JP2006038594A - Diacetylspermine measuring method and measuring reagent kit - Google Patents
Diacetylspermine measuring method and measuring reagent kit Download PDFInfo
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- JP2006038594A JP2006038594A JP2004217804A JP2004217804A JP2006038594A JP 2006038594 A JP2006038594 A JP 2006038594A JP 2004217804 A JP2004217804 A JP 2004217804A JP 2004217804 A JP2004217804 A JP 2004217804A JP 2006038594 A JP2006038594 A JP 2006038594A
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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 77
- 238000000034 method Methods 0.000 title claims abstract description 60
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 56
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- SVOAXTTVWPHOQW-UHFFFAOYSA-N 2-acetylsulfanylbutanedioic acid Chemical compound CC(=O)SC(C(O)=O)CC(O)=O SVOAXTTVWPHOQW-UHFFFAOYSA-N 0.000 description 1
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Abstract
Description
本発明は、金コロイド凝集反応を利用するジアセチルスペルミンの測定方法に関する。より詳細には、検体中のジアセチルスペルミンをｎｇ／ｍＬ単位で高感度にかつ短時間に測定することを可能にする技術に関する。 The present invention relates to a method for measuring diacetylspermine using a colloidal gold agglutination reaction. More specifically, the present invention relates to a technique that makes it possible to measure diacetylspermine in a specimen in units of ng / mL with high sensitivity and in a short time.
ジアセチルスペルミンは、尿中ポリアミン成分の１種であり、健常者尿中では尿中総ポリアミンの０．６％を占めるにすぎない微量成分である。しかし、癌患者の尿中においては、他の成分と比較して高い頻度で排泄量が増加し、しかも増加の割合も際立って高い。そのため、ジアセチルスペルミンは腫瘍マーカーとして注目されている（非特許文献１および２参照）。さらに、悪性腫瘍の治療効果の判定や予後予測の指標としても期待されている（非特許文献２）。 Diacetylspermine is a kind of urinary polyamine component, and is a trace component that only accounts for 0.6% of the total urine polyamine in the urine of healthy persons. However, in the urine of cancer patients, the amount of excretion increases more frequently than other components, and the rate of increase is remarkably high. Therefore, diacetylspermine has attracted attention as a tumor marker (see Non-Patent Documents 1 and 2). Furthermore, it is also expected as an index for determining the therapeutic effect of malignant tumors and predicting prognosis (Non-patent Document 2).
近年、臨床検査などの各種検査では自動化および測定時間の短縮が図られている。例えば、臨床検査においては、生体試料中の微量物質を測定するために免疫反応を利用する免疫測定方法が広く用いられている。免疫測定法としては、ＲＩＡ法、ＥＩＡ法、免疫比濁法、ラテックス凝集法、金属コロイド凝集法などの多くの方法がある。この中でもラテックス凝集法および金属コロイド凝集法は、反応液の分離や洗浄を行う必要がないため、自動化に適している。しかし、ジアセチルスペルミンなどの低分子物質（以下、ハプテンという）の測定においては、通常の免疫測定とは異なり競合法による免疫反応を利用するため、その測定を高感度かつ短時間に行うには困難が伴い、自動化の妨げとなっている。そのため、従来、生体試料中のｎｇ／ｍＬ濃度のハプテンを測定する場合、ＲＩＡ法やＥＩＡ法で測定されることが多く、自動化に適さず、簡便に測定できなかった。このように、ハプテン測定においても、高感度かつ短時間で測定が可能な、自動化に適した技術が望まれている。 In recent years, various tests such as clinical tests have been automated and shortened in measurement time. For example, in a clinical test, an immunoassay method using an immune reaction is widely used to measure a trace amount substance in a biological sample. There are many immunoassay methods such as RIA method, EIA method, immunoturbidimetric method, latex agglutination method, and metal colloid agglutination method. Among these, the latex agglutination method and the metal colloid agglomeration method are suitable for automation because it is not necessary to separate and wash the reaction solution. However, the measurement of low-molecular substances such as diacetylspermine (hereinafter referred to as “hapten”) uses an immune reaction by a competitive method, unlike normal immunoassay, so it is difficult to perform the measurement with high sensitivity and in a short time. As a result, automation has been hindered. Therefore, conventionally, when measuring a hapten having a concentration of ng / mL in a biological sample, it is often measured by an RIA method or an EIA method, which is not suitable for automation and cannot be easily measured. Thus, in hapten measurement, a technique suitable for automation that can measure with high sensitivity and in a short time is desired.
金属コロイド凝集法によるハプテンの測定用試薬として、血中ハロペリドール測定用試薬「マーキット（登録商標）Ｇハロペリドール」および血中ブロムペリドール測定用試薬「マーキット（登録商標）Ｇブロムペリドール」が、大日本製薬株式会社（製造、株式会社アズウェル）より販売されている。これらによれば、血液中のハロペリドールおよびブロムペリドールを自動分析機により、高感度かつ短時間に測定することができる（非特許文献３および４）。しかしながら、腫瘍マーカーとして注目されているジアセチルスペルミンを金属コロイド凝集法により測定する方法については未だ知られていない。 As reagents for measuring a hapten by a metal colloid aggregation method, a reagent for measuring blood haloperidol "Merkit (registered trademark) G haloperidol" and a reagent for measuring blood bromoperidol "Merkit (registered trademark) G bromperidol" It is sold by Nippon Pharmaceutical Co., Ltd. (Manufacturing, Aswell Co., Ltd.). According to these, haloperidol and bromperidol in blood can be measured with high sensitivity and in a short time by an automatic analyzer (Non-patent Documents 3 and 4). However, a method for measuring diacetylspermine, which is attracting attention as a tumor marker, by a metal colloid aggregation method is not yet known.
尿中のジアセチルスペルミンの測定については、ＥＬＩＳＡに用いるためのジアセチルスペルミン特異抗体の製法が報告されている（非特許文献５）。また、ジアセチルスペルミンの測定用試薬の一つとして、「尿中ジアセチルスペルミン測定試薬用ＥＬＩＳＡキット」が、株式会社トランスジェニック（販売、フナコシ株式会社）より市販されている。このキットは、ＥＬＩＳＡ法を行うための試薬であり、構成品も１０種と多く、測定操作方法は煩雑であり、自動化および短時間での測定は困難である。 Regarding the measurement of diacetylspermine in urine, a method for producing a diacetylspermine-specific antibody for use in ELISA has been reported (Non-patent Document 5). As one of the reagents for measuring diacetylspermine, “Urine diacetylspermine measuring reagent ELISA kit” is commercially available from Transgenic Co., Ltd. (sales, Funakoshi Co., Ltd.). This kit is a reagent for performing the ELISA method, and there are as many as 10 components, the measurement operation method is complicated, and automation and measurement in a short time are difficult.
以上のとおり公知の測定法では、ジアセチルスペルミンを高感度かつ短時間で測定を行うことができず、自動化も困難であった。
本発明は、測定感度が良好で、測定時間が短縮された、Ｂ／Ｆ分離を必要としない自動化に適したジアセチルスペルミンの測定法およびそのための測定キットを提供することを目的とする。 An object of the present invention is to provide a method for measuring diacetylspermine suitable for automation that does not require B / F separation and has a good measurement sensitivity and a short measurement time, and a measurement kit therefor.
上記の目的を達成するために鋭意検討した結果、まず、ジアセチルスペルミンを含有する検体と、塩とジアセチルスペルミン類似化合物結合蛋白質を含有する第１試薬とを混合した後、抗ジアセチルスペルミン抗体結合金コロイドを含有する第２試薬を添加して混合し、金コロイドの免疫反応凝集による吸光度変化を測定することによって、ｎｇ／ｍＬ濃度のジアセチルスペルミン測定を、高感度かつ短時間に行うことが可能であることを見出し、本発明を完成した。 As a result of intensive studies to achieve the above object, first, a sample containing diacetylspermine and a salt and a first reagent containing a diacetylspermine analog-binding protein are mixed, and then an anti-diacetylspermine antibody-binding gold colloid. It is possible to measure diacetylspermine at a concentration of ng / mL with high sensitivity and in a short time by adding and mixing a second reagent containing a mixture and measuring the change in absorbance due to immune reaction aggregation of colloidal gold. As a result, the present invention has been completed.
詳細には、本発明においては、予め検体とジアセチルスペルミン類似化合物結合蛋白質とを混合し、次いで抗ジアセチルスペルミン抗体結合金コロイドを添加することによって抗原抗体反応を開始し、検体中の被測定物質であるジアセチルスペルミンとジアセチルスペルミン類似化合物結合蛋白質との間で抗ジアセチルスペルミン抗体結合金コロイドが競合反応する、一段階のみの免疫競合反応とした。 Specifically, in the present invention, a specimen and a diacetylspermine analog-binding protein are mixed in advance, and then an antigen-antibody reaction is initiated by adding an anti-diacetylspermine antibody-binding gold colloid, The immunocompetitive reaction is a one-step immune reaction in which an anti-diacetylspermine antibody-binding gold colloid competes between a certain diacetylspermine and a diacetylspermine analog-binding protein.
本発明は、検体中のジアセチルスペルミンの量を測定する方法を提供し、この方法は、（ａ）該検体と、塩およびジアセチルスペルミン類似化合物結合蛋白質を含有する第１試薬とを混合する工程、（ｂ）該混合液に、抗ジアセチルスペルミン抗体結合金コロイドを含有する第２試薬を添加して混合する工程、および（ｃ）該ジアセチルスペルミン類似化合物結合蛋白質と抗ジアセチルスペルミン抗体結合金コロイドの免疫反応凝集による吸光度変化を、４５０〜５８０ｎｍで２回以上測定した吸光度、または、４５０ｎｍ〜５８０ｎｍを主波長および６２０ｎｍ〜８００ｎｍを副波長とする二波長で２回以上測定した吸光度より求める工程を含む。 The present invention provides a method for measuring the amount of diacetylspermine in a sample, the method comprising: (a) mixing the sample with a first reagent containing a salt and a diacetylspermine analog-binding protein; (B) adding and mixing a second reagent containing an anti-diacetylspermine antibody-binding gold colloid to the mixed solution; and (c) immunizing the diacetylspermine analog-binding protein and anti-diacetylspermine antibody-binding gold colloid. A step of determining the change in absorbance due to reaction aggregation from the absorbance measured twice or more at 450 to 580 nm, or the absorbance measured twice or more at two wavelengths having a main wavelength of 450 nm to 580 nm and a sub wavelength of 620 nm to 800 nm.
好適な実施態様では、上記工程（ａ）から（ｃ）が１５分以内で行われる。さらに好適な実施態様では、１０分以内で行われる。 In a preferred embodiment, steps (a) to (c) above are performed within 15 minutes. In a more preferred embodiment, it is performed within 10 minutes.
他の好適な実施態様では、上記混合液中に終濃度として、０．３〜１０％の塩、１０〜１０００ｍＭの緩衝剤、および５〜３０００ｎｇ／ｍＬのジアセチルスペルミン類似化合物結合蛋白質を含有するように、これら物質が第１試薬に含有される。 In another preferred embodiment, the mixture solution contains 0.3 to 10% salt, 10 to 1000 mM buffer, and 5 to 3000 ng / mL diacetylspermine analog-binding protein as final concentrations. In addition, these substances are contained in the first reagent.
好適な実施態様では、ジアセチルスペルミン類似化合物結合蛋白質は、アセチルスペルミン結合ウシ血清アルブミンである。 In a preferred embodiment, the diacetylspermine analog binding protein is acetylspermine-bound bovine serum albumin.
好適な実施態様では、上記第１試薬は、平均分子量５，０００から５００，０００の水溶性高分子物質を０．０１％〜１０％（Ｗ／Ｖ）含有する。 In a preferred embodiment, the first reagent contains 0.01% to 10% (W / V) of a water-soluble polymer substance having an average molecular weight of 5,000 to 500,000.
他の好適な実施態様では、抗ジアセチルスペルミン抗体結合金コロイドの抗ジアセチルスペルミン抗体は、アセチルスペルミン結合ウシ血清アルブミンを動物に投与することによって生成されたポリクローナル抗体またはモノクローナル抗体であり、そして上記ジアセチルスペルミン類似化合物結合蛋白質の蛋白質部分と反応しない。 In another preferred embodiment, the anti-diacetylspermine antibody-conjugated gold colloid anti-diacetylspermine antibody is a polyclonal or monoclonal antibody produced by administering acetylspermine-conjugated bovine serum albumin to an animal, and said diacetylspermine Does not react with the protein part of similar compound binding proteins.
好適な実施態様では、上記検体は、尿である。 In a preferred embodiment, the specimen is urine.
本発明はまた、上記の方法を実施するために使用される、塩およびジアセチルスペルミン類似化合物結合蛋白質を含有する第１試薬、および抗ジアセチルスペルミン抗体結合金コロイドを含有する第２試薬を含むジアセチルスペルミン測定用試薬キットを提供する。 The present invention also includes a first reagent containing a salt and a diacetylspermine analog-binding protein and a second reagent containing an anti-diacetylspermine antibody-binding gold colloid used to perform the above method. A reagent kit for measurement is provided.
本発明の検体中のジアセチルスペルミンの量を測定する方法は、（ａ）該検体と、塩およびジアセチルスペルミン類似化合物結合蛋白質を含有する第１試薬とを混合する工程、（ｂ）該混合液に抗ジアセチルスペルミン類似化合物抗体結合金コロイドを含有する第２試薬を添加して混合する工程、および（ｃ）該ジアセチルスペルミン類似化合物結合蛋白質と抗ジアセチルスペルミン抗体結合金コロイドの免疫凝集による吸光度変化を、４５０ｎｍ〜５８０ｎｍおよび６２０ｎｍ〜８００ｎｍの二波長で２回以上測定した吸光度変化量より求める工程を含み、この方法によれば、ｎｇ／ｍＬのジアセチルスペルミンが短時間に精度良く測定できる。また、洗浄などの工程が不要であるため、自動化に適している。したがって、本発明の方法は、ジアセチルスペルミン測定の臨床検査分野などでの迅速化および省力化に寄与することができる。 The method for measuring the amount of diacetylspermine in the sample of the present invention comprises: (a) a step of mixing the sample with a first reagent containing a salt and a diacetylspermine analog-binding protein; (b) Adding and mixing a second reagent containing an anti-diacetylspermine analog-antibody-binding gold colloid, and (c) changing the absorbance due to immunoaggregation of the diacetylspermine analog-binding protein and the anti-diacetylspermine antibody-binding gold colloid, This method includes a step of obtaining from an absorbance change amount measured twice or more at two wavelengths of 450 nm to 580 nm and 620 nm to 800 nm, and according to this method, ng / mL diacetylspermine can be accurately measured in a short time. Further, since a process such as washing is not necessary, it is suitable for automation. Therefore, the method of the present invention can contribute to speeding up and labor saving in the clinical laboratory field of diacetylspermine measurement.
本発明の検体中のジアセチルスペルミンの量を測定する方法は、（ａ）該検体と、塩およびジアセチルスペルミン類似化合物結合蛋白質を含有する第１試薬とを混合する工程、（ｂ）該混合液に、抗ジアセチルスペルミン抗体結合金コロイドを含有する第２試薬を添加して混合する工程、および（ｃ）該ジアセチルスペルミン類似化合物結合蛋白質と抗ジアセチルスペルミン抗体結合金コロイドの免疫反応凝集による吸光度変化を、４５０ｎｍ〜５８０で２回以上測定した吸光度、または、４５０ｎｍ〜５８０ｎｍを主波長および６２０ｎｍ〜８００ｎｍを副波長とする二波長で２回以上測定した吸光度より求める工程を含む。 The method for measuring the amount of diacetylspermine in the sample of the present invention comprises: (a) a step of mixing the sample with a first reagent containing a salt and a diacetylspermine analog-binding protein; (b) A step of adding and mixing a second reagent containing an anti-diacetylspermine antibody-binding gold colloid, and (c) a change in absorbance due to immune reaction aggregation of the diacetylspermine analog-binding protein and the anti-diacetylspermine antibody-binding gold colloid, A step of determining from the absorbance measured at least twice at 450 nm to 580 or the absorbance measured at least twice at two wavelengths having a main wavelength of 450 nm to 580 nm and a sub wavelength of 620 nm to 800 nm.
（検体）
本発明において、測定に供する検体としては、血清、血漿、尿、または髄液などの生体試料、あるいは環境中より得られたサンプルまたはその抽出物などが挙げられる。本発明においては、尿が特に好適である。
(Sample)
In the present invention, the sample to be measured includes biological samples such as serum, plasma, urine, or cerebrospinal fluid, samples obtained from the environment, or extracts thereof. In the present invention, urine is particularly suitable.
（第１試薬）
本発明の方法で使用される第１試薬は、塩およびジアセチルスペルミン類似化合物結合蛋白質を含有する。
(First reagent)
The first reagent used in the method of the present invention contains a salt and a diacetylspermine analog binding protein.
本発明において、ジアセチルスペルミン類似化合物結合蛋白質とは、ジアセチルスペルミン類似化合物と免疫原性蛋白質との結合物をいう。ジアセチルスペルミン類似化合物結合蛋白質（結合蛋白質）は、測定対象のジアセチルスペルミンと抗ジアセチルスペルミン抗体に対して競合反応し得るものであれば、ジアセチルスペルミン類似化合物として、どのような物質が蛋白質に結合したものであってもよい。蛋白質と結合させるジアセチルスペルミン類似化合物としては、結合蛋白質中のジアセチルスペルミン相当部分の化学構造が可能な限り本来のジアセチルスペルミンの構造に類似しているものを選択することが望ましい。また、ジアセチルスペルミン類似化合物としては、ジアセチルスペルミンと担体タンパク質との結合に伴って、ジアセチルスペルミン分子の一部が担体分子上の官能基と化学結合を生じることにより、必然的に遊離のジアセチルスペルミンとは一部異なる構造を有するようになった化合物も含む。本発明においては、ジアセチルスペルミン類似化合物として、Ｎ−アセチルスペルミンを用いることが好ましい。また、ジアセチルスペルミン類似化合物結合蛋白質には、蛋白質１分子当たり、４〜４０程度のジアセチルスペルミン類似化合物が結合していることが好ましい。 In the present invention, the diacetylspermine analog-binding protein refers to a conjugate of a diacetylspermine analog and an immunogenic protein. Diacetylspermine analog binding protein (binding protein) is a diacetylspermine analog that binds to the protein as long as it can compete with the target diacetylspermine and anti-diacetylspermine antibody. It may be. As the diacetylspermine-like compound to be bound to the protein, it is desirable to select a compound having a chemical structure of the portion corresponding to diacetylspermine in the binding protein as similar as possible to the original diacetylspermine structure. In addition, as a diacetylspermine-like compound, a part of the diacetylspermine molecule forms a chemical bond with a functional group on the carrier molecule in association with the binding of diacetylspermine and the carrier protein. Also includes compounds that have become partly different in structure. In the present invention, N-acetylspermine is preferably used as the diacetylspermine analog. In addition, it is preferable that about 4 to 40 diacetylspermine-like compounds are bound to the diacetylspermine-like compound binding protein per molecule of the protein.
ジアセチルスペルミン類似化合物結合蛋白質の蛋白質は、ハプテンを結合させるために当該技術分野で通常用いられる免疫原性蛋白質であり、各種動物由来のアルブミン、ヘモシアニン、サイログロブリン、フィブリノーゲン、酵素などから適宜選択される。これらの蛋白質は、天然に存在する形態でジアセチルスペルミン類似化合物を結合させることもできるが、該蛋白質のアミノ酸側鎖の一部を化学修飾して改変蛋白質とした後に、ジアセチルスペルミン類似化合物との結合に供してもよい。本発明においては、ウシ血清アルブミン（ＢＳＡ）のリシン残基の側鎖にメルカプトサクシニル基が付加されているメルカプトサクシニルＢＳＡが好ましい。 The protein of the diacetylspermine analog-binding protein is an immunogenic protein usually used in the art for binding a hapten, and is appropriately selected from various animal-derived albumins, hemocyanin, thyroglobulin, fibrinogen, enzymes, and the like. These proteins can be bound to diacetylspermine analogs in their naturally occurring form, but after modification of a part of the amino acid side chain of the protein to a modified protein, binding to diacetylspermine analogs You may use for. In the present invention, mercaptosuccinyl BSA in which a mercaptosuccinyl group is added to the side chain of a lysine residue of bovine serum albumin (BSA) is preferable.
上記ジアセチルスペルミン類似化合物結合蛋白質は、当業者が通常用いる方法により製造することができる。このような製造方法は、ジアセチルスペルミン類に存在するアミノ基、カルボキシル基またはチオール基などの官能基を利用して、ジアセチルスペルミン類似化合物と蛋白質とを直接または結合剤を介して化学結合させるものであり、ジアセチルスペルミン類似化合物類の構造に応じて種々のものが知られている（生化学実験法１１ エンザイムイムノアッセイ、Ｐ．Ｔｉｊｓｓｅｎ著、石川栄治編、２５２頁、１９８９年、東京化学同人）。化学結合を形成させるための試薬としては、アシル化剤、アルキル化剤などが挙げられる。好ましくは、カルボキシル基を活性化することにより得られるＮ−ヒドロキシスクシンイミドエステル、弱アルカリ条件下で用いられるマレイミド類などである。本発明においては、Ｎ−アセチルスペルミンの１級アミノ基の部分と二価性架橋剤ＧＭＢＳ（Ｎ−(４−マレイミドブチリルオキシ)スクシンイミド）のエステル部分とをアミド結合させ、生成した付加物のマレイミド部分を利用して、メルカプトサクシニルＢＳＡに結合させることが好ましい。 The diacetylspermine analog-binding protein can be produced by a method commonly used by those skilled in the art. Such a production method uses a functional group such as an amino group, a carboxyl group or a thiol group present in diacetylspermine to chemically bond a diacetylspermine-like compound and a protein directly or via a binder. There are various known compounds depending on the structure of diacetylspermine analogs (Biochemical Experimental Method 11 Enzyme Immunoassay, P. Tijssen, edited by Eiji Ishikawa, page 252, 1989, Tokyo Chemical Dojin). Examples of the reagent for forming a chemical bond include acylating agents and alkylating agents. Preferred are N-hydroxysuccinimide esters obtained by activating carboxyl groups, maleimides used under weak alkaline conditions, and the like. In the present invention, the primary amino group part of N-acetylspermine and the ester part of the divalent crosslinker GMBS (N- (4-maleimidobutyryloxy) succinimide) are amide-bonded, and Preferably, the maleimide moiety is used to bind to mercaptosuccinyl BSA.
第１試薬に含有されるジアセチルスペルミン類似化合物結合蛋白質は、反応最終液中に、蛋白質濃度として、５〜３０００ｎｇ／ｍＬ、好ましくは２０〜５００ｎｇ／ｍＬ、さらに好ましくは３０〜２００ｎｇ／ｍＬ含有される。ジアセチルスペルミン類似化合物結合蛋白質の量が少なすぎると、抗ジアセチルスペルミン抗体結合金コロイドとの凝集反応が生じ難い。一方、ジアセチルスペルミン類似化合物結合蛋白質の量が多すぎると、被測定物質のジアセチルスペルミンとジアセチルスペルミン類似化合物結合蛋白質との競合反応が生じ難い。 The diacetylspermine analog-binding protein contained in the first reagent is contained in the final reaction solution as a protein concentration of 5-3000 ng / mL, preferably 20-500 ng / mL, more preferably 30-200 ng / mL. . If the amount of the diacetylspermine analog-binding protein is too small, an agglutination reaction with the anti-diacetylspermine antibody-binding gold colloid is difficult to occur. On the other hand, when the amount of the diacetylspermine analog-binding protein is too large, a competitive reaction between the substance to be measured, diacetylspermine and the diacetylspermine analog-binding protein, hardly occurs.
第１試薬は、塩も含む。塩としては、塩化ナトリウムや塩化カリウムなどの無機塩類が挙げられる。これらは、単独で用いても、組み合わせて用いてもよい。これらの無機塩類の濃度は、終濃度として、通常０．３〜１０％、好ましくは１〜７％、より好ましくは２〜５％である。塩濃度が０．３％よりも少ないとハプテンとの競合反応が進行し難い。一方、１０％より多すぎると反応抑制が強くなり好ましくない。 The first reagent also includes a salt. Examples of the salt include inorganic salts such as sodium chloride and potassium chloride. These may be used alone or in combination. The concentration of these inorganic salts is usually 0.3 to 10%, preferably 1 to 7%, more preferably 2 to 5% as the final concentration. When the salt concentration is less than 0.3%, the competitive reaction with the hapten hardly proceeds. On the other hand, if it is more than 10%, the reaction suppression becomes strong, which is not preferable.
また、十分な測定感度が得られるという点において、第１試薬は、凝集促進剤として、平均分子量５，０００から５００，０００の水溶性高分子を含有することが好ましい。含有量は、通常０．０１％〜１０％（Ｗ／Ｖ）、好ましくは０．０１〜５．０％（Ｗ／Ｖ）より好ましくは０．１〜３．０％（Ｗ／Ｖ）、さらに好ましくは１．０〜２．０％（Ｗ／Ｖ）である。水溶性高分子物質としては、ポリエチレングリコール（ＰＥＧ）、ポリビニルピロリドン、カルボキシメチルセルロース、デキストラン、デキストラン硫酸、コンドロイチン硫酸、ヒアルロン酸、ポリアクリル酸、プルランなどが挙げられる。 In addition, the first reagent preferably contains a water-soluble polymer having an average molecular weight of 5,000 to 500,000 as an aggregation accelerator in that sufficient measurement sensitivity can be obtained. The content is usually 0.01% to 10% (W / V), preferably 0.01 to 5.0% (W / V), more preferably 0.1 to 3.0% (W / V), More preferably, it is 1.0 to 2.0% (W / V). Examples of the water-soluble polymer substance include polyethylene glycol (PEG), polyvinyl pyrrolidone, carboxymethyl cellulose, dextran, dextran sulfate, chondroitin sulfate, hyaluronic acid, polyacrylic acid, and pullulan.
第１試薬には、ｐＨの維持のために適切な緩衝剤が含有され得る。緩衝剤としては、例えば、リン酸緩衝液、トリス塩酸緩衝液、コハク酸緩衝液、あるいはグリシルグリシン、ＭＥＳ（２−(Ｎ−モノホリノ)エタンスルホン酸）、ＨＥＰＥＳ（Ｎ−２−ヒドロキシエチル−ピペラジン−Ｎ’−エタンスルホン酸）、ＴＥＳ（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）、ＭＯＰＳ（３−(Ｎ−モノホリノ)プロパンスルホン酸）、ＰＩＰＥＳ（ピペラジン−１，４−ビス(２−エタンスルホン酸)）などのグッド緩衝液が好適に用いられる。ｐＨは、好ましくは５．０〜９．０、より好ましくは５．５〜８．０、さらに好ましくは６．０〜７．０である。緩衝剤の使用濃度は、終濃度として、好ましくは１０〜１０００ｍＭ、より好ましくは５０〜４００ｍＭである。 The first reagent may contain a suitable buffer for maintaining the pH. Examples of the buffer include phosphate buffer, Tris-HCl buffer, succinate buffer, glycylglycine, MES (2- (N-monophorino) ethanesulfonic acid), HEPES (N-2-hydroxyethyl- Piperazine-N′-ethanesulfonic acid), TES (N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid), MOPS (3- (N-monophorino) propanesulfonic acid), PIPES (piperazine-1,4) Good buffer solution such as -bis (2-ethanesulfonic acid)) is preferably used. The pH is preferably 5.0 to 9.0, more preferably 5.5 to 8.0, and still more preferably 6.0 to 7.0. The use concentration of the buffer is preferably 10 to 1000 mM, more preferably 50 to 400 mM, as the final concentration.
さらに、第１試薬は、アジ化ナトリウム、有機酸類、糖類、アミノ酸類、ＥＤＴＡなどのキレート剤、ＤＴＴなどのＳＨ試薬、各種動物由来のアルブミン、動物血清、γ−グロブリン、ヒトＩｇＧやＩｇＭに対する抗体、非イオンおよびイオン性の界面活性剤などを含有してもよい。これらの添加濃度は適宜選択できる。 Furthermore, the first reagent is sodium azide, organic acids, saccharides, amino acids, chelating agents such as EDTA, SH reagent such as DTT, albumins derived from various animals, animal serum, γ-globulin, antibodies against human IgG and IgM , Nonionic and ionic surfactants may be contained. These addition concentrations can be selected as appropriate.
（第２試薬）
本発明の方法で使用される第２試薬は、抗ジアセチルスペルミン抗体結合金コロイドを含有する。
(Second reagent)
The second reagent used in the method of the present invention contains an anti-diacetylspermine antibody-conjugated gold colloid.
上記抗ジアセチルスペルミン抗体結合金コロイドは、金コロイドに抗ジアセチルスペルミン抗体を結合または吸着させたものであり、当業者が通常用いる方法により製造され得る。抗体はそのまま結合させても、化学処理や酵素処理後に結合させてもよい。また、金コロイドと抗ジアセチルスペルミン抗体とは、ビオチン・アビジンのような親和性のある物質や修飾ＰＥＧのような化合物を介して結合してもよい。通常、抗ジアセチルスペルミン抗体を金コロイドに結合させた後、アルブミンなどの蛋白質でブロッキング処理を行う。そのブロッキング剤としては、ウシ血清アルブミン（ＢＳＡ）が好ましい。 The anti-diacetylspermine antibody-bound gold colloid is obtained by binding or adsorbing an anti-diacetylspermine antibody to a gold colloid, and can be produced by methods commonly used by those skilled in the art. The antibody may be bound as it is or after chemical treatment or enzyme treatment. Further, the colloidal gold and the anti-diacetylspermine antibody may be bound via an affinity substance such as biotin / avidin or a compound such as modified PEG. Usually, an anti-diacetylspermine antibody is bound to a colloidal gold, followed by a blocking treatment with a protein such as albumin. As the blocking agent, bovine serum albumin (BSA) is preferable.
抗ジアセチルスペルミン抗体は、測定対象のジアセチルスペルミンに対して特異的な免疫反応性を有するものであれば、ポリクローナル抗体またはモノクローナル抗体のいずれでもよい。通常は、測定対象のジアセチルスペルミン類似化合物と担体との結合物を動物に投与することにより産生させたポリクローナル抗体またはモノクローナル抗体であり、ジアセチルスペルミン類似化合物結合蛋白質の蛋白質部分と反応しない抗体である。ジアセチルスペルミン類似化合物結合蛋白質の蛋白質部分と反応する抗体が含まれている場合は、吸着処理などによって除くことができる。また、抗ジアセチルスペルミン抗体が、測定対象のジアセチルスペルミンに類似の化合物と反応し、測定値に影響を及ぼす場合には、その類似化合物と反応する抗体を除くことができる。このような類似化合物としては、例えば、Ｎ１−アセチルスペルミジン、Ｎ８−アセチルスペルミジン、Ｎ−アセチルスペルミン、Ｎ１，Ｎ８−ジアセチルスペルミジンなどが挙げられる。これらの類似化合物と反応する抗体を除くためには、例えば、各種のポリアミン誘導体を結合させた樹脂を用いた親和性クロマトグラフィーを利用することができる。特に好適な実施態様においては、非特許文献５に記載の方法に準じて、目的の抗体の親和性精製を行うことができる。 The anti-diacetylspermine antibody may be either a polyclonal antibody or a monoclonal antibody as long as it has specific immunoreactivity to the diacetylspermine to be measured. Usually, it is a polyclonal antibody or a monoclonal antibody produced by administering a conjugate of a diacetylspermine analogue compound to be measured and a carrier to an animal, and is an antibody that does not react with the protein portion of the diacetylspermine analogue-binding protein. When an antibody that reacts with the protein portion of the diacetylspermine analog-binding protein is contained, it can be removed by adsorption treatment or the like. Further, when an anti-diacetylspermine antibody reacts with a compound similar to diacetylspermine to be measured and affects the measured value, the antibody reacting with the similar compound can be excluded. Examples of such similar compounds include N 1 -acetylspermidine, N 8 -acetylspermidine, N-acetylspermine, N 1 , N 8 -diacetylspermidine, and the like. In order to remove antibodies that react with these similar compounds, for example, affinity chromatography using a resin bound with various polyamine derivatives can be used. In a particularly preferred embodiment, affinity purification of the target antibody can be performed according to the method described in Non-Patent Document 5.
抗ジアセチルスペルミン抗体を製造するための上記化合物における担体としては、該化合物が免疫原となり得るものであればいずれでもよく、例えば、蛋白質やポリペプチドなどが挙げられる。具体的には、アルブミン、グロブリン、ヘモシアニン、サイログロブリン、エデスチンなどが挙げられる。その中でも、好ましいのはアルブミンであり、特に好ましいのはＢＳＡである。また、抗ジアセチルスペルミン抗体を製造するためのハプテンと担体との結合物として、上記第１試薬に含有されるジアセチルスペルミン類似化合物結合蛋白質、具体的には、Ｎ−アセチルスペルミンを１級アミノ基の部分で二価性架橋剤の一端にアミド結合させたのち、架橋剤の他端で担体蛋白質と結合させたものも使用され得る。本発明においては、二価性架橋剤としてＧＭＢＳを利用し、そして担体タンパク質として、ＢＳＡのリシン残基をメルカプトサクシニル化したＢＳＡ誘導体を利用することが特に好ましい。 The carrier for the above-mentioned compound for producing an anti-diacetylspermine antibody may be any carrier as long as the compound can serve as an immunogen, and examples thereof include proteins and polypeptides. Specific examples include albumin, globulin, hemocyanin, thyroglobulin, and edestin. Among them, albumin is preferable, and BSA is particularly preferable. Further, as a conjugate of a hapten and a carrier for producing an anti-diacetylspermine antibody, a diacetylspermine-like compound-binding protein contained in the first reagent, specifically, N-acetylspermine is a primary amino group. It is also possible to use an amide bond at one end of the bivalent crosslinker and a bond to the carrier protein at the other end of the crosslinker. In the present invention, it is particularly preferable to use GMBS as a bivalent cross-linking agent and to use a BSA derivative obtained by mercaptosuccinylating a lysine residue of BSA as a carrier protein.
ポリクローナル抗体は、上記のジアセチルスペルミン類似化合物と担体との結合物を抗原として使用し、この結合物を適切なアジュバントと混合して、ウサギ、モルモット、羊、山羊などのヒト以外の動物に非経口的に投与（免疫）し、血清を採取して当業者が通常用いる方法で処理することによって製造し得る。 Polyclonal antibodies are obtained by using a conjugate of the above-mentioned diacetylspermine analogue compound and a carrier as an antigen, mixing this conjugate with an appropriate adjuvant, and parenterally to non-human animals such as rabbits, guinea pigs, sheep, goats, etc. Can be prepared (immunized), and serum can be collected and processed by methods commonly used by those skilled in the art.
モノクローナル抗体は、上記のように免疫された動物の脾臓細胞を採取し、当業者が通常用いるミルシュタインらの方法によって、ミエローマ細胞との細胞融合、抗体産生細胞スクリーニング、およびクローニングを行い、抗ジアセチルスペルミン抗体を産生する細胞株を樹立し、これを培養することにより製造することができる。 Monoclonal antibodies are obtained by collecting spleen cells of an immunized animal as described above, performing cell fusion with myeloma cells, screening for antibody-producing cells, and cloning by methods commonly used by those skilled in the art. It can be produced by establishing a cell line that produces a spermine antibody and culturing it.
このようにして得られた抗ジアセチルスペルミン抗体を、上述のように金コロイドに結合または吸着させることにより、抗ジアセチルスペルミン抗体結合金コロイドが得られる。こうして得られた抗ジアセチルスペルミン抗体結合金コロイドは、特定のジアセチルスペルミンおよびジアセチルスペルミン類似化合物結合蛋白質と特異的に結合する。 The anti-diacetylspermine antibody-bound gold colloid is obtained by binding or adsorbing the anti-diacetylspermine antibody thus obtained to the gold colloid as described above. The thus obtained anti-diacetylspermine antibody-binding gold colloid specifically binds to a specific diacetylspermine and a diacetylspermine analog-binding protein.
上記抗ジアセチルスペルミン抗体結合金コロイドは、反応液中での終濃度として、５４０ｎｍ（光路長１ｃｍ）での吸光度が１．５〜３．０となる金コロイド量であることが好ましい。金コロイドはその粒子径によって極大吸収が多少変化するが、５４０ｎｍ付近で極大吸収を示すため、この５４０ｎｍでの吸光度に基づいて金コロイド濃度を規定することができる。より好ましい金コロイド量は、５４０ｎｍでの吸光度で、１．９〜２．８であり、さらに好ましい金コロイド量は２．１〜２．６である。反応液中の抗ジアセチルスペルミン結合金コロイド量が、５４０ｎｍでの吸光度で１．５より少ないと反応が進行し難く、一方、３．０より多いと試薬コストや測定精度面で好ましくない。 The anti-diacetylspermine antibody-bound gold colloid preferably has a gold colloid amount such that the absorbance at 540 nm (optical path length 1 cm) is 1.5 to 3.0 as the final concentration in the reaction solution. Although the maximum absorption of gold colloid varies slightly depending on the particle diameter, it exhibits a maximum absorption near 540 nm. Therefore, the gold colloid concentration can be defined based on the absorbance at 540 nm. A more preferable amount of colloidal gold is 1.9 to 2.8 in terms of absorbance at 540 nm, and a more preferable amount of colloidal gold is 2.1 to 2.6. If the amount of the anti-diacetylspermine-bonded gold colloid in the reaction solution is less than 1.5 in terms of absorbance at 540 nm, the reaction hardly proceeds. On the other hand, if it exceeds 3.0, it is not preferable in terms of reagent cost and measurement accuracy.
第２試薬には、必要に応じて、アジ化ナトリウム、糖類、有機酸類、アミノ酸類、緩衝剤、ＥＤＴＡなどのキレート剤、各種動物由来のアルブミンや界面活性剤などをさらに含有させてもよい。これらの添加濃度は適宜選択できる。 The second reagent may further contain sodium azide, saccharides, organic acids, amino acids, buffers, chelating agents such as EDTA, albumins derived from various animals, surfactants, and the like, if necessary. These addition concentrations can be selected as appropriate.
（ジアセチルスペルミン量の測定方法）
本発明の方法による検体中のジアセチルスペルミン量の測定は、検体と上記第１試薬とを混合し、一定の時間後、上記第２試薬を添加して混合し、金コロイドの凝集による吸光度を、４５０ｎｍ〜５８０ｎｍおよび６２０ｎｍ〜８００ｎｍの二波長で２回以上測定し、吸光度変化量を求めることによって行われる。
(Measurement method of diacetylspermine content)
Measurement of the amount of diacetylspermine in the sample by the method of the present invention is performed by mixing the sample and the first reagent, and after a certain time, adding and mixing the second reagent, and measuring the absorbance due to the aggregation of gold colloid, The measurement is performed twice or more at two wavelengths of 450 nm to 580 nm and 620 nm to 800 nm, and the change in absorbance is obtained.
吸光度の測定は、好ましくは、第２試薬の添加後２分以内に一回目の吸光度測定を行い、一回目の測定より８分以内に二回目の吸光度測定を行って、一回目と二回目との吸光度差を求める。あるいは、第２試薬添加後、２分以内より吸光度測定を開始し、測定開始後８分より短い時間内での吸光度変化（時間当たり）を求めてもよい。検体と第１試薬とを混合してから全ての測定が終了するまでに要する時間は、好ましくは１５分以内であり、より好ましくは１０分以内である。測定温度は、好ましくは２０〜４５℃であり、より好ましくは３０〜４０℃、さらに好ましくは３７℃である。 Preferably, the absorbance is measured by measuring the first absorbance within 2 minutes after the addition of the second reagent, and measuring the second absorbance within 8 minutes from the first measurement. Obtain the difference in absorbance. Alternatively, the absorbance measurement may be started within 2 minutes after the addition of the second reagent, and the change in absorbance (per hour) within a time shorter than 8 minutes after the start of measurement may be obtained. The time required from the mixing of the specimen and the first reagent to the end of all measurements is preferably within 15 minutes, more preferably within 10 minutes. Measurement temperature becomes like this. Preferably it is 20-45 degreeC, More preferably, it is 30-40 degreeC, More preferably, it is 37 degreeC.
通常、ジアセチルスペルミン標準品（２濃度以上）を試料と同様の測定操作で測定して検量線を作成し、試料中のジアセチルスペルミン濃度を求める。 Usually, a standard curve of diacetylspermine (2 concentrations or more) is measured by the same measurement procedure as that of the sample to prepare a calibration curve, and the concentration of diacetylspermine in the sample is obtained.
（ジアセチルスペルミン測定用試薬キット）
本発明のジアセチルスペルミン測定用試薬キットは、塩およびジアセチルスペルミン類似化合物結合蛋白質を含有する第１試薬、および抗ジアセチルスペルミン抗体結合金コロイドを含有する第２試薬を含む。第２試薬中の抗ジアセチルスペルミン抗体結合金コロイドは、好ましくは、終濃度として５４０ｎｍでの吸光度が１．５〜３．０となるように含まれる。また、本発明のジアセチルスペルミン測定用試薬キットは、検量線作成用のジアセチルスペルミンの標準品を含んでいてもよい。本発明のジアセチルスペルミン測定用測定キットは、上記測定方法において使用することが好ましく、特に好ましくは、尿中ジアセチルスペルミンの測定に使用される。
(Diacetylspermine measurement reagent kit)
The reagent kit for measuring diacetylspermine of the present invention includes a first reagent containing a salt and a diacetylspermine analog-binding protein, and a second reagent containing an anti-diacetylspermine antibody-binding gold colloid. The anti-diacetylspermine antibody-bound gold colloid in the second reagent is preferably included so that the absorbance at 540 nm is 1.5 to 3.0 as the final concentration. The reagent kit for measuring diacetylspermine of the present invention may contain a standard product of diacetylspermine for preparing a calibration curve. The measurement kit for measuring diacetylspermine of the present invention is preferably used in the above-described measurement method, and particularly preferably used for measuring urinary diacetylspermine.
以下、実施例に基づいて本発明をさらに具体的に説明するが、本発明はこれらによって限定されるものではない。なお、以下の実施例において、％は、特に記載がない限り、Ｗ／Ｖ％を表す。 EXAMPLES Hereinafter, although this invention is demonstrated further more concretely based on an Example, this invention is not limited by these. In the following examples,% represents W / V% unless otherwise specified.
実施例１：アセチルスペルミン結合ＢＳＡの調製
アセチルスペルミン結合ＢＳＡは、非特許文献５に記載の方法に準じて作製した。まず、担体蛋白質であるＢＳＡ ２００ｍｇとＳ−アセチルメルカプト無水コハク酸 １５ｍｇとを０．１Ｍリン酸緩衝液（ｐＨ７．０）中で混合し、室温で３０分間攪拌して、Ｓ−アセチルメルカプトコハク酸(ＡＭＳ)−ＢＳＡ複合体を作製した。これとは別に、二価性架橋試薬であるＧＭＢＳ ４５ｍｇとＮ−アセチルスペルミン・３塩酸塩 １４７ｍｇとをテトラヒドロフラン／５０ｍＭリン酸緩衝液（ｐＨ７．０）（１：１（ｖ／ｖ））中で室温にて３０分間混合して、アセチルスペルミン−ＧＭＳ結合物を作製した。次いで、ＡＭＳ−ＢＳＡ ２００ｍｇにヒドロキシルアミン塩酸塩４．２ｍｇを加え、メルカプトスクシニル−ＢＳＡ（ＭＳ−ＢＳＡ）とした。次いで、アセチルスペルミン−ＧＭＢ結合物とＭＳ−ＢＳＡとを混合して、Ｎ−アセチルスペルミンがアシルアミド結合した免疫抗原アセチルスペルミン−ＧＭＢ−ＢＳＡを作製した。このようにしてアセチルスペルミンを担体にアシルアミド結合させることにより、ジアセチルスペルミン類似化合物を側鎖として多数持つ免疫抗原を作製した。
Example 1: Preparation of acetylspermine-bound BSA Acetylspermine-bound BSA was prepared according to the method described in Non-Patent Document 5. First, 200 mg of BSA as a carrier protein and 15 mg of S-acetylmercaptosuccinic anhydride were mixed in 0.1 M phosphate buffer (pH 7.0) and stirred at room temperature for 30 minutes to obtain S-acetylmercaptosuccinic acid. (AMS) -BSA complex was prepared. Separately, 45 mg of GMBS, which is a bivalent crosslinking reagent, and 147 mg of N-acetylspermine trihydrochloride are added in tetrahydrofuran / 50 mM phosphate buffer (pH 7.0) (1: 1 (v / v)). The mixture was mixed at room temperature for 30 minutes to produce an acetylspermine-GMS conjugate. Subsequently, 4.2 mg of hydroxylamine hydrochloride was added to 200 mg of AMS-BSA to obtain mercaptosuccinyl-BSA (MS-BSA). Next, the acetylspermine-GMB conjugate and MS-BSA were mixed to prepare an immunity antigen acetylspermine-GMB-BSA in which N-acetylspermine was acylamide-linked. In this way, an immunized antigen having a large number of diacetylspermine-like compounds as side chains was prepared by linking acetylspermine to an acylamide on a carrier.
アセチルスペルミン結合ＢＳＡの濃度は、１ｍｇ／ｍＬのＢＳＡ溶液の２８０ｎｍにおける吸光度（光路長１ｃｍ）、Ａ２８０を０．６６として、Ａ２８０値に基づいて算出した。 The concentration of acetylspermine-bound BSA was calculated based on the A 280 value, assuming that the absorbance at 280 nm (optical path length 1 cm) of a 1 mg / mL BSA solution and A 280 was 0.66.
実施例２：抗ジアセチルスペルミン抗体の調製
（抗体作成）
上記実施例１で得たアセチルスペルミン結合ＢＳＡとアジュバント（初回免疫：コンプリートアジュバント、追加免疫：インコンプリートアジュバント）とを等量混和したエマルジョンを、ウサギ（New Zealand White種）背部皮下に注射することにより免疫を行った。初回免疫は１ｍｇ／羽で、そして追加免疫は１ｍｇ／羽の投与量で２週間おきに５〜６回行った。
Example 2: Preparation of anti-diacetylspermine antibody (antibody production)
By injecting an emulsion of equal amounts of acetylspermine-conjugated BSA obtained in Example 1 above and an adjuvant (primary immunization: complete adjuvant, booster immunization: incomplete adjuvant) into the back of a rabbit (New Zealand White) by subcutaneous injection Immunized. The initial immunization was 1 mg / fowl, and booster immunizations were carried out 5-6 times every 2 weeks at a dose of 1 mg / fowl.
（抗体精製）
上記の方法で免疫したウサギから採血し、得られた抗血清から、段階的な親和性精製によって高度にジアセチルスペルミン特異的な抗体を分画精製した。親和性精製については、非特許文献５に記載の方法に準じた。すなわち、抗血清をＮ１−アセチルスペルミン結合カルボキシトヨパールに吸着させ、樹脂に吸着する成分を溶出・回収し、この成分をさらにＮ８−アセチルスペルミジン結合カルボキシトヨパールに吸着させ、この樹脂に吸着しない成分を溶出し、部分精製抗体を得た。次いで、得られた部分精製抗体を、再度アセチルスペルミン結合カルボキシトヨパールに吸着させ、２ｍＭのＮ１−アセチルスペルミジンを添加してもなお溶出されず、アセチルスペルミン結合カルボキシトヨパールに吸着したまま残留する成分を回収して精製抗体とした。この抗体は、ジアセチルスペルミンの類似物質として尿中に約３０倍も多く存在して尿中ジアセチルスペルミンの定量を妨害する可能性があるＮ１−アセチルスペルミジンとの交差反応性が０．１％以下であった。
(Antibody purification)
Blood was collected from rabbits immunized by the above method, and highly diacetylspermine-specific antibodies were fractionated and purified from the obtained antiserum by stepwise affinity purification. Affinity purification was in accordance with the method described in Non-Patent Document 5. That is, anti-serum N 1 - adsorbed on spermine binding carboxymethyl Toyopearl, the component adsorbed on the resin elution and recovery, and the component further N 8 - adsorbed on acetyl spermidine binding carboxymethyl Toyopearl, adsorbed to the resin Unpurified components were eluted to obtain partially purified antibodies. Subsequently, the partially purified antibody thus obtained is adsorbed again on acetylspermine-conjugated carboxytoyopearl, and is not eluted even when 2 mM N 1 -acetylspermidine is added, and remains adsorbed on acetylspermine-conjugated carboxytoyopearl. The components were recovered and used as purified antibodies. This antibody has a cross-reactivity with N 1 -acetylspermidine of not more than 0.1%, which is about 30 times as much as diacetylspermine in the urine and may interfere with the determination of urinary diacetylspermine. Met.
実施例３：金コロイド液の調製
９５℃の蒸留水１Ｌに１０％塩化金酸溶液２ｍＬを攪拌しながら加え、１分後に２％クエン酸ナトリウム溶液１０ｍＬを加え、さらに２０分間攪拌した後３０℃に冷却した。冷却後、０．１Ｍ炭酸カリウム溶液でｐＨ７．１に調節した。
Example 3: Preparation of colloidal gold solution 2 mL of 10% chloroauric acid solution was added to 1 L of distilled water at 95 ° C with stirring, and after 1 minute, 10 mL of 2% sodium citrate solution was added, and the mixture was further stirred for 20 minutes and then 30 ° C. Cooled to. After cooling, the pH was adjusted to 7.1 with 0.1 M potassium carbonate solution.
実施例４：第１試薬の調製
上記実施例１で調製したジアセチルスペルミン結合ＢＳＡ、塩化ナトリウム、およびポリエチレングリコール２００００（ＰＥＧ）をそれぞれ種々の濃度で含む、０．２％ＥＤＴＡ、０．３％トリトンＸ−１００、０．１％ＢＳＡ、０．５％ウサギ血清、および０．１％アジ化ナトリウムを含む２００ｍＭ ＭＥＳ（ｐＨ６．２）を調製し、アセチルスペルミン結合ＢＳＡの濃度、塩化ナトリウム濃度、およびＰＥＧ濃度がそれぞれ異なる種々の第１試薬を調製した。
Example 4: Preparation of first reagent 0.2% EDTA, 0.3% Triton containing diacetylspermine-conjugated BSA prepared in Example 1 above, sodium chloride, and polyethylene glycol 20000 (PEG) at various concentrations, respectively. A 200 mM MES (pH 6.2) containing X-100, 0.1% BSA, 0.5% rabbit serum, and 0.1% sodium azide is prepared, and the concentration of acetylspermine-conjugated BSA, sodium chloride concentration, and Various first reagents having different PEG concentrations were prepared.
実施例５：抗ジアセチルスペルミン抗体結合金コロイド試薬（第２試薬）の調製
上記実施例２で調製した抗ジアセチルスペルミン抗体を、０．０５％アジ化ナトリウムを含む１０ｍＭ ＨＥＰＥＳ（ｐＨ７．１）で希釈し、４０μｇ／ｍＬの濃度にした。この液１００ｍＬを上記実施例３で調製した金コロイド液１Ｌに加え、冷蔵下で２時間攪拌した。この混合物に、５．４６％マンニトール、０．５％ＢＳＡ、および０．０５％アジ化ナトリウムを含む１０ｍＭ ＨＥＰＥＳ（ｐＨ７．１）を１１０ｍＬ添加し、３７℃で９０分間攪拌し、８，０００回転で４０分間遠心分離し、上清を除去した。得られた残渣に、３％マンニトール、０．１％ＢＳＡ、および０．０５％アジ化ナトリウムを含む５ｍＭ ＨＥＰＥＳ（ｐＨ７．５）（Ａ溶液）を約１Ｌ加え、抗体結合金コロイドを分散させた後、８，０００回転で４０分間遠心分離し、上清を除去した。残渣をＡ溶液に分散させ、この抗ジアセチルスペルミン抗体結合金コロイドの分散液を精製水で３０倍希釈したときに５４０ｎｍでの吸光度が１．０となるようにＡ溶液を添加し、抗体結合金コロイド原液とした。
Example 5: Preparation of anti-diacetylspermine antibody-conjugated gold colloid reagent (second reagent) The anti-diacetylspermine antibody prepared in Example 2 above was diluted with 10 mM HEPES (pH 7.1) containing 0.05% sodium azide. To a concentration of 40 μg / mL. 100 mL of this solution was added to 1 L of the colloidal gold solution prepared in Example 3 and stirred for 2 hours under refrigeration. To this mixture, 110 mL of 10 mM HEPES (pH 7.1) containing 5.46% mannitol, 0.5% BSA, and 0.05% sodium azide was added, stirred at 37 ° C. for 90 minutes, and rotated at 8,000 rpm. And centrifuged for 40 minutes and the supernatant was removed. About 1 L of 5 mM HEPES (pH 7.5) (solution A) containing 3% mannitol, 0.1% BSA, and 0.05% sodium azide was added to the obtained residue to disperse the antibody-bound gold colloid. Thereafter, the mixture was centrifuged at 8,000 rpm for 40 minutes, and the supernatant was removed. The residue was dispersed in the solution A, and when the anti-diacetylspermine antibody-bound gold colloid dispersion was diluted 30-fold with purified water, the solution A was added so that the absorbance at 540 nm was 1.0. A colloid stock solution was prepared.
さらに、抗体結合金コロイド原液をＡ溶液で希釈して、種々の抗体結合金コロイド濃度を有する抗ジアセチルスペルミン抗体結合金コロイド試薬を調製した。 Furthermore, the antibody-bound gold colloid stock solution was diluted with the solution A to prepare anti-diacetylspermine antibody-bound gold colloid reagents having various antibody-bound gold colloid concentrations.
実施例６：ジアセチルスペルミン添加尿の調製
ジアセチルスペルミン１ｍｇ／ｍＬを健常人ヒト尿で希釈し、種々の濃度のジアセチルスペルミンを含有する尿を調製した。
Example 6: Preparation of diacetylspermine-added urine Diacetylspermine 1 mg / mL was diluted with normal human urine to prepare urine containing various concentrations of diacetylspermine.
実施例７：第１試薬および第２試薬における各成分の濃度の検討
本実施例においては、第１試薬および第２試薬における各成分の濃度の検討を以下のように行った。まず、上記実施例６で調製したジアセチルスペルミン添加尿検体２０μＬおよび上記実施例４の第１試薬２００μＬを混合・攪拌し、３７℃で約５分加温した。次いで、上記実施例５で調製した第２試薬５０μＬを添加・攪拌した後、日立７０７０形自動分析装置により主波長５４６ｎｍおよび副波長６６０ｎｍで測光ポイント１８から３１の二ポイント測定を行って、二ポイント間の吸光度差を求めた。測定時間は約１０分間であった。
Example 7: Examination of the concentration of each component in the first reagent and the second reagent In this example, the concentration of each component in the first reagent and the second reagent was examined as follows. First, 20 μL of the diacetylspermine-added urine sample prepared in Example 6 and 200 μL of the first reagent of Example 4 were mixed and stirred, and heated at 37 ° C. for about 5 minutes. Next, 50 μL of the second reagent prepared in Example 5 above was added and stirred, and then two-point measurement of photometry points 18 to 31 was performed at a main wavelength of 546 nm and a sub-wavelength of 660 nm using Hitachi 7070 type automatic analyzer. The difference in absorbance between them was determined. The measurement time was about 10 minutes.
（塩濃度およびＰＥＧ濃度についての検討）
５０ｎｇ／ｍＬのアセチルスペルミン結合ＢＳＡを含む第１試薬中の塩として、塩化ナトリウムを０％〜７％の間で変化させ、尿中ジアセチルスペルミンを測定したときの吸光度変化を検討した。結果を表１に示す。なお、このとき、第１試薬中のＰＥＧ濃度は１．６％または１．８％であり、アセチルスペルミン結合ＢＳＡ量は５０ｎｇ／ｍＬであり、そして第２試薬中の抗ジアセチルスペルミン抗体結合金コロイドの測定液中での吸光度は（５４０ｎｍ（光路長１ｃｍ））は２．２２であった。
(Examination of salt concentration and PEG concentration)
As a salt in the first reagent containing 50 ng / mL of acetylspermine-bound BSA, sodium chloride was changed between 0% and 7%, and changes in absorbance were measured when urinary diacetylspermine was measured. The results are shown in Table 1. At this time, the PEG concentration in the first reagent is 1.6% or 1.8%, the amount of acetylspermine-bound BSA is 50 ng / mL, and the anti-diacetylspermine antibody-bound gold colloid in the second reagent The absorbance in the measurement solution was (540 nm (optical path length: 1 cm)) was 2.22.
塩化ナトリウム濃度が０％の場合は、ジアセチルスペルミン濃度に依存した吸光度変化が認められず、測定できなかった。塩化ナトリウム濃度が１％〜５％と増加するに従って、ジアセチルスペルミンが０Ｍのときの吸光度差が小さくなった。塩化ナトリウム濃度が６％および７％の場合は、ＰＥＧ濃度を１．８％にすることにより、ジアセチルスペルミンが０Ｍのときの吸光度差が大きくなった。このように、塩濃度が高すぎると、抗原抗体反応が抑制されるため、凝集促進のためにＰＥＧなどの水溶性高分子物質を多く使用する必要が生じ、さらに好ましい塩濃度および好ましいＰＥＧ（水溶性高分子物質）濃度の厳密な設定が必要であった。これらのことから、塩化ナトリウム濃度が約２％の場合、ジアセチルスペルミン濃度に依存して明瞭に吸光度差が変化することがわかった。 When the sodium chloride concentration was 0%, no change in absorbance depending on the diacetylspermine concentration was observed, and measurement was not possible. As the sodium chloride concentration increased from 1% to 5%, the difference in absorbance when diacetylspermine was 0 M was reduced. When the sodium chloride concentration was 6% and 7%, the difference in absorbance when diacetylspermine was 0 M was increased by setting the PEG concentration to 1.8%. As described above, when the salt concentration is too high, the antigen-antibody reaction is suppressed. Therefore, it is necessary to use a large amount of a water-soluble polymer substance such as PEG for promoting aggregation, and more preferable salt concentration and preferable PEG (water solution It was necessary to set the concentration precisely. From these facts, it was found that when the sodium chloride concentration was about 2%, the absorbance difference clearly changed depending on the diacetylspermine concentration.
（抗ハプテン抗体結合金コロイド量についての検討）
２％の塩化ナトリウム、１．６％のＰＥＧ、および１００ｎｇ／ｍＬのアセチルスペルミン結合ＢＳＡを含む第１試薬を用い、そして測定液における５４０ｎｍ（光路長１ｃｍ）の吸光度が１．５〜３．０となるような量で抗ハプテン抗体結合金コロイド（抗ジアセチルスペルミン抗体結合金コロイド）を含む第２試薬を用いて、尿中ジアセチルスペルミンを測定した。結果を表２に示す。
(Study on the amount of colloidal gold conjugated with anti-hapten antibody)
A first reagent containing 2% sodium chloride, 1.6% PEG, and 100 ng / mL acetylspermine-conjugated BSA was used, and the absorbance at 540 nm (optical path length 1 cm) in the measurement solution was 1.5 to 3.0. Urinary diacetylspermine was measured using a second reagent containing an anti-hapten antibody-bound gold colloid (anti-diacetylspermine antibody-bound gold colloid) in such an amount as follows. The results are shown in Table 2.
ジアセチルスペルミンが０Ｍのときの吸光度差が、抗ジアセチルスペルミン抗体結合金コロイド量の増加に従って大きくなった。金コロイド量が少ない場合は、ジアセチルスペルミン濃度に依存的な吸光度差の変化（競合反応の進行）が小さいため好ましくない。金コロイド量が多い場合は使用可能であるが、試薬のコストや測定精度の面で好ましくない。これらの結果から、第２試薬中の金コロイド量は、測定液における５４０ｎｍ（光路長１ｃｍ）の吸光度が約２．５となる場合が好適であることがわかった。 The difference in absorbance when diacetylspermine was 0 M was increased as the amount of anti-diacetylspermine antibody-bound gold colloid was increased. A small amount of colloidal gold is not preferable because the change in absorbance difference depending on the diacetylspermine concentration (competitive reaction progress) is small. Although it can be used when the amount of colloidal gold is large, it is not preferable in terms of reagent cost and measurement accuracy. From these results, it was found that the amount of colloidal gold in the second reagent is suitable when the absorbance at 540 nm (optical path length 1 cm) in the measurement solution is about 2.5.
（ジアセチルスペルミン類似化合物結合蛋白質の量についての検討）
第１試薬中の競合物質であるハプテン結合蛋白質（アセチルスペルミン結合ＢＳＡ）量を５０ｎｇ／ｍＬ〜２００ｎｇ／ｍＬまで変化させて、尿中ジアセチルスペルミンを測定した。結果を表３に示す。このときの抗ジアセチルスペルミン抗体結合金コロイド量は、測定反応液中で５４０ｎｍ（光路長１ｃｍ）の吸光度が２．５となる濃度であった。
(Study on the amount of diacetylspermine analog binding protein)
Urinary diacetylspermine was measured by changing the amount of hapten-binding protein (acetylspermine-bound BSA), which is a competitor in the first reagent, from 50 ng / mL to 200 ng / mL. The results are shown in Table 3. The amount of the anti-diacetylspermine antibody-bound gold colloid at this time was such a concentration that the absorbance at 540 nm (optical path length 1 cm) was 2.5 in the measurement reaction solution.
アセチルスペルミン結合ＢＳＡ量に依存して、ジアセチルスペルミンが０Ｍのときの吸光度差が大きくなったが、いずれの濃度においても尿中ジアセチルスペルミン濃度に応じた吸光度差の低下が認められ、測定可能であることがわかった。 Depending on the amount of acetylspermine-bound BSA, the difference in absorbance when diacetylspermine was 0 M was increased, but at any concentration, a decrease in the absorbance difference according to the urinary diacetylspermine concentration was observed and measurable. I understood it.
実施例８：尿中ジアセチルスペルミンの測定
１００ｎｇ／ｍＬのアセチルスペルミン結合ＢＳＡ、２％の塩化ナトリウム、および１．８％のＰＥＧを含む第１試薬と、測定反応液中で５４０ｎｍ（１ｃｍ）の吸光度が２．５となる濃度の抗ジアセチルスペルミン抗体結合金コロイドを含む第２試薬とを用いて、尿中ジアセチルスペルミンを測定した。結果を図１に示す。図１から明らかなように、尿中ジアセチルスペルミン濃度に応じた吸光度差の低下が認められた。このように、ジアセチルスペルミンをｎｇ／ｍＬからμｇ／ｍＬまでの範囲で測定できることがわかった。
Example 8: Measurement of urinary diacetylspermine A first reagent containing 100 ng / mL acetylspermine-conjugated BSA, 2% sodium chloride, and 1.8% PEG, and absorbance at 540 nm (1 cm) in the measurement reaction solution Urinary diacetylspermine was measured using a second reagent containing an anti-diacetylspermine antibody-bound gold colloid at a concentration of 2.5. The results are shown in FIG. As is clear from FIG. 1, a decrease in the absorbance difference according to the urinary diacetylspermine concentration was observed. Thus, it was found that diacetylspermine can be measured in the range from ng / mL to μg / mL.
本発明の方法によれば、ｎｇ／ｍＬからμｇ／ｍＬまでの範囲のジアセチルスペルミンが短時間に精度良く測定できる。また、洗浄などの工程が不要であるため、自動化に適している。したがって、本発明の方法は、ジアセチルスペルミン測定の臨床検査分野などでの迅速化および省力化に寄与することができる。さらに、ジアセチルスペルミンは、腫瘍マーカーとしてだけではなく、悪性腫瘍の治療効果の判定や予後予測の指標としても期待されているため、本発明の方法は、臨床検査分野において非常に有用であり得る。 According to the method of the present invention, diacetylspermine in the range from ng / mL to μg / mL can be measured accurately in a short time. Further, since a process such as washing is not necessary, it is suitable for automation. Therefore, the method of the present invention can contribute to speeding up and labor saving in the clinical laboratory field of diacetylspermine measurement. Furthermore, since diacetylspermine is expected not only as a tumor marker but also as an indicator for determining the therapeutic effect of malignant tumors and predicting prognosis, the method of the present invention can be very useful in the clinical laboratory field.
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| JP2008292326A (en) * | 2007-05-24 | 2008-12-04 | Bl:Kk | Immunoassay in the presence of hyaluronic acid and products used therefor |
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| WO2009131536A1 (en) * | 2008-04-24 | 2009-10-29 | Svanova Biotech Ab | Molecular assebly comprising gold and a linker for detection of (bio) chemical entities |
| CN105527434A (en) * | 2015-12-31 | 2016-04-27 | 辽宁迈迪生物科技有限公司 | A kit used for detecting N1,N<12>-diacetylspermine (DAS) |
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| WO2009131536A1 (en) * | 2008-04-24 | 2009-10-29 | Svanova Biotech Ab | Molecular assebly comprising gold and a linker for detection of (bio) chemical entities |
| CN105527434A (en) * | 2015-12-31 | 2016-04-27 | 辽宁迈迪生物科技有限公司 | A kit used for detecting N1,N<12>-diacetylspermine (DAS) |
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