなく、長期間保存することができるため、本発明により沈降が抑制された担体粒子を測定に用いることにより、誤差を含まない、安定した性能が得られる測定を行えることが確かめられた。 From these facts, in the method for suppressing sedimentation of carrier particles on which a substance that specifically binds to a substance to be measured is immobilized according to the present invention, the carrier particles can be stored for a long period of time without sedimentation. It was confirmed that by using carrier particles with suppressed sedimentation in measurements, it is possible to perform measurements that do not include errors and provide stable performance.
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