JPH11304803A - Analysis method of protein in sample and kit thereof - Google Patents
Analysis method of protein in sample and kit thereofInfo
- Publication number
- JPH11304803A JPH11304803A JP15835398A JP15835398A JPH11304803A JP H11304803 A JPH11304803 A JP H11304803A JP 15835398 A JP15835398 A JP 15835398A JP 15835398 A JP15835398 A JP 15835398A JP H11304803 A JPH11304803 A JP H11304803A
- Authority
- JP
- Japan
- Prior art keywords
- protein
- sample
- ion
- solution
- dye
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
【0001】[0001]
【発明の属する技術分野】本発明は、試料中に微量に含
まれるタンパク質を簡便に定性・定量することができる
分析方法及び分析キットに関する。分析化学、生命科
学、医療の分野、特に臨床検査分野において有用であ
る。[0001] The present invention relates to an analysis method and an analysis kit which can easily and qualitatively and quantitatively determine a minute amount of a protein contained in a sample. It is useful in the fields of analytical chemistry, life science, and medicine, especially in the field of clinical testing.
【0002】[0002]
【従来の技術】従来の最も簡便なタンパク質分析手段と
して、タンパク質と結合するとその色調を変える色素を
担体に含浸させた試験片が挙げられる。しかし、この試
験片は吸収可能な試料が数μl 〜数十μl にすぎず、タ
ンパク質を僅かしか含まない試料については適用困難で
あった。その上、試料中の他の物質や非結合態色素の影
響を受け、誤結果を招く場合もあった。2. Description of the Related Art Conventionally, the simplest means for protein analysis is a test piece in which a carrier is impregnated with a dye that changes its color tone when bound to a protein. However, this test piece can absorb only a few μl to several tens μl of a sample, and it is difficult to apply a sample containing only a small amount of protein. In addition, other substances and unbound dyes in the sample may be affected, resulting in erroneous results.
【0003】そのために近年、試料を濃縮して感度を上
げ、かつ試料中の妨害物質の影響を低減させるいくつか
の技術が提案されている。[0003] For this purpose, in recent years, several techniques have been proposed for concentrating a sample to increase the sensitivity and reducing the influence of interfering substances in the sample.
【0004】例えば、木下ら〔分析化学 23巻,1543-1
544頁,1974年〕は、試料中のタンパク質をメンブラン
フィルタ上に濃縮し、その後フィルタをフルオレセイン
イソチオシアネート(FITC)で蛍光染色し、洗浄後
にスポットの蛍光強度を測定する技術を報告している。
しかしながら、この技術においては、タンパク質とFI
TCとの反応に30分も要し、また妨害物質を除去する
ために染色後には洗浄を必要とするという問題があっ
た。For example, Kinoshita et al. [Analytical Chemistry 23, 1543-1]
544, 1974] report a technique in which a protein in a sample is concentrated on a membrane filter, the filter is then stained with fluorescein isothiocyanate (FITC), and after washing, the fluorescence intensity of the spot is measured.
However, in this technique, protein and FI
There was a problem that the reaction with TC required as long as 30 minutes and that washing was required after staining to remove interfering substances.
【0005】また、藤田ら〔分析化学 44巻,733-738
頁,1995年〕は、タンパク質と色素−金属錯体との沈殿
会合体をフィルタで物理的に補集した後、水酸化ナトリ
ウム水溶液で再溶解し、この溶液の吸光度を測定する技
術を報告している。しかしながら、この技術において
は、沈殿物の目詰まりのため定量性が決して良好とは言
えず、またフィルタ上の着色を観察するものではなく、
再溶解液を測定するものであるため、簡易迅速な分析に
適さないものであった。Also, Fujita et al. [Analytical Chemistry 44, 733-738]
Pp. 1995] reported a technique for physically collecting a precipitated aggregate of a protein and a dye-metal complex with a filter, re-dissolving with an aqueous sodium hydroxide solution, and measuring the absorbance of the solution. I have. However, in this technique, the quantitativeness is not always good due to clogging of the precipitate, and the coloring on the filter is not observed,
Since the re-dissolved solution was measured, it was not suitable for simple and rapid analysis.
【0006】[0006]
【発明が解決しようとする課題】以上のように、尿中タ
ンパク質分析のようなタンパク質を僅かしか含まない試
料を濃縮して感度を上げるこれまでの技術は、洗浄工
程、発色工程、抽出工程等の工程を必須とするために操
作が煩雑であり、色測定まで時間がかかり、定量性にも
問題があった。そこで、本発明は、工程数を極力減ら
し、色測定を短時間で可能とし、定量性に優れた、簡易
迅速なタンパク質の分析手段を提供することを目的とす
る。As described above, the conventional techniques for increasing the sensitivity by concentrating a sample containing only a small amount of protein, such as the analysis of protein in urine, include a washing step, a coloring step, and an extracting step. Since the step (1) is indispensable, the operation is complicated, it takes time to measure the color, and there is a problem in the quantitativeness. Therefore, an object of the present invention is to provide a simple and rapid protein analysis means which minimizes the number of steps, enables color measurement in a short time, and has excellent quantitative properties.
【0007】[0007]
【課題を解決するための手段】本発明は、a)タンパク
質を含有する可能性がある試料と、タンパク質とイオン
会合体を形成する色素を混合し、前記イオン会合体を沈
殿させずにイオン会合体溶液を形成させる工程;b)前
記イオン会合体溶液を疎水性フィルタに通すことによ
り、前記イオン会合体溶液中に溶解した前記会合体を前
記フィルタに吸着させる工程;及び、c)前記フィルタ
上の前記会合体を定性・定量的に分析する工程を順次行
う、試料中のタンパク質の分析方法である。According to the present invention, there is provided a method comprising the steps of: a) mixing a sample which may contain a protein with a dye which forms an ion aggregate with the protein, and precipitating the ion aggregate without precipitating the ion aggregate; Forming a combined solution; b) passing the ion-associated solution through a hydrophobic filter so that the aggregate dissolved in the ion-associated solution is adsorbed to the filter; and c) on the filter. A method for analyzing a protein in a sample, wherein the step of qualitatively and quantitatively analyzing the above aggregate is sequentially performed.
【0008】また、本発明は、タンパク質とイオン会合
体を形成する色素及び疎水性フィルタを含み、場合によ
りイオン会合体溶液を疎水性フィルタに通すための装置
を含む、前記方法に使用されるタンパク質分析キットで
ある。イオン会合体溶液を疎水性フィルタに通すための
装置としては、例えば、前記フィルタを固定可能な開口
部を、その一端に有するシリンダと、シリンダ内壁に摺
動して前記一端の方向に移動しうる、外部から操作可能
なピストンが挙げられ、ピストンの移動により、イオン
会合体溶液中のイオン会合体が該フィルタに吸着する。[0008] The present invention also relates to a protein used in the above method, which comprises a dye for forming an ion associate with the protein and a hydrophobic filter, and optionally a device for passing the ion associate solution through the hydrophobic filter. This is an analysis kit. As an apparatus for passing the ion association solution through a hydrophobic filter, for example, an opening capable of fixing the filter can be moved in the direction of the one end by sliding the cylinder at one end thereof and the inner wall of the cylinder. And an externally operable piston. The movement of the piston causes the ion association in the ion association solution to be adsorbed to the filter.
【0009】[0009]
【発明の実施の態様】本発明で分析可能な試料は、例え
ば、生体試料(血液、血清、血漿、尿、髄液、精液、唾
液、汗、腹水、羊水及び涙等の体液、脳、肝臓及び腎臓
等の臓器の抽出液、毛髪、爪及び皮膚等の組織の抽出液
並びに糞便等)、食品(穀物、野菜、果物、魚介類、加
工食品等)、飲料(水、茶、牛乳、果汁、酒類等)が挙
げられるが、特に限定されない。また、必要に応じて希
釈や濃縮を行う。BEST MODE FOR CARRYING OUT THE INVENTION Samples that can be analyzed in the present invention include, for example, biological samples (blood, serum, plasma, urine, cerebrospinal fluid, semen, body fluids such as saliva, sweat, ascites, amniotic fluid, and tears), brain, and liver. And extracts of organs such as kidneys, extracts of tissues such as hair, nails and skin and feces, etc.), foods (cereals, vegetables, fruits, seafood, processed foods, etc.), beverages (water, tea, milk, fruit juice) , Alcoholic beverages, etc.), but is not particularly limited. Further, dilution and concentration are performed as needed.
【0010】本発明で使用される色素としては、それと
タンパク質との会合体が沈殿しにくい性質になるような
色素が好ましい。具体的には、溶解度の高いイオン会合
体を形成するようなイオン性基を有する色素が好まし
い。[0010] The dye used in the present invention is preferably a dye which causes an association between the dye and the protein to be less likely to precipitate. Specifically, a dye having an ionic group that forms an ion associate having high solubility is preferable.
【0011】特に陰イオン性基を有する色素が好まし
く、タンパク質の正荷電部位と結合して電気的な中和を
行うことができる。例えば、スルホン基や水酸基等の親
水性基を有する色素が挙げられる。[0011] Dyes having an anionic group are particularly preferred, and can be electrically neutralized by binding to a positively charged site of a protein. For example, a dye having a hydrophilic group such as a sulfone group or a hydroxyl group may be used.
【0012】また、会合体の疎水性フィルタへの吸着性
を高めるため、色素が疎水性基を有することが好まし
く、特に芳香環を有する色素が好ましい。Further, in order to enhance the adsorptivity of the aggregate to the hydrophobic filter, the dye preferably has a hydrophobic group, and particularly preferably a dye having an aromatic ring.
【0013】このようなものとして、例えば、トリフェ
ニルメタン系色素(例えば、ブロモクロロフェノールブ
ルー、テトラブロモフェノールブルー、ブロモフェノー
ルブルー、クマシーブリリアントブルー、ブロモクレゾ
ールパープル)、アゾ色素(例えば、バッファローブラ
ック、アシッドオレンジ12、メチルオレンジ、オレン
ジG、アゾスルファチアゾール)、キサンテン系色素
(例えば、エオシン、エリスロシン)等が挙げられる。Examples of such dyes include triphenylmethane dyes (eg, bromochlorophenol blue, tetrabromophenol blue, bromophenol blue, Coomassie brilliant blue, bromocresol purple), azo dyes (eg, buffalo black, Acid Orange 12, methyl orange, orange G, azosulfathiazole), xanthene dyes (eg, eosin, erythrosine) and the like.
【0014】この中でも特に、ブロモクロロフェノール
ブルー(BCPB)、クマシーブリリアントブルー(C
BB)、テトラブロモフェノールブルー(TBPB)、
ブロモクレゾールパープル(BCP)、ブロモフェノー
ルブルー(BPB)やエオシンが好ましい。Among them, bromochlorophenol blue (BCPB) and Coomassie brilliant blue (C
BB), tetrabromophenol blue (TBPB),
Bromocresol purple (BCP), bromophenol blue (BPB) and eosin are preferred.
【0015】前記色素は、驚くべきことにほぼすべての
種類のタンパク質と結合し、イオン会合体を形成する。
本発明でのイオン会合体の形成機序は定かではないが、
静電引力型イオン会合や疎水構造型イオン会合のいずれ
かであると考えられる〔本水,「総合論文 イオン会合
を利用する分離・分析法の設計」,分析化学 38巻,14
7-170頁,1989年〕。[0015] The dyes surprisingly bind to almost all types of proteins and form ion associates.
Although the formation mechanism of the ion aggregate in the present invention is not clear,
It is considered to be either electrostatic attraction type ion association or hydrophobic structure type ion association [Honshu, "Comprehensive paper Design of separation and analysis method using ion association", Analytical Chemistry 38, 14
7-170, 1989].
【0016】本発明では、このイオン会合体を溶媒中に
完全に溶解させ、沈殿を生じさせないようにする。条件
は、当業者により容易に実験的に設定可能である。In the present invention, this ion associate is completely dissolved in a solvent so as not to cause precipitation. Conditions can be easily set experimentally by those skilled in the art.
【0017】例えば、タンパク質含有溶液として、ヒト
血清アルブミン(HSA)水溶液を用いた場合の条件例
を表1に示す。For example, Table 1 shows examples of conditions when an aqueous solution of human serum albumin (HSA) is used as the protein-containing solution.
【0018】[0018]
【表1】 [Table 1]
【0019】試料と色素を混合する際に、色素は、色素
を含有する溶液の形態で混合してもよく、または粉末や
固形状の形態で混合してもよい。When the sample and the dye are mixed, the dye may be mixed in the form of a solution containing the dye, or may be mixed in the form of a powder or a solid.
【0020】試料と色素の混合によりイオン会合体は瞬
時に形成される。このイオン会合体の色調は、色素のそ
れと相違することが好ましい。例えば、表1の条件下
で、BCPB、TBPBやBPBを用いた場合、タンパ
ク質添加前は黄色であるのに対し添加後は青色となり、
目視でタンパク質の定性・定量分析が可能となる。An ion associate is instantaneously formed by mixing the sample and the dye. The color tone of this ion associate is preferably different from that of the dye. For example, when BCPB, TBPB or BPB is used under the conditions of Table 1, the protein is yellow before the protein is added, but becomes blue after the protein is added.
Qualitative and quantitative analysis of proteins becomes possible by visual observation.
【0021】例えば、図1に示したように、BCPBは
pKa2 であるpH4.1以下では−1価(HBCPB
- )となり黄色を示すのであるが、タンパク質と会合す
ることにより−2価(BCPB2-)となって青色を示す
ようになる。For example, as shown in FIG. 1, BCPB is -1 valent (HBCPB) at pH 4.1 or lower which is pKa 2.
- ), Which shows yellow, but becomes -2 (BCPB 2- ) by associating with the protein and shows blue.
【0022】以上のように色調の変化が黄色から青色の
ように吸収波長が離れていると、色調の変化を目視にて
判別しやすいので特に好ましい。As described above, it is particularly preferable that the change in the color tone is different from the absorption wavelength, such as from yellow to blue, because the change in the color tone can be easily visually recognized.
【0023】なお、このような場合には、緩衝剤を添加
し、溶液中のpHを色素のpKaよりも低く設定するの
が好ましい。In such a case, it is preferable to add a buffer and set the pH in the solution to be lower than the pKa of the dye.
【0024】このイオン会合体溶液を疎水性フィルタに
通すことにより、イオン会合体溶液中のイオン会合体が
疎水性フィルタに吸着される。By passing the ion-associated solution through a hydrophobic filter, the ion-associated in the ion-associated solution is adsorbed on the hydrophobic filter.
【0025】このイオン会合体が疎水性フィルタに吸着
される原理は定かではないが、電気的に中和された会合
体が溶液中において疎水性フィルタに疎水結合的に吸着
すると考えられる。Although the principle of the adsorption of the ion aggregate on the hydrophobic filter is not clear, it is considered that the electrically neutralized aggregate is hydrophobically adsorbed on the hydrophobic filter in the solution.
【0026】なお、溶媒から疎水性フィルタへの分配比
が小さい色素を選択することにより、フィルタ上には会
合していない色素が殆ど吸着されないので、会合してい
ない色素の色の影響を回避することが可能となる。By selecting a dye having a small distribution ratio from the solvent to the hydrophobic filter, the unassociated dye is hardly adsorbed on the filter, so that the effect of the color of the unassociated dye is avoided. It becomes possible.
【0027】これにより、フィルタ上の会合していない
色素を除去するための洗浄操作が不要となる。例えば、
表1に示したすべての組み合わせにおいて、洗浄工程は
不要である(フィルタにはニトロセルロースフィルタを
使用)。This eliminates the need for a washing operation for removing unassociated dye on the filter. For example,
In all combinations shown in Table 1, a washing step is not required (a nitrocellulose filter is used for the filter).
【0028】ところで、本発明においては、混合する試
料と色素の量の比によってイオン会合体が疎水性フィル
タに吸着する量が変わってくる。In the present invention, the amount of the ion aggregate adsorbed on the hydrophobic filter changes depending on the ratio of the amount of the sample to the amount of the dye to be mixed.
【0029】これを実験的に示したのが図2である。な
お、この実験の操作の詳細は、後の参考例2に記載し
た。FIG. 2 shows this experimentally. The details of the operation of this experiment are described in Reference Example 2 later.
【0030】この図2において、横軸は混合した試料中
のタンパク質濃度を、左の縦軸は吸光度の差を、右の縦
軸は吸光度を表す。In FIG. 2, the horizontal axis represents the protein concentration in the mixed sample, the left vertical axis represents the difference in absorbance, and the right vertical axis represents the absorbance.
【0031】そして、この図2中の「○」は、イオン会
合体に特異的な602nmにおける、疎水性フィルタに
通す前のイオン会合体溶液の吸光度から疎水性フィルタ
を通した後のろ液の吸光度を差し引いた値(吸光度の
差)、つまり、疎水性フィルタに吸着したイオン会合体
の量を表す。In FIG. 2, “○” indicates the absorbance of the ion association solution before passing through the hydrophobic filter at 602 nm specific to the ion association and the filtrate after passing through the hydrophobic filter. A value obtained by subtracting the absorbance (difference in absorbance), that is, the amount of the ion aggregate adsorbed on the hydrophobic filter.
【0032】また、「■」は、イオン会合体に特異的な
602nmにおける、疎水性フィルタに通す前のイオン
会合体溶液の吸光度、つまり、イオン会合体溶液中に存
在するイオン会合体の量を表す。The symbol “■” represents the absorbance of the ion association solution before passing through the hydrophobic filter at 602 nm specific to the ion association, that is, the amount of the ion association present in the ion association solution. Represent.
【0033】この図より、試料と色素を混合したイオン
会合体溶液中に形成されるイオン会合体の量は、試料中
のタンパク質濃度が高くなるにつれて増加してゆくこと
が分かる。From this figure, it can be seen that the amount of ion associate formed in the ion associate solution obtained by mixing the sample and the dye increases as the protein concentration in the sample increases.
【0034】また、疎水性フィルタに吸着するイオン会
合体の量は、試料中のタンパク質濃度が約30mg/l
までの間は試料中のタンパク質濃度に応じて増加する
が、この濃度以上では逆に低下してゆくことが分かる。Further, the amount of the ion-associated substance adsorbed on the hydrophobic filter is determined when the protein concentration in the sample is about 30 mg / l.
Up to this point, it increases in accordance with the protein concentration in the sample, but at or above this concentration, it decreases.
【0035】また、イオン会合体溶液を疎水性フィルタ
に通し乾燥した後の疎水性フィルタ上の着色の強さをデ
ンシトメータで測定する実験を行った。この実験の結果
を図3に示した。また、実験の操作の詳細を後の参考例
3に記載した。Further, an experiment was conducted in which the coloring strength on the hydrophobic filter after passing the ion-associated solution through a hydrophobic filter and drying was measured with a densitometer. The result of this experiment is shown in FIG. The details of the experiment are described in Reference Example 3 below.
【0036】図3において、横軸は色素と混合した試料
中のタンパク質の濃度を、縦軸はイオン会合体に特異的
な602nmにおける吸光度の高さ(吸光度のピークの
面積)を表す。In FIG. 3, the horizontal axis represents the concentration of the protein in the sample mixed with the dye, and the vertical axis represents the level of absorbance at 602 nm (area of the peak of absorbance) specific to the ion associate.
【0037】この図3から、この実験系において、疎水
性フィルタ上のイオン会合体の量を表す602nmにお
ける吸光度の高さは、試料中のタンパク質濃度が約50
mg/lまでの間は試料中のタンパク質濃度に応じて増
加するが、この濃度以上では逆に低下してゆくことが分
かる。From FIG. 3, it can be seen that, in this experimental system, the absorbance at 602 nm, which indicates the amount of ion association on the hydrophobic filter, is approximately 50% at the protein concentration in the sample.
It can be seen that the concentration increases up to mg / l in accordance with the protein concentration in the sample, but decreases above this concentration.
【0038】つまり、本発明において、試料中のタンパ
ク質と色素の会合体の形成時に、色素の量に対してタン
パク質の量が一定範囲にある場合は、形成されたイオン
会合体は疎水性フィルタに吸着するが、一定範囲を越え
タンパク質量が多くなってくると、形成されるイオン会
合体の量は増えるにもかかわらず疎水性フィルタには吸
着しなくなってくる。That is, in the present invention, when the amount of the protein is within a certain range relative to the amount of the dye at the time of forming the aggregate of the protein and the dye in the sample, the formed ion aggregate is applied to the hydrophobic filter. Although the protein is adsorbed, when the amount of protein increases beyond a certain range, it does not adsorb to the hydrophobic filter although the amount of ion aggregate formed increases.
【0039】本発明において、使用される疎水性フィル
タの材質として、例えば、ニトロセルロース、オクタデ
シルシリル化シリカ、オクチルシリル化シリカ、エチル
シリル化シリカ、メチルシリル化シリカ、フェニルシリ
ル化シリカ、フルオロカーボン化シリカ、グラファイト
カーボン化シリカ等が挙げられる。このようなフィルタ
は市販されており、例えば、混合セルロースエステルフ
ィルタA020A025A(ニトロセルロース、ポアサイズ0.
2μm 、アドバンテック東洋社製)が使用しうる。In the present invention, examples of the material of the hydrophobic filter used include nitrocellulose, octadecylsilylated silica, octylsilylated silica, ethylsilylated silica, methylsilylated silica, phenylsilylated silica, fluorocarbonized silica, and graphite. And carbonized silica. Such a filter is commercially available, for example, a mixed cellulose ester filter A020A025A (nitrocellulose, pore size 0.1 mL).
2 μm, manufactured by Advantech Toyo).
【0040】フィルタ上の着色の測定は、目視により行
っても、デンシトメータやスポットメータ等の機器を用
いてもよい。また、着色強度は、例えば、可視部の吸光
度、透過率、蛍光強度を測定することによりなされる。The coloration on the filter may be measured visually or using a device such as a densitometer or a spot meter. The coloring intensity is determined, for example, by measuring the absorbance, transmittance, and fluorescence intensity in the visible region.
【0041】なお、本発明において、フィルタ上の着色
は、疎水性フィルタにイオン会合体溶液を通した直後の
疎水性フィルタがぬれている時と、疎水性フィルタが乾
燥している時では色が異なる。In the present invention, the color on the filter is different between when the hydrophobic filter is wet immediately after passing the ion association solution through the hydrophobic filter and when the hydrophobic filter is dry. different.
【0042】これを実験的に示したのが図4である。な
お、この実験の詳細は、後の参考例4に記載した。FIG. 4 shows this experimentally. The details of this experiment are described in Reference Example 4 below.
【0043】この図4において、横軸は疎水性フィルタ
上の着色の吸光度測定時の波長を、縦軸はその時の吸光
度の値を表す。そして、この図4中の実線は、イオン会
合体溶液を疎水性フィルタに通した直後の疎水性フィル
タがぬれている時の吸光度を、破線はこの疎水性フィル
タを乾燥した時の吸光度を表す。In FIG. 4, the horizontal axis represents the wavelength at the time of measuring the absorbance of the coloring on the hydrophobic filter, and the vertical axis represents the value of the absorbance at that time. The solid line in FIG. 4 shows the absorbance when the hydrophobic filter is wet immediately after passing the ion-associated solution through the hydrophobic filter, and the broken line shows the absorbance when the hydrophobic filter is dried.
【0044】この図より、疎水性フィルタ上の着色は、
疎水性フィルタがぬれている時に比べて乾燥している時
は、HBCPB- に特異的な434nm近辺(黄色)の
吸光度が減少し、かつイオン会合体に特異的な602n
m近辺(青色)の吸光度が増加していることが分かる。From this figure, the coloring on the hydrophobic filter is
When dried than when hydrophobic filter is wet, HBCPB - the absorbance decreases near specific 434 nm (yellow), and the specific ion associate 602n
It can be seen that the absorbance in the vicinity of m (blue) has increased.
【0045】よって、フィルタ上の着色の測定は、フィ
ルタを乾燥した後に行うことが好ましい。フィルタの乾
燥は、放置、風を当てること、又は加温等により行えば
よい。放置により乾燥する場合は、通常は20分間以上
放置すればよい。Therefore, it is preferable to measure the coloring on the filter after drying the filter. Drying of the filter may be performed by leaving, blowing, heating or the like. In the case of drying by leaving, it is usually sufficient to leave it for 20 minutes or more.
【0046】上記の参考例2、参考例3、及び参考例4
の結果より、本発明における試料中のタンパク質と色素
の会合体の形成、形成されたイオン会合体の疎水性フィ
ルタへの吸着、及び着色について、以下のような作用が
推察される。Reference Example 2, Reference Example 3, and Reference Example 4
From the results of (1), the following effects are presumed on the formation of the aggregate of the protein and the dye in the sample, the adsorption of the formed ion aggregate to the hydrophobic filter, and the coloring in the present invention.
【0047】色素はpKa2 以下のpHでは1価(HB
CPB- )となっているが、タンパク質と会合すること
により色素は多価(BCPB2-)となる。この時に、色
素の量に対してタンパク質の量が一定以上の場合は、タ
ンパク質に会合している色素は多価(BCPB2-)であ
って、タンパク質の限定された特定の部位に会合する。The dye is monovalent (HB) at a pH of pKa 2 or less.
CPB − ), but the dye becomes polyvalent (BCPB 2− ) by associating with the protein. At this time, if the amount of the protein is at least a certain amount relative to the amount of the dye, the dye associated with the protein is multivalent (BCPB 2− ) and associates with a specific limited site of the protein.
【0048】また、色素の量に対してタンパク質の量が
一定以下の場合、つまりタンパク質に対して色素が過剰
に存在する場合は、多価の色素(BCPB2-)が特定の
部位に会合したタンパク質に、更に過剰に存在する1価
の色素(HBCPB- )が非選択的に会合して多重会合
体を形成する。When the amount of the protein is less than a certain amount relative to the amount of the dye, that is, when the amount of the dye is excessive relative to the amount of the protein, the polyvalent dye (BCPB 2− ) is associated with a specific site. The monovalent dye (HBCPB − ), which is present in excess, further non-selectively associates with the protein to form a multi-aggregate.
【0049】そして、色素の量に対してタンパク質の量
が一定以上の場合に形成される、多価の色素とのイオン
会合体は、疎水性フィルタとの親和性が低く僅かしか吸
着しない。An ion associate with a polyvalent dye, which is formed when the amount of the protein is at least a certain amount relative to the amount of the dye, has a low affinity for the hydrophobic filter and is slightly adsorbed.
【0050】しかし、色素の量に対してタンパク質の量
が一定以下の場合に形成される多重会合体は、多価の色
素に加えて1価の色素が多重に会合しているため疎水性
が高くなり、疎水的親和性によって疎水性フィルタへの
吸着率が高くなる。However, the multi-aggregate formed when the amount of the protein is less than a certain amount relative to the amount of the dye has a hydrophobic property because a monovalent dye is multiply associated with a polyvalent dye. And the adsorption rate to the hydrophobic filter is increased by the hydrophobic affinity.
【0051】以上のことは、試料中のタンパク質濃度が
増加するにつれ形成されるイオン会合体量が増してゆく
にもかかわらず、疎水性フィルタに吸着するイオン会合
体の量が一定のタンパク質濃度を境に増加から減少に転
じることより推察される。(図2及び図3を参照のこ
と。)As described above, although the amount of ion aggregates formed increases as the protein concentration in the sample increases, the amount of ion aggregates adsorbed on the hydrophobic filter decreases to a constant protein concentration. It is inferred from the fact that the situation changes from increasing to decreasing. (See FIGS. 2 and 3)
【0052】そして、疎水性フィルタに吸着した多重会
合体は、多価の色素(BCPB2-)〔青色〕が会合した
上に1価の色素(HBCPB- )〔黄色〕が多数会合し
ているため黄緑色を示すのであるが、これが乾燥状態に
置かれることによってpH又は塩濃度等の環境が変わ
り、1価の色素(HBCPB- )のプロトン解離、又は
タンパク質の特定の部位への会合を引き起こし、1価の
色素(HBCPB- )が多価の色素(BCPB2-)に変
換され多価の色素の色(青色)に変わる。The multi-aggregate adsorbed on the hydrophobic filter has a polyvalent dye (BCPB 2− ) [blue] associated therewith and a large number of monovalent dyes (HBCPB − ) [yellow] associated therewith. although indicate yellow-green because this changes the environment, such as pH or salt concentration by being placed in a dry state, monovalent dye (HBCPB -) cause proton dissociation or association to a particular site of the protein of The monovalent dye (HBCPB − ) is converted to a polyvalent dye (BCPB 2− ) and changes to a multivalent dye color (blue).
【0053】上記のことは、乾燥により、1価の色素
(HBCPB- )に特異的な434nm近辺(黄色)の
吸光度が減少し、イオン会合体に特異的な602nm近
辺(青色)の吸光度が増加することより推察される。
(図4を参照のこと)[0053] The above means that, upon drying, a monovalent dye (HBCPB -) absorbance of the specific 434nm vicinity (yellow) is reduced, increasing absorbance near 602nm specific to ion associate (blue) Inferred from doing
(See FIG. 4)
【0054】以上詳述したように、本発明の試料中のタ
ンパク質の分析方法、及びタンパク質分析キットでは、
分析を行う試料中のタンパク質の量に対して色素の量等
を調整することにより、試料中のタンパク質が特定の濃
度範囲にある時のみ、疎水性フィルタ上に着色を生じさ
せることができる。つまり、試料中のタンパク質が特定
の濃度範囲にあることを検出することができる。As described in detail above, the method for analyzing proteins in a sample and the protein analysis kit of the present invention
By adjusting the amount of the dye and the like with respect to the amount of the protein in the sample to be analyzed, coloring can be caused on the hydrophobic filter only when the protein in the sample is in a specific concentration range. That is, it is possible to detect that the protein in the sample is in a specific concentration range.
【0055】そして、色素の量等を調整して、着色を生
じる濃度範囲を段階的に変えていった複数の濃度の色素
よりなる分析キットを用いて1つの試料を分析すること
により、その試料中のタンパク質の定量分析を行うこと
ができる。Then, one sample is analyzed by using an analysis kit comprising a plurality of concentrations of dyes in which the concentration range in which coloring occurs is changed stepwise by adjusting the amount of the dyes and the like. Quantitative analysis of the protein in it can be performed.
【0056】本発明のタンパク質分析キットであるが、
図5に本発明の方法を実施するための分析キットの例
(分析キット1)を示す。分析に際し、まず疎水性フィ
ルタ2をシリンダ3の開口部に透き間なく固定する。次
に、試料と色素を混合したイオン会合体溶液を、予めシ
リンダ3内部に注入しておき、ピストン5を開口部4の
方向に移動させて(すなわち、レバー6を押して)、イ
オン会合体溶液をシリンダ外部に押し出す。この際、こ
のイオン会合体溶液中に含まれているイオン会合体が疎
水性フィルタ2に吸着される。あるいは、試料に色素を
添加したイオン会合体溶液中に開口部4を導入し、ピス
トン5を開口部4と反対側に移動させる(すなわち、レ
バー6を引く)ことにより、シリンダ3内部にイオン会
合体溶液を吸引して、疎水性フィルタ2にイオン会合体
を吸着させる態様にしてもよい。The protein analysis kit of the present invention,
FIG. 5 shows an example of an analysis kit (analysis kit 1) for performing the method of the present invention. In the analysis, first, the hydrophobic filter 2 is fixed to the opening of the cylinder 3 without any gap. Next, the ion association solution in which the sample and the dye are mixed is injected into the cylinder 3 in advance, and the piston 5 is moved in the direction of the opening 4 (that is, the lever 6 is pushed) to thereby obtain the ion association solution. To the outside of the cylinder. At this time, the ion association contained in the ion association solution is adsorbed on the hydrophobic filter 2. Alternatively, the opening 4 is introduced into the ion association solution obtained by adding a dye to the sample, and the piston 5 is moved to the side opposite to the opening 4 (that is, the lever 6 is pulled), so that the ion association is formed inside the cylinder 3. A mode may be adopted in which the combined solution is sucked and the ion association is adsorbed on the hydrophobic filter 2.
【0057】〔参考例1〕(最適pHの決定)7.45
×10-4M ブロモクロロフェノールブルー(BCPB)
〔東京化成社製〕水溶液8ml及び2N NaCl水溶液1
mlを混合し、蒸留水を加え、全容を500mlとした。次
に、430nmでの色素のみの吸光度、590nmでの色素
のみの吸光度及び0.0504gのHSAを混合してイ
オン会合体を形成させたときの600nmでの吸光度を、
pHを変えて測定した。その結果を図1に示す。この結
果から、この条件下ではpH2.5付近とするのが最適
である。Reference Example 1 (Determination of optimum pH) 7.45
× 10 -4 M bromochlorophenol blue (BCPB)
[Tokyo Kasei Co., Ltd.] 8ml aqueous solution and 2N NaCl aqueous solution 1
Then, distilled water was added thereto to make the total volume 500 ml. Next, the absorbance of the dye alone at 430 nm, the absorbance of the dye alone at 590 nm, and the absorbance at 600 nm when 0.0504 g of HSA was mixed to form an ion associate,
The measurement was performed while changing the pH. The result is shown in FIG. From these results, it is optimal that the pH is around 2.5 under these conditions.
【0058】〔参考例2〕 (疎水性フィルタに吸着するイオン会合体の量の検討
(1)) (1)色素溶液の調製 BCPB(東京化成社製)2.034mgを蒸留水に溶
解し全量100mlとして色素溶液〔35μM BCPB
溶液〕を調製した。[Reference Example 2] (Study of the amount of ion associate adsorbed on hydrophobic filter (1)) (1) Preparation of dye solution 2.034 mg of BCPB (manufactured by Tokyo Chemical Industry Co., Ltd.) was dissolved in distilled water, and 100 ml of the dye solution [35 μM BCPB
Solution] was prepared.
【0059】(2)緩衝液の調製 モノクロロ酢酸189mgを蒸留水90mlに溶解し、
塩酸でpHを3.1に調整した後、全量を100mlと
し、緩衝液〔0.02M モノクロロ酢酸−塩酸緩衝液
(pH3.1)〕を調製した。(2) Preparation of Buffer Solution 189 mg of monochloroacetic acid was dissolved in 90 ml of distilled water.
After adjusting the pH to 3.1 with hydrochloric acid, the total volume was adjusted to 100 ml to prepare a buffer [0.02 M monochloroacetic acid-hydrochloric acid buffer (pH 3.1)].
【0060】(3)試料の調製 HSAを、10、20、30、40、50、100、1
50、200、250、300、350、400mg/lと
なるように、生理食塩水(0.9% 塩化ナトリウム水溶
液)に溶解し、12種類の試料を調製した。(3) Preparation of Sample HSA was prepared at 10, 20, 30, 40, 50, 100, 1
It was dissolved in physiological saline (0.9% aqueous sodium chloride solution) at a concentration of 50, 200, 250, 300, 350, and 400 mg / l to prepare 12 types of samples.
【0061】(4)測定 前記色素溶液1mlと前記緩衝液1mlを混合し、更に前記
の各試料8mlを混合してイオン会合体溶液を調製した。(4) Measurement 1 ml of the dye solution and 1 ml of the buffer were mixed, and 8 ml of each sample was mixed to prepare an ion-associated solution.
【0062】次に、これら12種類のイオン会合体溶液
それぞれの602nmにおける吸光度を、U−2000
型分光光度計(日立製作所社製)で測定した。Next, the absorbance at 602 nm of each of these 12 types of ion association solutions was measured using U-2000.
Was measured with a spectrophotometer (manufactured by Hitachi, Ltd.).
【0063】その後、これら12種類のイオン会合体溶
液の10mlを、それぞれニトロセルロースフィルタ(ポ
アサイズ:0.2μm 、直径:25mm、製品番号:A020
A025A、アドバンテック東洋社製)に上から注いで通し
た。Thereafter, 10 ml of each of these 12 kinds of ion association solutions was added to a nitrocellulose filter (pore size: 0.2 μm, diameter: 25 mm, product number: A020).
A025A, manufactured by Advantech Toyosha).
【0064】ここで得られた12種類のろ液の602n
mにおける吸光度を先と同様にして測定した。602 n of the 12 kinds of filtrates obtained here
The absorbance at m was measured in the same manner as above.
【0065】そして、疎水性フィルタに吸着したイオン
会合体の量を算出するため、イオン会合体に特異的な6
02nmにおける、疎水性フィルタに通す前のイオン会
合体溶液の吸光度から疎水性フィルタを通した後のろ液
の吸光度を差し引いた。この結果を図2に示した。Then, in order to calculate the amount of the ion-associated substance adsorbed on the hydrophobic filter, a specific amount of the ion-associated substance was determined.
At 02 nm, the absorbance of the filtrate after passing through the hydrophobic filter was subtracted from the absorbance of the ion association solution before passing through the hydrophobic filter. The result is shown in FIG.
【0066】〔参考例3〕 (疎水性フィルタに吸着するイオン会合体の量の検討
(2)) (1)色素溶液の調製 BCPB(東京化成社製)2.034mgを蒸留水に溶
解し全量100mlとして色素溶液〔35μM BCPB
溶液〕を調製した。[Reference Example 3] (Study of the amount of ion associate adsorbed on the hydrophobic filter (2)) (1) Preparation of dye solution BCPB (manufactured by Tokyo Chemical Industry Co., Ltd.) (2.034 mg) was dissolved in distilled water and the total amount was dissolved. 100 ml of the dye solution [35 μM BCPB
Solution] was prepared.
【0067】(2)緩衝液の調製 モノクロロ酢酸189mgを蒸留水90mlに溶解し、
塩酸でpHを3.0に調整した後、全量を100mlと
し、緩衝液〔0.02M モノクロロ酢酸−塩酸緩衝液
(pH3.0)〕を調製した。(2) Preparation of Buffer Solution 189 mg of monochloroacetic acid was dissolved in 90 ml of distilled water.
After adjusting the pH to 3.0 with hydrochloric acid, the total volume was adjusted to 100 ml to prepare a buffer [0.02 M monochloroacetic acid-hydrochloric acid buffer (pH 3.0)].
【0068】(3)試料の調製 HSAを、5、10、15、20、25、50、75、
100、125、150mg/lとなるように、生理食塩水
(0.9% 塩化ナトリウム水溶液)に溶解し、10種類
の試料を調製した。(3) Preparation of Sample HSA was prepared in 5, 10, 15, 20, 25, 50, 75,
It was dissolved in physiological saline (0.9% aqueous sodium chloride solution) so as to be 100, 125, and 150 mg / l, and ten kinds of samples were prepared.
【0069】(4)測定 前記色素溶液1mlと前記緩衝液1mlを混合し、これに前
記の各試料1mlを混合し、更に蒸留水2mlを混合してイ
オン会合体溶液を調製した。(4) Measurement 1 ml of the dye solution and 1 ml of the buffer were mixed, 1 ml of each sample was mixed with the mixture, and 2 ml of distilled water was further mixed to prepare an ion association solution.
【0070】次に、これら10種類のイオン会合体溶液
5mlそれぞれをシリンジ内に吸引した後、シリンジの先
にニトロセルロースフィルタ(ポアサイズ:0.2μm
、直径:25mm、製品番号:A020A025A、アドバンテッ
ク東洋社製)を付け、シリンジ内のイオン会合体溶液を
ピストンで押してフィルタに通した。Next, 5 ml of each of these 10 types of ion associated solution was sucked into a syringe, and a nitrocellulose filter (pore size: 0.2 μm) was placed in front of the syringe.
, Diameter: 25 mm, product number: A020A025A, manufactured by Advantech Toyo), and the ion-associated solution in the syringe was pushed by a piston and passed through a filter.
【0071】これら10種類のフィルタを40℃の乾燥
器で10分間乾燥した後、フィルタ上の着色の602n
mにおける吸光度の高さ(吸光度のピークの面積)をC
S−9300PC型デンシトメータ(島津製作所社製)
で測定した。この結果を図3に示した。After drying these 10 types of filters in a dryer at 40 ° C. for 10 minutes, the colored 602 n
The absorbance height at m (area of the absorbance peak) is C
S-9300PC densitometer (manufactured by Shimadzu Corporation)
Was measured. The result is shown in FIG.
【0072】〔参考例4〕 (乾燥による疎水性フィルタ上の着色のスペクトル変化
の検討) (1)色素溶液の調製 BCPB(東京化成社製)2.034mgを蒸留水に溶
解し全量100mlとして色素溶液〔35μM BCPB
溶液〕を調製した。REFERENCE EXAMPLE 4 (Study of Coloring Spectrum Change on Hydrophobic Filter by Drying) (1) Preparation of Dye Solution BCPB (manufactured by Tokyo Chemical Industry Co., Ltd.) (2.034 mg) was dissolved in distilled water to make a total volume of 100 ml. Solution [35 μM BCPB
Solution] was prepared.
【0073】(2)緩衝液の調製 モノクロロ酢酸189mgを蒸留水90mlに溶解し、
塩酸でpHを3.0に調整した後、全量を100mlと
し、緩衝液〔0.02M モノクロロ酢酸−塩酸緩衝液
(pH3.0)〕を調製した。(2) Preparation of Buffer Solution 189 mg of monochloroacetic acid was dissolved in 90 ml of distilled water.
After adjusting the pH to 3.0 with hydrochloric acid, the total volume was adjusted to 100 ml to prepare a buffer [0.02 M monochloroacetic acid-hydrochloric acid buffer (pH 3.0)].
【0074】(3)試料の調製 HSAを50mg/lになるように、生理食塩水(0.9%
塩化ナトリウム水溶液)に溶解して試料を調製した。(3) Preparation of Samples HSA was adjusted to 50 mg / l with physiological saline (0.9%
(Aqueous sodium chloride solution) to prepare a sample.
【0075】(4)測定 前記色素溶液1mlと前記緩衝液1mlを混合し、これに前
記の試料1mlを混合し、更に蒸留水2mlを混合してイオ
ン会合体溶液を調製した。(4) Measurement 1 ml of the dye solution and 1 ml of the buffer were mixed, 1 ml of the sample was mixed with the mixture, and 2 ml of distilled water was further mixed to prepare an ion association solution.
【0076】次に、このイオン会合体溶液5mlをシリン
ジ内に吸引した後、シリンジの先にニトロセルロースフ
ィルタ(ポアサイズ:0.2μm 、直径:25mm、製品
番号:A020A025A、アドバンテック東洋社製)を付け、
シリンジ内のイオン会合体溶液をピストンで押してフィ
ルタに通した。Next, 5 ml of this ion association solution was sucked into a syringe, and a nitrocellulose filter (pore size: 0.2 μm, diameter: 25 mm, product number: A020A025A, manufactured by Advantech Toyo) was attached to the tip of the syringe. ,
The ion association solution in the syringe was pushed by a piston and passed through a filter.
【0077】このフィルタを40℃の乾燥器で10分間
乾燥した後、フィルタ上の着色の吸光度スペクトルをC
S−9300PC型デンシトメータ(島津製作所社製)
でスキャンして測定した。この結果を図4に示した。After drying this filter in a dryer at 40 ° C. for 10 minutes, the absorbance spectrum of the color on the filter
S-9300PC densitometer (manufactured by Shimadzu Corporation)
Was scanned and measured. The result is shown in FIG.
【0078】[0078]
【発明の効果】本発明によれば、抽出工程、発色工程、
洗浄工程が原則的に不要であるため工程数が非常に少な
く、またイオン会合の原理を採用しているので色測定が
短時間で可能であり、更に沈殿させないでフィルタに吸
着させるので定量性に優れている、簡易迅速なタンパク
質の分析手段が提供される。According to the present invention, an extraction step, a coloring step,
Since the washing step is basically unnecessary, the number of steps is very small.The principle of ion association is adopted, so that color measurement can be performed in a short time. An excellent, simple and rapid means of analyzing proteins is provided.
【0079】そして、本発明の分析方法及び分析キット
においては、試料中のタンパク質が特定の濃度範囲にあ
ることを検出することができる。In the analysis method and the analysis kit of the present invention, it is possible to detect that the protein in the sample is in a specific concentration range.
【0080】更に、複数の濃度の色素よりなる分析キッ
トを用いることにより、試料中のタンパク質の定量分析
を行うこともできる。Further, by using an analysis kit comprising a plurality of dyes at different concentrations, quantitative analysis of proteins in a sample can be performed.
【0081】[0081]
【実施例】〔実施例1〕 (試料中のタンパク質の分析(1)) (1)色素含有溶液の調製 BCPB(東京化成社製)を372.5μM となるよう
に1M モノクロロ酢酸−塩酸緩衝液(pH2.05)に
溶解し、色素含有溶液を調製した。[Example 1] (Analysis of protein in sample (1)) (1) Preparation of dye-containing solution BCPB (manufactured by Tokyo Chemical Industry Co., Ltd.) was adjusted to 372.5 μM with 1 M monochloroacetic acid-hydrochloric acid buffer. (PH 2.05) to prepare a dye-containing solution.
【0082】(2)試料の調製 HSAを、1.0、2.5、5.0、10、25、50、
100、200、250、500mg/lになるように、生
理食塩水(0.9% 塩化ナトリウム水溶液)に溶解し、
10種類の試料を調製した。また、HSAを50mg/lと
なるように尿に溶解した試料も調製した。(2) Preparation of Sample HSA was prepared at 1.0, 2.5, 5.0, 10, 25, 50,
Dissolve in physiological saline (0.9% sodium chloride aqueous solution) so as to be 100, 200, 250, 500 mg / l,
Ten samples were prepared. A sample in which HSA was dissolved in urine so as to be 50 mg / l was also prepared.
【0083】(3)測定 前記色素含有溶液0.5mlと前記の各試料2mlを混合し
た。その後、これら11種類の混合液の0.5mlを、そ
れぞれニトロセルロースフィルタ(ポアサイズ:0.2
μm 、アドバンテック東洋社製)に上から注いで通し
た。(3) Measurement 0.5 ml of the dye-containing solution and 2 ml of each sample were mixed. Thereafter, 0.5 ml of each of these 11 kinds of mixed liquids was added to a nitrocellulose filter (pore size: 0.2
μm, manufactured by Advantech Toyo).
【0084】このフィルタの色を目視により測定した。
フィルタ上に青色のスポットが検出された場合、試料中
にタンパク質が存在すると判定した。The color of this filter was measured visually.
If a blue spot was detected on the filter, it was determined that protein was present in the sample.
【0085】その結果、2.5mg/lの濃度まで、目視で
判定可能であった。また、フィルタ上にUVランプを照
射し、その蛍光(赤色)を目視により測定した。その結
果、すべての濃度において目視で判定可能であった。As a result, it was possible to determine visually up to a concentration of 2.5 mg / l. Further, a UV lamp was irradiated on the filter, and the fluorescence (red) was visually measured. As a result, it was possible to determine visually at all concentrations.
【0086】そして、フィルタの色の測定及び蛍光色の
測定のいずれの手段においても、尿試料中のHSAが確
認された。HSA in the urine sample was confirmed by both means of measuring the color of the filter and measuring the fluorescent color.
【0087】〔実施例2〕 (試料中のタンパク質の分析(2)) (1)色素溶液の調製 BCPB(東京化成社製)2.034mgを蒸留水に溶
解し全量100mlとして色素溶液〔35μM BCPB
溶液〕を調製した。[Example 2] (Analysis of protein in sample (2)) (1) Preparation of dye solution 2.034 mg of BCPB (manufactured by Tokyo Chemical Industry Co., Ltd.) was dissolved in distilled water to make a total volume of 100 ml, and the dye solution [35 µM BCPB]
Solution] was prepared.
【0088】(2)緩衝液の調製 モノクロロ酢酸189mgを蒸留水90mlに溶解し、
塩酸でpHを3.0に調整した後、全量を100mlと
し、緩衝液〔0.02M モノクロロ酢酸−塩酸緩衝液
(pH3.0)〕を調製した。(2) Preparation of Buffer Solution 189 mg of monochloroacetic acid was dissolved in 90 ml of distilled water.
After adjusting the pH to 3.0 with hydrochloric acid, the total volume was adjusted to 100 ml to prepare a buffer [0.02 M monochloroacetic acid-hydrochloric acid buffer (pH 3.0)].
【0089】(3)試料の調製 HSAを、1.25、3.75、6.25、10、1
2.5、16.25、18.75、22.5、25、3
7.5、50、75、100mg/lとなるように、生理食
塩水(0.9% 塩化ナトリウム水溶液)に溶解し、13
種類の試料を調製した。また、対照として、生理食塩水
よりなる試料も用意した。(3) Preparation of Sample HSA was prepared at 1.25, 3.75, 6.25, 10, 1
2.5, 16.25, 18.75, 22.5, 25, 3
Dissolve in physiological saline (0.9% aqueous sodium chloride solution) to a concentration of 7.5, 50, 75, or 100 mg / l.
Different types of samples were prepared. In addition, a sample composed of physiological saline was prepared as a control.
【0090】(4)測定 前記色素溶液1mlと前記緩衝液1mlを混合し、これに前
記の各試料1mlを混合し、更に蒸留水2mlを混合してイ
オン会合体溶液を調製した。(4) Measurement 1 ml of the dye solution and 1 ml of the buffer were mixed, 1 ml of each of the samples was mixed, and 2 ml of distilled water was further mixed to prepare an ion association solution.
【0091】次に、これら14種類のイオン会合体溶液
5mlそれぞれをシリンジ内に吸引した後、シリンジの先
にニトロセルロースフィルタ(ポアサイズ:0.2μm
、直径:25mm、製品番号:A020A025A、アドバンテッ
ク東洋社製)を付け、シリンジ内のイオン会合体溶液を
ピストンで押してフィルタに通した。Next, 5 ml of each of these 14 kinds of ion association solutions was sucked into a syringe, and a nitrocellulose filter (pore size: 0.2 μm) was placed in front of the syringe.
, Diameter: 25 mm, product number: A020A025A, manufactured by Advantech Toyo), and the ion-associated solution in the syringe was pushed by a piston and passed through a filter.
【0092】これら14種類のフィルタを40℃の乾燥
器で10分間乾燥した。これらのフィルタの色を目視に
より測定した。フィルタ上の着色を図6に示した。These 14 types of filters were dried in a dryer at 40 ° C. for 10 minutes. The color of these filters was measured visually. The coloring on the filter is shown in FIG.
【0093】この結果、試料中のタンパク質濃度が3.
75mg/l〜22.5mg/lの場合に青色の着色が検出され
た。つまり、3.75mg/l〜22.5mg/lの試料中のタ
ンパク質の存在が確認された。As a result, the protein concentration in the sample was 3.
Blue coloration was detected at 75 mg / l to 22.5 mg / l. That is, the presence of the protein in the sample of 3.75 mg / l to 22.5 mg / l was confirmed.
【0094】〔実施例3〕 (試料中のタンパク質の分析(3)) (1)色素溶液の調製 BCPB(東京化成社製)の、10.17mg、14.
53mg、15.98mg、17.43mg、20.3
4mg、21.79mg、23.24mg、29.05
mg、又は40.67mgを蒸留水に溶解し全量100
mlとして9種類の色素溶液〔175μM 、250μM
、275μM 、300μM 、350μM、375μM 、
400μM 、500μM 、又は700μM BCPB溶
液〕を調製した。[Example 3] (Analysis of protein in sample (3)) (1) Preparation of dye solution 10.17 mg of BCPB (manufactured by Tokyo Chemical Industry Co., Ltd.);
53 mg, 15.98 mg, 17.43 mg, 20.3
4 mg, 21.79 mg, 23.24 mg, 29.05
mg or 40.67 mg dissolved in distilled water to give a total amount of 100 mg.
9 dye solutions [175 μM, 250 μM
, 275 μM, 300 μM, 350 μM, 375 μM,
400 μM, 500 μM, or 700 μM BCPB solution].
【0095】(2)緩衝液の調製 モノクロロ酢酸1.89gを蒸留水90mlに溶解し、
塩酸でpHを3.1に調整した後、全量を100mlと
し、緩衝液〔0.2M モノクロロ酢酸−塩酸緩衝液(p
H3.1)〕を調製した。(2) Preparation of Buffer Solution 1.89 g of monochloroacetic acid was dissolved in 90 ml of distilled water.
After adjusting the pH to 3.1 with hydrochloric acid, the total volume was adjusted to 100 ml, and the buffer [0.2 M monochloroacetic acid-hydrochloric acid buffer (p
H3.1)] was prepared.
【0096】(3)試料の調製 HSAを、10、25、50、75、100、125、
150、175、200、300、400mg/lとなるよ
うに、生理食塩水(0.9% 塩化ナトリウム水溶液)に
溶解し、11種類の試料を調製した。また、対照とし
て、生理食塩水よりなる試料も用意した。(3) Preparation of sample HSA was prepared in 10, 25, 50, 75, 100, 125,
It dissolved in physiological saline (0.9% sodium chloride aqueous solution) so that it might become 150, 175, 200, 300, and 400 mg / l, and 11 types of samples were prepared. In addition, a sample composed of physiological saline was prepared as a control.
【0097】(4)測定 前記9種類の色素溶液1mlのそれぞれと前記緩衝液1ml
を混合したものを12組用意した。この1組ずつに前記
の12種類の試料の1種類ずつをそれぞれ1ml混合し、
更に蒸留水2mlを混合して合計108種類のイオン会合
体溶液を調製した。(4) Measurement 1 ml of each of the nine dye solutions and 1 ml of the buffer
Were prepared as a set. 1 ml of each of the above 12 kinds of samples is mixed with each of the sets,
Further, 2 ml of distilled water was mixed to prepare a total of 108 kinds of ion associated solution.
【0098】次に、これら108種類のイオン会合体溶
液5mlそれぞれをシリンジ内に吸引した後、シリンジの
先にニトロセルロースフィルタ(ポアサイズ:0.2μ
m 、直径:25mm、製品番号:A020A025A、アドバンテ
ック東洋社製)を付け、シリンジ内のイオン会合体溶液
をピストンで押してフィルタに通した。Next, after sucking 5 ml of each of these 108 kinds of ion associated solution into a syringe, a nitrocellulose filter (pore size: 0.2 μm) was placed in front of the syringe.
m, diameter: 25 mm, product number: A020A025A, manufactured by Advantech Toyo), and the ion-associated solution in the syringe was pushed by a piston to pass through a filter.
【0099】これら108種類のフィルタを40℃の乾
燥器で10分間乾燥した。このフィルタの色を目視によ
り測定した。These 108 types of filters were dried in a dryer at 40 ° C. for 10 minutes. The color of this filter was measured visually.
【0100】これらのフィルタ上の着色を図7に示し
た。この図7において、横軸は試料中のタンパク質濃度
を、縦軸は色素溶液中のBCPB濃度を表す。そして、
この図7中の「●」は疎水性フィルタ上の着色が青色で
あることを、「◎」は疎水性フィルタ上の着色がうすい
水色であることを、そして「○」は疎水性フィルタが着
色されていないことを表す。The coloring on these filters is shown in FIG. In FIG. 7, the horizontal axis represents the protein concentration in the sample, and the vertical axis represents the BCPB concentration in the dye solution. And
In FIG. 7, “●” indicates that the color on the hydrophobic filter is blue, “◎” indicates that the color on the hydrophobic filter is light blue, and “「 ”indicates that the hydrophobic filter is colored. It is not done.
【0101】また、図8には、CS−9300PC型デ
ンシトメータ(島津製作所社製)で測定した、これら1
08種類のフィルタ上の着色の602nmにおける吸光
度の高さ(吸光度のピークの面積)を示した。FIG. 8 shows the results of measurement using a CS-9300PC densitometer (manufactured by Shimadzu Corporation).
The height of the absorbance (area of the peak of the absorbance) at 602 nm of the coloring on the 08 types of filters is shown.
【0102】これらの結果より、本発明においては、分
析を行う試料中のタンパク質の量に対して色素の量を調
整することにより、試料中のタンパク質が特定の濃度範
囲にある時のみ、疎水性フィルタ上に着色を生じさせる
ことができる。つまり、試料中のタンパク質が特定の濃
度範囲にあることを検出することができることが確かめ
られた。From these results, in the present invention, by adjusting the amount of the dye with respect to the amount of the protein in the sample to be analyzed, only when the protein in the sample is in a specific concentration range, the hydrophobicity is increased. Coloring can occur on the filter. That is, it was confirmed that it was possible to detect that the protein in the sample was in a specific concentration range.
【図1】pHと、色素(430nm、590nm)及びイオ
ン会合体(600nm)の吸光度との関係を示した図であ
る。FIG. 1 is a diagram showing a relationship between pH and absorbance of a dye (430 nm, 590 nm) and an ion associate (600 nm).
【図2】試料中のタンパク質濃度と疎水性フィルタに吸
着するイオン会合体等の量の関係を示した図である。FIG. 2 is a diagram showing the relationship between the protein concentration in a sample and the amount of ion aggregates and the like adsorbed on a hydrophobic filter.
【図3】試料中のタンパク質濃度と疎水性フィルタに吸
着するイオン会合体の量の関係を示した図である。FIG. 3 is a graph showing the relationship between the protein concentration in a sample and the amount of ion associate adsorbed on a hydrophobic filter.
【図4】疎水性フィルタがぬれている時と乾燥している
時の着色のスペクトルを示した図である。FIG. 4 is a diagram showing coloring spectra when the hydrophobic filter is wet and when it is dry.
【図5】本発明に係るタンパク質分析キットである。FIG. 5 is a protein analysis kit according to the present invention.
【図6】各タンパク質濃度の試料における疎水性フィル
タの着色を示した図(写真)である。FIG. 6 is a diagram (photograph) showing coloring of a hydrophobic filter in a sample having each protein concentration.
【図7】試料中のタンパク質濃度及び色素溶液中のBC
PB濃度の両方を変えて分析を行った時の疎水性フィル
タの着色を示した図である。FIG. 7: Protein concentration in sample and BC in dye solution
It is a figure showing coloring of a hydrophobic filter at the time of performing analysis by changing both PB concentrations.
【図8】試料中のタンパク質濃度及び色素溶液中のBC
PB濃度の両方を変えて分析を行った時の疎水性フィル
タの着色の吸光度の高さを示した図である。FIG. 8: Protein concentration in sample and BC in dye solution
It is the figure which showed the height of the light absorbency of the coloring of the hydrophobic filter at the time of performing analysis by changing both PB concentrations.
1 分析キット 2 疎水性フィルタ 3 シリンダ 4 開口部 5 ピストン 6 レバー DESCRIPTION OF SYMBOLS 1 Analysis kit 2 Hydrophobic filter 3 Cylinder 4 Opening 5 Piston 6 Lever
Claims (2)
試料と、タンパク質とイオン会合体を形成する色素を混
合し、該イオン会合体を沈殿させずにイオン会合体溶液
を形成させる工程; b)該イオン会合体溶液を疎水性フィルタに通すことに
より、該イオン会合体溶液中に溶解した該会合体を該フ
ィルタに吸着させる工程;及び c)該フィルタ上の該会合体を定性・定量的に分析する
工程を順次行うことを特徴とする試料中のタンパク質の
分析方法。1. a) mixing a sample which may contain a protein and a dye which forms an ion aggregate with the protein, and forming an ion aggregate solution without precipitating the ion aggregate; b A) a step of adsorbing the aggregate dissolved in the ion aggregate solution on the filter by passing the ion aggregate solution through a hydrophobic filter; and c) qualitatively and quantitatively determining the aggregate on the filter. A method for analyzing a protein in a sample, comprising sequentially performing the steps of:
素;及び疎水性フィルタを含む、請求項1記載の方法に
使用されるタンパク質分析キット。2. A protein analysis kit for use in the method according to claim 1, comprising a dye that forms an ion aggregate with a protein; and a hydrophobic filter.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP15835398A JP4039739B2 (en) | 1998-02-23 | 1998-05-21 | Method for analyzing protein in sample and kit thereof |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP5899298 | 1998-02-23 | ||
JP10-58992 | 1998-02-23 | ||
JP15835398A JP4039739B2 (en) | 1998-02-23 | 1998-05-21 | Method for analyzing protein in sample and kit thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
JPH11304803A true JPH11304803A (en) | 1999-11-05 |
JP4039739B2 JP4039739B2 (en) | 2008-01-30 |
Family
ID=26400013
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP15835398A Expired - Fee Related JP4039739B2 (en) | 1998-02-23 | 1998-05-21 | Method for analyzing protein in sample and kit thereof |
Country Status (1)
Country | Link |
---|---|
JP (1) | JP4039739B2 (en) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2004004117A (en) * | 2003-07-31 | 2004-01-08 | Menicon Co Ltd | Method for detecting contamination of protein on contact lens |
WO2008066003A1 (en) * | 2006-11-28 | 2008-06-05 | Nomura Micro Science Co., Ltd. | Method for quantitative determination of nickel and/or copper and equipment to be used in the method |
JP2009058511A (en) * | 2007-08-29 | 2009-03-19 | Cem Corp | Automated protein analyzer |
US7968344B2 (en) | 2007-08-29 | 2011-06-28 | Cem Corporation | Automated protein analyzer |
WO2019093495A1 (en) * | 2017-11-10 | 2019-05-16 | 株式会社明治 | Method for detecting protein |
-
1998
- 1998-05-21 JP JP15835398A patent/JP4039739B2/en not_active Expired - Fee Related
Cited By (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2004004117A (en) * | 2003-07-31 | 2004-01-08 | Menicon Co Ltd | Method for detecting contamination of protein on contact lens |
WO2005012915A1 (en) | 2003-07-31 | 2005-02-10 | Menicon Co., Ltd. | Method of detecting protein dirt on contact lens |
EP1650567A1 (en) * | 2003-07-31 | 2006-04-26 | Menicon Co., Ltd. | Method of detecting protein dirt on contact lens |
EP1650567A4 (en) * | 2003-07-31 | 2007-09-05 | Menicon Co Ltd | Method of detecting protein dirt on contact lens |
JPWO2008066003A1 (en) * | 2006-11-28 | 2010-03-04 | 野村マイクロ・サイエンス株式会社 | Nickel and / or copper determination method and apparatus used therefor |
WO2008066003A1 (en) * | 2006-11-28 | 2008-06-05 | Nomura Micro Science Co., Ltd. | Method for quantitative determination of nickel and/or copper and equipment to be used in the method |
JP2009058511A (en) * | 2007-08-29 | 2009-03-19 | Cem Corp | Automated protein analyzer |
US7968344B2 (en) | 2007-08-29 | 2011-06-28 | Cem Corporation | Automated protein analyzer |
US8147759B2 (en) | 2007-08-29 | 2012-04-03 | Cem Corporation | Automated protein analyzer |
US8663993B2 (en) | 2007-08-29 | 2014-03-04 | Cem Corporation | Automated protein analyzer |
US8852948B2 (en) | 2007-08-29 | 2014-10-07 | Cem Corporation | Colorimetric protein analysis method |
US9091632B2 (en) | 2007-08-29 | 2015-07-28 | Cem Corporation | Automated protein analyzer |
WO2019093495A1 (en) * | 2017-11-10 | 2019-05-16 | 株式会社明治 | Method for detecting protein |
JPWO2019093495A1 (en) * | 2017-11-10 | 2020-11-26 | 株式会社明治 | Protein detection method |
US11781991B2 (en) | 2017-11-10 | 2023-10-10 | Meiji Co., Ltd. | Method for detecting protein |
Also Published As
Publication number | Publication date |
---|---|
JP4039739B2 (en) | 2008-01-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
TWI521206B (en) | Organic colored microparticles, including their diagnostic kit and in vitro diagnostic methods | |
Heller et al. | A chemical investigation of the Shroud of Turin | |
US11353450B2 (en) | Analyte detection using Raman spectroscopy | |
JP6248030B2 (en) | Analyte detection or quantification method, analyte detection or quantification kit, and lateral flow chromatographic test strip for analyte detection or quantification | |
JPS6361622B2 (en) | ||
JP4039739B2 (en) | Method for analyzing protein in sample and kit thereof | |
JP2012194013A (en) | Immunohistochemical staining method and reaction reagent | |
ES2909503T3 (en) | Cellulose microparticles hydrophilic dyes | |
KR20200028445A (en) | Organic colored particulates, diagnostic kits and in vitro diagnostic methods | |
CN105929181B (en) | Method for detecting heroin in biological detection material based on nano material | |
JP5493069B2 (en) | Method and kit for measuring protein in sample | |
Wang et al. | Determination of γ-globulin at nanogram levels by its enhancement effect on the resonance light scattering of functionalized HgS nanoparticles | |
JP4391171B2 (en) | Protein measuring method and measuring kit | |
JPS6345563A (en) | Cell analysis | |
DE102011054396B4 (en) | Use of broadband absorbing and emitting NIR fluorophores with large Stokes shift as dyes in core-shell nanoparticles for biomarker analysis and as components of FRET systems and method based thereon | |
JP3674529B2 (en) | Rapid and simple detection method of urinary disease marker protein using absorption label | |
Luo et al. | Discrimination of wet or dried arterial and venous blood for forensic applications via eosin fluorescence lifetime | |
JP6881564B2 (en) | Particle surface condition evaluation method and evaluation system | |
CN104849228B (en) | Using based on the DNA imaging cells art of UV light come the in-vitro method that detects and/or diagnose cancer | |
JPS62135769A (en) | Method and reagent for measuring ratio between protein and dna in cell | |
JP5584993B2 (en) | Method and kit for measuring protein in sample | |
RU2180104C1 (en) | Method of simultaneous determination of lysosomal cationic proteins in granulocytes and dna, rna in lymphocytes in single tissue preparation | |
JPH07243972A (en) | Quantitative determination method for trace protein in lipid | |
CN115901629A (en) | Detection probe based on surface enhanced Raman scattering, preparation method and application | |
JPH11337543A (en) | Simplified measurement method of colored material on carrier |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20050428 |
|
A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20060718 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20060725 |
|
A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20060922 |
|
A521 | Written amendment |
Effective date: 20061215 Free format text: JAPANESE INTERMEDIATE CODE: A523 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20070424 |
|
A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20070618 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20071016 |
|
A61 | First payment of annual fees (during grant procedure) |
Effective date: 20071106 Free format text: JAPANESE INTERMEDIATE CODE: A61 |
|
FPAY | Renewal fee payment (prs date is renewal date of database) |
Year of fee payment: 3 Free format text: PAYMENT UNTIL: 20101116 |
|
R150 | Certificate of patent (=grant) or registration of utility model |
Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
FPAY | Renewal fee payment (prs date is renewal date of database) |
Year of fee payment: 4 Free format text: PAYMENT UNTIL: 20111116 |
|
FPAY | Renewal fee payment (prs date is renewal date of database) |
Year of fee payment: 5 Free format text: PAYMENT UNTIL: 20121116 |
|
FPAY | Renewal fee payment (prs date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20121116 Year of fee payment: 5 |
|
FPAY | Renewal fee payment (prs date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20121116 Year of fee payment: 5 |
|
FPAY | Renewal fee payment (prs date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20131116 Year of fee payment: 6 |
|
LAPS | Cancellation because of no payment of annual fees |