JP4189301B2 - Centrifuge tube and centrifuge method - Google Patents

Centrifuge tube and centrifuge method Download PDF

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JP4189301B2
JP4189301B2 JP2003388642A JP2003388642A JP4189301B2 JP 4189301 B2 JP4189301 B2 JP 4189301B2 JP 2003388642 A JP2003388642 A JP 2003388642A JP 2003388642 A JP2003388642 A JP 2003388642A JP 4189301 B2 JP4189301 B2 JP 4189301B2
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centrifuge tube
centrifuge
groove
bulge
sample
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禎宏 早川
庸助 岩田
直樹 浅川
信 牟田
栄一 山本
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Shimadzu Corp
Eisai R&D Management Co Ltd
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Eisai R&D Management Co Ltd
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Description

本発明は生体試料をＨＰＬＣ（高速液体クロマトグラフ）などの分析装置で分析するのに先立ち、試料から不溶成分を除去するための前処理として行う遠心分離方法、及び遠心分離機で使用する遠沈管に関するものである。 The present invention relates to a centrifugation method used as a pretreatment for removing insoluble components from a sample prior to analyzing a biological sample with an analyzer such as HPLC (high performance liquid chromatograph), and a centrifuge used in a centrifuge. it relates to the tube.

薬物の動態測定の分野では、血清や血漿などの生体物質を測定試料とするが、これら生体試料中にはタンパク質が変性したり、繊維質成分などによる不溶成分が生じる。特に、生体試料を冷凍保存したあと、凍結を融解した試料には不溶成分の発生が顕著である。これらの試料をＨＰＬＣなどを使って分離分析する際には、装置内部での目詰まりなどを回避するために不溶成分を除去しなければならない。   In the field of drug kinetics measurement, biological materials such as serum and plasma are used as measurement samples. In these biological samples, proteins are denatured or insoluble components such as fibrous components are generated. In particular, generation of insoluble components is significant in samples that have been frozen and thawed after the biological sample has been stored frozen. When these samples are separated and analyzed using HPLC or the like, insoluble components must be removed in order to avoid clogging inside the apparatus.

これまで、血液や血漿・血清などの試料を不溶成分と分離するために試料を遠沈管に入れ、遠心分離機にかけていた。遠心分離したあと、上澄み液を採取しＨＰＬＣなどで測定することが行われている。   Until now, in order to separate samples such as blood, plasma and serum from insoluble components, the samples were put in a centrifuge tube and subjected to a centrifuge. After centrifugation, the supernatant is collected and measured by HPLC or the like.

しかし、遠心分離した不溶成分と上澄み液の比重が似通っているため、遠沈管に衝撃を与えると、遠心分離したにも拘わらず不溶成分と上澄み液が界面で混ざり合い、測定に供しうる最終的な試料の体積が減ってしまう。そのため、遠心分離後の遠沈管を持ち運んだりして取り扱う際には細心の注意を要した。
そこで、本発明は、前処理のための遠心分離をした試料の上澄み液と不溶成分との分離を維持するのが容易な遠心分離方法と、そのために使用する遠沈管を提供することを目的とするものである。 However, since the specific gravity of the centrifuged insoluble component is similar to that of the supernatant, when the centrifuge tube is subjected to an impact, the insoluble component and the supernatant are mixed at the interface despite the centrifugal separation, and can be used for measurement The volume of a small sample is reduced. For this reason, extreme care was required when carrying and handling the centrifuge tube after centrifugation.
Accordingly, an object of the present invention is to provide a centrifugation method that can easily maintain the separation of the supernatant and insoluble components of a sample that has been centrifuged for pretreatment, and a centrifuge tube used therefor. To do.

遠沈管に試料液を入れ、遠心分離機で遠心分離する方法において、本発明の方法では、遠沈管の底部に不溶成分を捕捉する手段を遠沈管と一体に形成しておいた状態で遠心分離を行う。
そのために、本発明の遠沈管は、その底部に不溶成分を捕捉する手段を備えている。 In the method of putting a sample solution in a centrifuge tube and centrifuging with a centrifuge, in the method of the present invention, the centrifuge is centrifuged in a state in which means for capturing insoluble components is formed integrally with the centrifuge tube at the bottom of the centrifuge tube. I do.
For this purpose, the centrifuge tube of the present invention is provided with means for capturing insoluble components at the bottom thereof.

そのような遠沈管の一形態は、不溶成分を捕捉する手段として遠沈管の底面に形成され、不溶成分が入り込める大きさの溝を備えたものである。この遠沈管に試料を入れて遠心分離を行うと、遠心分離時に不溶成分が遠沈管底部のその溝に入り込んで捕捉される。   One form of such a centrifuge tube is provided with a groove formed on the bottom surface of the centrifuge tube as a means for capturing the insoluble component and having a size that allows the insoluble component to enter. When a sample is placed in the centrifuge tube and centrifuged, insoluble components enter the groove at the bottom of the centrifuge tube and are captured during centrifugation.

このようにして、本発明によれば、遠心分離終了後、試料中の不溶成分は遠沈管底部にたまって捕捉され、浮き上がりにくくなり、持ち運び時に注意を必要としなくなる。   In this way, according to the present invention, after completion of the centrifugation, the insoluble component in the sample accumulates on the bottom of the centrifuge tube and is captured and becomes difficult to lift, so that no caution is required when carrying it.

以上のように、本発明では遠沈管の底部に不溶成分を捕捉する手段を遠沈管と一体に形成しておいた状態で遠心分離を行うようにしたので、不溶成分と遠心分離された上澄み液との再拡散による混合が起きにくくなるので、遠心分離後も細心の注意を払わずに遠沈管を取り扱うことができるようになる。 As described above, in the present invention, the centrifugal separation is performed in a state where the means for capturing the insoluble component at the bottom of the centrifuge tube is formed integrally with the centrifuge tube. Mixing due to re-diffusion is difficult, so that the centrifuge tube can be handled even after centrifugation without paying close attention.

図１は遠沈管の底部に溝を設けた第１の実施例を表わしたものであり、（Ａ）はその概略断面図、（Ｂ）はその遠沈管に血液などの試料を入れ、遠心分離を行った後の状態を示した断面図である。   1A and 1B show a first embodiment in which a groove is provided at the bottom of a centrifuge tube. FIG. 1A is a schematic cross-sectional view of the first embodiment, and FIG. It is sectional drawing which showed the state after performing.

遠沈管２は上部が開口し、底をもつ円筒状で、ガラス又はプラスチック製である。その底部には中央部が盛り上がる隆起部４が形成されていることにより、底部側方に溝６が形成されている。図示は省略されているが、上部開口部の外側面には遠心分離機に装着するためのつば部が設けられている。   The centrifuge tube 2 has a cylindrical shape with an open top and a bottom, and is made of glass or plastic. A groove 6 is formed on the side of the bottom by forming a raised portion 4 with a raised center at the bottom. Although not shown in the drawings, a collar portion for mounting on the centrifuge is provided on the outer surface of the upper opening.

この遠沈管２に血液などの試料８を入れ、遠心分離を行うと、（Ｂ）に示されるように、不溶成分８ａが底部に移動して溝６内に入る。溝に入った不溶成分８ａは溝の隙間に捕捉されることにより、試料の上澄み液に再拡散していきにくくなる。   When a sample 8 such as blood is put into the centrifuge tube 2 and centrifuged, the insoluble component 8a moves to the bottom and enters the groove 6 as shown in FIG. The insoluble component 8a that has entered the groove is trapped in the gap of the groove, so that it becomes difficult to re-diffuse into the supernatant of the sample.

図２は図１の実施例を改良したものである。遠沈管２ａの底部には図１と同様に中央部に隆起部４が形成されていることにより、側方に溝６が形成されている。この実施例では溝６の上部には遠沈管の内側壁に底部隆起部４を取り囲むように周方向に環状に延び、内側に突出した隆起１０が形成されている。その隆起１０と底部隆起部４によって溝６の上部入口が狭くなっている。
１２は遠沈管上部開口部の外側面に設けられ、この遠沈管２ａを遠心分離機に装着するためのつば部である。 FIG. 2 is a modification of the embodiment of FIG. In the bottom of the centrifuge tube 2a, a ridge 4 is formed at the center as in FIG. 1, so that a groove 6 is formed on the side. In this embodiment, a ridge 10 is formed on the inner wall of the centrifuge tube in the circumferential direction so as to surround the bottom ridge 4 and project inwardly at the top of the groove 6. The ridge 10 and bottom ridge 4 narrow the upper entrance of the groove 6.
A collar 12 is provided on the outer surface of the upper opening of the centrifuge tube and is used to mount the centrifuge tube 2a on the centrifuge.

図２の遠沈管に試料８を入れて遠心分離を行うと、その不溶成分８ａは溝６内に入るが、その溝６の上部入口が狭くなっていることによって不溶成分８ａが上澄み液中に再拡散していくのが一層有効に阻止される。   When the sample 8 is placed in the centrifuge tube of FIG. 2 and centrifuged, the insoluble component 8a enters the groove 6, but the upper inlet of the groove 6 is narrowed so that the insoluble component 8a is in the supernatant. Re-diffusion is more effectively prevented.

図３は図２の遠沈管をさらに改良した実施例である。図２の遠沈管と比較すると、底部中央部の隆起部４にも遠沈管内側に突出する隆起１４がさらに形成されている点で異なる。隆起１４は隆起１０に対向するように環状に延びて形成されている。   FIG. 3 shows an embodiment in which the centrifuge tube of FIG. 2 is further improved. Compared with the centrifuge tube of FIG. 2, it is different in that a bulge 14 that protrudes to the inside of the centrifuge tube is further formed on the bulge 4 at the center of the bottom. The ridge 14 extends in an annular shape so as to face the ridge 10.

この実施例の遠沈管では、隆起１０と１４が対向する方向に設けられていることにより、溝６の上部入口がさらに狭められ、内部に捕捉された試料の不溶成分８ａが上澄み液に再拡散するのがよりいっそう有効に阻止される。   In the centrifuge tube of this embodiment, since the ridges 10 and 14 are provided in the opposing direction, the upper inlet of the groove 6 is further narrowed, and the insoluble component 8a of the sample trapped inside is re-diffused into the supernatant. Is more effectively prevented.

図１〜図３の実施例では、遠沈管の底部に溝を設けることにより不溶成分がその溝中に残留し、遠心分離された上澄み液との再拡散による混合が起きにくくなる。
また、図１〜図３の実施例では、遠心分離後に上澄み液を試料として分析に供した後、溝６に入り込んだ不溶成分８ａを洗浄するのが容易ではなくなるので、遠沈管を使い捨て式にするのが好ましい。 In the embodiment shown in FIGS. 1 to 3, by providing a groove at the bottom of the centrifuge tube, insoluble components remain in the groove, and mixing due to re-diffusion with the centrifuged supernatant liquid is less likely to occur.
Also, in the embodiment of FIGS. 1 to 3, since it is not easy to wash the insoluble component 8a that has entered the groove 6 after the supernatant is used as a sample after centrifugation, the centrifuge tube is made disposable. It is preferable to do this.

図４は参考例を表わしたものである。この参考例は、（Ａ）に示されるように、上部が開口し、底部に底をもつ円筒状遠沈管２０に、不溶成分を捕捉する手段として粒状物２２を入れたものである。粒状物２２はガラスビーズなどであり、試料として処理される血液などよりも比重が大きい材質からなり、試料の不溶成分を隙間に捕捉できるように適当な隙間を形成する大きさ、例えば１〜２ｍｍの直径をもつものが好ましい。図示は省略されているが、上部開口部の外側面には遠心分離機に装着するためのつば部が設けられている。 FIG. 4 shows a reference example . In this reference example , as shown in (A), a granular centrifuge 22 is placed in a cylindrical centrifuge tube 20 having an open top and a bottom at the bottom as a means for capturing insoluble components. The granular material 22 is a glass bead or the like and is made of a material having a specific gravity greater than that of blood or the like processed as a sample, and has a size that forms an appropriate gap so that insoluble components of the sample can be captured in the gap, for example, 1 to 2 mm Are preferred. Although not shown in the drawings, a collar portion for mounting on the centrifuge is provided on the outer surface of the upper opening.

（Ｂ）に示されるように、粒状物２２を入れた遠沈管に試料８を入れ、遠心分離を行うと、その不溶成分８ａは粒状物２２の隙間に捕捉される。
この実施例でも、同じように粒状物の間隙に不溶成分が残留し、上澄み液との再拡散による混合がおきにくくなる。 As shown in (B), when the sample 8 is placed in a centrifuge tube containing the particulate matter 22 and centrifuged, the insoluble component 8a is trapped in the gap between the particulate matter 22.
In this embodiment as well, insoluble components remain in the gaps between the granular materials, and mixing by re-diffusion with the supernatant becomes difficult.

図５は他の参考例を表わしたものである。（Ａ）に示されるように、上部に開口をもち、底部に底をもつ通常の遠沈管２０に、フィルタ２４を入れたものである。フィルタ２４は溶液を透過し、不溶成分を濾過する大きさの孔をもったものである。ここでも図示は省略されているが、遠沈管２０には上部開口部の外側面には遠心分離機に装着するためのつば部が設けられている。 FIG. 5 shows another reference example . As shown in (A), a filter 24 is placed in a normal centrifuge tube 20 having an opening at the top and a bottom at the bottom. The filter 24 has pores of a size that allows the solution to pass therethrough and filters insoluble components. Although not shown here, the centrifuge tube 20 is provided with a collar portion on the outer surface of the upper opening for mounting on the centrifuge.

フィルタ２４は、（Ｃ）に示されるように、その外形寸法が遠沈管２０の内径に対応して内径の壁面を摺動できる大きさであり、円筒状の側壁２６の底面にフィルタ２４が取りつけられたものである。このように側壁２６を有することにより、フィルタ２４が傾くことなしに遠沈管２０の内面に沿って底部方向に移動することができる。フィルタ２４と側壁２６の材質は、遠心分離後にフィルタ２４が浮き上らないように、処理される試料の血液などと比重と等しいか、それよりも大きいものを選択する。   As shown in (C), the filter 24 is sized such that its outer dimension can slide on the inner wall surface corresponding to the inner diameter of the centrifuge tube 20, and the filter 24 is attached to the bottom surface of the cylindrical side wall 26. It is what was done. By having the side wall 26 in this way, the filter 24 can move in the bottom direction along the inner surface of the centrifuge tube 20 without tilting. The material of the filter 24 and the side wall 26 is selected so as to be equal to or larger than the specific gravity of blood or the like of the sample to be processed so that the filter 24 does not float after centrifugation.

フィルタ２４は遠沈管２０に試料８を入れた後に、その上部に投入する。遠心分離により、（Ｂ）に示されるように試料の不溶成分８ａが底部に移動するとともにフィルタ２４も底部に移動し、その際上澄み液はフィルタ２４を透過するが、不溶成分８ａは透過せずに捕捉されるので、不溶成分８ａは遠沈管の底部とフィルタ２４の間に保持される。   The filter 24 is put into the upper part of the centrifuge tube 20 after the sample 8 is put therein. By centrifugation, the insoluble component 8a of the sample moves to the bottom and the filter 24 also moves to the bottom as shown in (B). At this time, the supernatant passes through the filter 24, but does not pass through the insoluble component 8a. Therefore, the insoluble component 8a is held between the bottom of the centrifuge tube and the filter 24.

図６はさらに他の参考例を表わしたものである。（Ａ）に示されるように、上部に開口をもち、底部に底をもつ通常の遠沈管２０に、不溶成分捕捉用器具３０を入れたものである。不溶成分捕捉用器具３０は、上部に漏斗状に開いた開口部３２をもち下部にも開口部３４をもつとともに、上下の開口部３２，３４がそれらの開口面より小さい断面積の穴３６で連通している。ここでも図示は省略されているが、遠沈管２０には上部開口部の外側面には遠心分離機に装着するためのつば部が設けられている。 FIG. 6 shows still another reference example . As shown in (A), an insoluble component capturing device 30 is placed in a normal centrifuge tube 20 having an opening at the top and a bottom at the bottom. The insoluble component capturing device 30 has an opening 32 opened in a funnel shape at the top and an opening 34 at the bottom, and the upper and lower openings 32, 34 are holes 36 having a cross-sectional area smaller than those opening surfaces. Communicate. Although not shown here, the centrifuge tube 20 is provided with a collar portion on the outer surface of the upper opening for mounting on the centrifuge.

不溶成分捕捉用器具３０を遠沈管２０に入れ、その上から試料８を入れて遠心分離を行うと、遠心分離時に試料８中の不溶成分８が連通穴３６を通って下部の開口部３４に移動して捕捉される。   When the insoluble component capturing device 30 is placed in the centrifuge tube 20 and the sample 8 is placed on the centrifuge tube 20 and centrifuged, the insoluble component 8 in the sample 8 passes through the communication hole 36 into the lower opening 34 at the time of centrifugation. Moved and captured.

この不溶成分捕捉用器具３０は上部の開口部３２が漏斗状に開いた形状をしており、かつ連通穴３６の断面積が小さいので、回収できずに無駄になる上澄み液の割合が少なくてすみ、貴重な試料をより有効に使用できるようになる。   The insoluble component capturing device 30 has a shape in which the upper opening 32 is opened in a funnel shape, and since the cross-sectional area of the communication hole 36 is small, the ratio of the supernatant liquid that cannot be recovered and is wasted is small. In short, valuable samples can be used more effectively.

本発明の遠心分離方法及びそれに使用する機器は、生体試料その他の試料を分析などの試料として供するための前処理として利用することができる。   The centrifugation method of the present invention and the equipment used therefor can be used as a pretreatment for providing a biological sample or other sample as a sample for analysis or the like.

第１の実施例を表わしたものであり、（Ａ）はその概略垂直断面図、（Ｂ）は遠心分離を行った後の状態を示した概略垂直断面図である。1A and 1B show a first embodiment, in which FIG. 1A is a schematic vertical sectional view thereof, and FIG. 1B is a schematic vertical sectional view showing a state after centrifugation. 同実施例を改良した遠沈管を示す垂直断面図である。It is a vertical sectional view which shows the centrifuge tube which improved the Example. 同実施例をさらに改良した遠沈管を示す垂直断面図である。It is a vertical sectional view which shows the centrifuge tube which improved the Example further. 参考例を表わしたものであり、（Ａ）はその概略垂直断面図、（Ｂ）は遠心分離を行った後の状態を示した垂直断面図である。 It shows a reference example , (A) is a schematic vertical cross-sectional view thereof, (B) is a vertical cross-sectional view showing a state after centrifugation. 他の参考例を表わしたものであり、（Ａ）はその概略垂直断面図、（Ｂ）は遠心分離を行った後の状態を示した概略垂直断面図、（Ｃ）はフイルタ部分を示す垂直断面図である。 It represents another reference example , (A) is a schematic vertical sectional view thereof, (B) is a schematic vertical sectional view showing a state after being centrifuged, and (C) is a vertical sectional view showing a filter portion. It is sectional drawing. さらに他の参考例を表わしたものであり、（Ａ）はそこで用いる器具の概略垂直断面図、（Ｂ）は遠心分離を行った後の状態を示した概略垂直断面図である。 Further, another reference example is shown, (A) is a schematic vertical sectional view of an instrument used therein, and (B) is a schematic vertical sectional view showing a state after centrifugal separation.

符号の説明Explanation of symbols

２，２Ａ，２ｂ，２０， 遠沈管
４ 隆起部
６ 溝
８ 試料
８ａ 試料の不溶成分
１０，１４ 隆起 2, 2A, 2b, 20, centrifuge tube 4 ridge 6 groove 8 sample 8a sample insoluble component 10, 14 bulge

Claims (4)

遠心分離機で用いる遠沈管において、
その底部に不溶成分を捕捉する手段を備え、
前記手段は、遠沈管の底面に形成され、前記不溶成分が入り込める大きさの溝であり、該溝は底面中央部に隆起部が形成されていることにより底部周縁に沿って形成されているものであることを特徴とする遠沈管。 In centrifuge tubes used in centrifuges,
Equipped with means for capturing insoluble components at the bottom ,
The means is a groove formed on the bottom surface of the centrifuge tube and having a size capable of containing the insoluble component, and the groove is formed along the bottom periphery by forming a raised portion at the center of the bottom surface. centrifuge tube, characterized in that it. 該遠沈管の内側壁で前記溝の上部には前記隆起部を取り囲んで周方向に環状に延び、内側に突出した第１の隆起が形成されており、第１の隆起と前記隆起部によって前記溝の上部入口が狭くなっている請求項１に記載の遠沈管。A first bulge is formed on the inner wall of the centrifuge tube so as to surround the bulge portion in the upper portion of the groove and annularly extend in the circumferential direction and project inward. The first bulge and the bulge portion The centrifuge tube according to claim 1, wherein an upper entrance of the groove is narrow. 前記隆起部の遠沈管内側には第１の隆起に対向するように環状に延びた第２の隆起が形成されている請求項２に記載の遠沈管。  The centrifuge tube according to claim 2, wherein a second bulge extending in an annular shape so as to face the first bulge is formed inside the centrifuge tube of the bulge portion. 遠沈管に試料液を入れ、遠心分離機で遠心分離する方法において、
遠沈管として請求項１から３のいずれか一項に記載の遠沈管を用いることを特徴とする遠心分離方法。 In a method of putting a sample solution in a centrifuge tube and centrifuging with a centrifuge,
Centrifugation method, which comprises using a centrifuge tube according to any one of claims 1 to 3 as a centrifuge tube.
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