JP2018141739A - Biological sample pretreatment tool - Google Patents
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本発明は、生体試料中のヘモグロビンを定量する際に、測定誤差の原因となるビリルビン等の夾雑物質を除去するための前処理器具に関する。 The present invention relates to a pretreatment instrument for removing contaminants such as bilirubin that cause measurement errors when quantifying hemoglobin in a biological sample.
糖尿病診断項目の一つである糖化ヘモグロビン（以下、ＨｂＡ１ｃと略すことがある）とは、血液中の酸素を運搬する役割を担うヘモグロビン（以下、Ｈｂと略すことがある）に糖（グルコース）が結合した糖化ヘモグロビンの内、ヘモグロビンβ鎖のＮ末端側に位置するバリン残基が糖化された物質を指し、総ヘモグロビン量に対するＨｂＡ１ｃ濃度が過去１〜２ヶ月間の平均血糖値を反映することから、糖尿病の長期的な経過を観察するのに利用されている。 Glycated hemoglobin (hereinafter sometimes abbreviated as HbA1c), which is one of the diagnostic items for diabetes, refers to hemoglobin (hereinafter abbreviated as Hb) that plays a role in transporting oxygen in blood. Among the bound glycated hemoglobin, the valine residue located on the N-terminal side of the hemoglobin β chain refers to a glycated substance, and the HbA1c concentration relative to the total hemoglobin amount reflects the average blood glucose level over the past 1-2 months It is used to observe the long-term course of diabetes.
近年、医療現場ではＰＯＣＴという言葉が注目を集めている。ＰＯＣＴとはＰｏｉｎｔ Ｏｆ Ｃａｒｅ Ｔｅｓｔｉｎｇの略であり、医療従事者が被験者の傍らで行う臨床検査のことをいう。ＰＯＣＴは大規模病院の中央検査室等で行う臨床検査とは異なり、その場で瞬時に検査結果が得られることから、糖尿病診断においてもＰＯＣＴが広まりつつある。 In recent years, the term POCT has attracted attention in the medical field. POCT is an abbreviation for Point Of Care Testing, and refers to a clinical test performed by a medical worker alongside a subject. Unlike clinical tests performed in a central laboratory of a large-scale hospital or the like, POCT can obtain test results instantly, so POCT is also spreading in diabetes diagnosis.
ＨｂＡ１ｃ濃度の測定を目的としたＰＯＣＴの中に、イムノクロマト法を利用した技術が提案されている。イムノクロマト法とは毛細管現象を利用した免疫測定法であり、イムノクロマト法を利用した妊娠検査薬やインフルエンザ検査薬などで用いられる分析技術であり世界的に普及している。従来のイムノクロマト法は目視判定（定性評価）が一般的であったが、近年、クロマトリーダー等の分析装置を利用し、検体中に含まれる物質の濃度を定量化する技術が開発されつつある。 A technique using an immunochromatography method has been proposed in POCT for the purpose of measuring the HbA1c concentration. The immunochromatography method is an immunoassay method using a capillary phenomenon, and is an analytical technique used in pregnancy test drugs and influenza test drugs using the immunochromatography method and is widely used worldwide. Conventional immunochromatography is generally performed by visual determination (qualitative evaluation), but in recent years, a technique for quantifying the concentration of a substance contained in a sample using an analyzer such as a chromatographic reader is being developed.
しかし、血液中には、ビリルビンやアスコルビン酸等の還元物質が存在し、これらの物質の存在により、ヘモグロビンおよび糖化ヘモグロビンの測定値は大きく影響を受け、測定値に誤差を生じることがある。また、ビリルビン等は色素としても作用することから、測定波長によっては誤差の原因となりうる。 However, there are reducing substances such as bilirubin and ascorbic acid in the blood, and the presence of these substances greatly affects the measured values of hemoglobin and glycated hemoglobin, and may cause an error in the measured values. In addition, bilirubin or the like also acts as a pigment, and may cause an error depending on the measurement wavelength.
ビリルビンの影響を回避する方法として、両性界面活性剤を測定試薬に添加する方法が知られている。例えば、特許文献１には、ビリルビンの影響を回避する目的で第１試薬に両性界面活性剤を添加することが記載されている。また、特許文献２には、酵素反応により生成される過酸化水素をペルオキシターゼ及び被酸化性発色剤で検出する生体成分の測定法において、第一試薬又は第一試薬と第二試薬両方に、両性界面活性剤とフェロシアン化合物を存在させる生体成分の測定方法が記載されている。 As a method for avoiding the influence of bilirubin, a method of adding an amphoteric surfactant to a measurement reagent is known. For example, Patent Document 1 describes that an amphoteric surfactant is added to the first reagent for the purpose of avoiding the influence of bilirubin. Further, in Patent Document 2, in a method for measuring a biological component in which hydrogen peroxide generated by an enzyme reaction is detected with a peroxidase and an oxidizable color former, both the first reagent and both the first reagent and the second reagent are amphoteric. A method for measuring biological components in the presence of a surfactant and a ferrocyan compound is described.
本発明は、生体試料中のヘモグロビン濃度を定量的に測定する際に、測定誤差の原因となるビリルビン等の測定阻害物質を選択的、効率的に除去することができる生体試料の前処理器具を提供することを課題とする。 The present invention provides a pretreatment instrument for a biological sample that can selectively and efficiently remove measurement-inhibiting substances such as bilirubin that cause measurement errors when quantitatively measuring the hemoglobin concentration in the biological sample. The issue is to provide.
本発明者は、前記課題を解決するために鋭意検討した結果、特定の構成を有する前処理器具を用いて生体試料を処理することにより、生体試料中の夾雑物質を選択的かつ瞬時に除去できることを見出し、本発明を完成した。 As a result of intensive studies to solve the above problems, the present inventor can selectively and instantaneously remove contaminants in a biological sample by processing the biological sample using a pretreatment instrument having a specific configuration. The present invention has been completed.
すなわち、本発明は以下の構成を有する。
（１）生体試料を前処理するための器具であって、前記器具は前記生体試料の入口と出口を有するハウジングケースと前記ハウジングケース内に夾雑物質の除去材料が充填されたことを特徴とする前処理器具。
（２）前記夾雑物質の除去材料が、活性炭、活性炭素繊維、酸化チタン、ジビニルベンゼン／グリシジルメタクリレート共重合体および疎水性シリカからなる群から選ばれる１種以上であることを特徴とする（１）に記載の前処理器具。
（３）前記夾雑物質の除去材料が、繊維、織布、不織布、粒子および粉末からなる群から選ばれる１以上の形態であることを特徴とする（１）または（２）に記載の前処理器具。
（４）前記夾雑物質の除去材料の全細孔容積が０．２５〜１．００ｃｃ／ｇであることを特徴とする（１）〜（３）のいずれかに記載の前処理器具。
（５）前記夾雑物質は、ビリルビンおよび／または乳ビであることを特徴とする（１）〜（４）のいずれかに記載の前処理器具。
That is, the present invention has the following configuration.
(1) An instrument for preprocessing a biological sample, wherein the instrument has a housing case having an inlet and an outlet for the biological sample, and the housing case is filled with a contaminant removal material. Pre-processing equipment.
(2) The contaminant removal material is at least one selected from the group consisting of activated carbon, activated carbon fiber, titanium oxide, divinylbenzene / glycidyl methacrylate copolymer and hydrophobic silica (1 ) The pretreatment instrument described in.
(3) The pretreatment according to (1) or (2), wherein the contaminant removal material is in one or more forms selected from the group consisting of fibers, woven fabrics, nonwoven fabrics, particles, and powders. Instruments.
(4) The pretreatment instrument according to any one of (1) to (3), wherein a total pore volume of the contaminant removal material is 0.25 to 1.00 cc / g.
(5) The pretreatment instrument according to any one of (1) to (4), wherein the contaminant is bilirubin and / or milk.
本発明によれば、特定の構成を有する前処理器具に生体試料を接触させることにより、生体試料中の夾雑物質を選択的、効率的、速やかに除去できるので、夾雑物質の影響を受けずにヘモグロビン濃度を定量的に測定することができる。また、夾雑物質の影響が低減されるので、ヘモグロビンの極大吸収波長域での測定が可能となり、生体試料中のヘモグロビン濃度が低い場合にも高精度に測定することができる。 According to the present invention, the contaminants in the biological sample can be selectively, efficiently, and quickly removed by bringing the biological sample into contact with the pretreatment instrument having a specific configuration, so that it is not affected by the contaminants. The hemoglobin concentration can be measured quantitatively. In addition, since the influence of contaminants is reduced, measurement in the maximum absorption wavelength region of hemoglobin is possible, and measurement can be performed with high accuracy even when the hemoglobin concentration in the biological sample is low.
本発明において、生体試料は、血液、血しょう、血清、便、尿などのヘモグロビンを含有する体液等を意味するが、それらの中には、ヘモグロビンを定量的に分析、特に吸光光度法を利用して分析（測定）する際に、測定誤差を与える多様な夾雑物質（測定阻害物質）が含まれている。 In the present invention, the biological sample means a body fluid containing hemoglobin such as blood, plasma, serum, stool, urine, etc., and among these, hemoglobin is quantitatively analyzed, particularly using a spectrophotometric method. When analyzing (measurement), various contaminants (measurement inhibitory substances) that give measurement errors are included.
本発明は、生体試料を検出装置（イムノクロマト試験片）等に供する前に予め、生体試料中の夾雑物質を除去するための器具であって、前記器具は前記生体試料の入口と出口を有するハウジングケースと前記ハウジングケース内に夾雑物質の除去材料が充填されたものであることを特徴とする。以下、本発明の前処理器具の詳細について、図１を参照して説明する。 The present invention is an instrument for removing contaminants in a biological sample in advance before subjecting the biological sample to a detection device (immunochromatographic test strip) or the like, the instrument having a housing having an inlet and an outlet for the biological sample The housing and the housing case are filled with a contaminant removing material. Hereinafter, the details of the pretreatment instrument of the present invention will be described with reference to FIG.
［実施形態１］
図１は、シリンジフィルタタイプの前処理器具の断面図を示す。図１において、１は、生体試料を導入するための試料入口である。また、２は、夾雑物質の除去材料である。また、３は、処理された生体試料への除去材料自体や除去材料からの溶出物のコンタミを防止するためのメンブレンフィルタである。さらに、４は、処理された生体試料を取り出すための試料出口である。なお、３は目的、用途により有っても無くてもよい。また、コンタミ防止を達成できるものであれば、他のものを代用してもよい。
[Embodiment 1]
FIG. 1 shows a cross-sectional view of a syringe filter type pretreatment instrument. In FIG. 1, 1 is a sample inlet for introducing a biological sample. Reference numeral 2 denotes a contaminant removal material. Reference numeral 3 denotes a membrane filter for preventing contamination of the removal material itself and the eluate from the removal material on the treated biological sample. Furthermore, 4 is a sample outlet for taking out the processed biological sample. Note that 3 may or may not be present depending on the purpose and application. Moreover, as long as contamination prevention can be achieved, other ones may be substituted.
試料入口１は、シリンジが液密に接続できるような構造を有していればよく、必要によりメス型ルアロック構造を有していてもよい。一方、試料出口４は、イムノクロマト試験片に直接試料を適量滴下できるように形状等を調節するのが好ましい。イムノクロマト試験片を用いた測定においては、その大きさにもよるが、凡そ１００μＬ〜５００μＬ程度を滴下できる形状とすればよい。 The sample inlet 1 only needs to have a structure that allows the syringe to be liquid-tightly connected, and may have a female luer lock structure as necessary. On the other hand, it is preferable to adjust the shape and the like of the sample outlet 4 so that an appropriate amount of the sample can be dropped directly onto the immunochromatographic test piece. In the measurement using an immunochromatographic test piece, although it depends on its size, it may be shaped so that about 100 μL to 500 μL can be dropped.
本発明において、夾雑物質の除去材料は、活性炭、活性炭素繊維（以下、活性炭と活性炭素繊維をあわせて単に活性炭と称することがある）、酸化チタン、ジビニルベンゼン／グリシジルメタクリレート共重合体および疎水性シリカからなる群から選ばれる１種以上であることが好ましい。これらの中で、活性炭が入手のしやすさやコストの面より好ましい。また、活性炭の中でも水処理用のものがより好ましい。 In the present invention, the contaminant removal material is activated carbon, activated carbon fiber (hereinafter, activated carbon and activated carbon fiber may be simply referred to as activated carbon), titanium oxide, divinylbenzene / glycidyl methacrylate copolymer, and hydrophobicity. It is preferably at least one selected from the group consisting of silica. Among these, activated carbon is preferable from the viewpoint of availability and cost. Among activated carbons, those for water treatment are more preferred.
本発明において、夾雑物質の除去材料は、繊維、織布、不織布、粒子および粉末からなる群から選ばれる１以上の形態であることが好ましい。前処理器具をコンパクトにできることから、不織布や粒子、粉末の形態であることがより好ましい。ここで、酸化チタンおよび疎水性シリカは粒子状または粉末状のものが入手でき、それ以外は繊維状、不織布状、粒子状または粉末状のいずれも入手および調製可能である。 In the present invention, the contaminant removal material is preferably in one or more forms selected from the group consisting of fibers, woven fabrics, nonwoven fabrics, particles and powders. Since the pretreatment instrument can be made compact, it is more preferably in the form of a nonwoven fabric, particles or powder. Here, the titanium oxide and the hydrophobic silica can be obtained in the form of particles or powder, and other than that, any of fiber, nonwoven fabric, particles, or powder can be obtained and prepared.
前記活性炭は、その原料（前駆体）は特に限定されるものではなく、例えばフェノール、セルロース、レーヨン、ポリアクリロニトリル系、ピッチ系、アラミド、ポリイミド、ポリアミド、ポリアミドイミド、ポリフェニレンベンゾビスオキサゾール、ポリビニルアルコール、ポリエーテルスルホン、ポリスルホン、ポリフェニレンオキサイドなどを挙げることができる。これらの材料を用いて、繊維、織布、不織布、粒子、粉末に成形した後、炭化、賦活化、必要により精製することで製造することができる。 The raw material (precursor) of the activated carbon is not particularly limited. For example, phenol, cellulose, rayon, polyacrylonitrile, pitch, aramid, polyimide, polyamide, polyamideimide, polyphenylenebenzobisoxazole, polyvinyl alcohol, Examples thereof include polyethersulfone, polysulfone, and polyphenylene oxide. These materials can be used to produce fibers, woven fabrics, nonwoven fabrics, particles, and powders, and then carbonized, activated, and purified as necessary.
本発明において、夾雑物質の除去材料は、多孔質体を用いる場合、その全細孔容積は０．２５〜１．００ｃｃ／ｇの範囲内であることが好ましい。全細孔容積が小さすぎると、生体試料中の夾雑物質を十分に除去できないことがあり、大きすぎると、単繊維の強度が著しく低下し、形態を保てないことがある。なお、全細孔容積は、例えば比表面積・細孔分布測定装置Ｇｅｍｉｎｉ２３７５（Ｍｉｃｒｏｍｅｒｉｔｉｃｓ社製）を用いて測定することができる。 In the present invention, when a porous material is used as the contaminant removal material, the total pore volume is preferably in the range of 0.25 to 1.00 cc / g. If the total pore volume is too small, contaminants in the biological sample may not be sufficiently removed, and if it is too large, the strength of the single fiber may be significantly reduced and the form may not be maintained. The total pore volume can be measured using, for example, a specific surface area / pore distribution measuring device Gemini 2375 (manufactured by Micromeritics).
本発明において、夾雑物質の除去材料は、その比表面積は５〜２５００ｍ２／ｇの範囲内であることが好ましい。比表面積が小さすぎると、生体試料中の夾雑物質を十分に除去できないことがあるとか、前処理器具を必要以上に大きくする必要がある。また、比表面積が大きすぎると、単繊維の強度が著しく低下し、形態を保てないことがある。比表面積とは、液体窒素温度での窒素ガス吸着等温線によるＢＥＴ法により求められる比表面積を意味し、例えば比表面積・細孔分布測定装置Ｇｅｍｉｎｉ２３７５（Ｍｉｃｒｏｍｅｒｉｔｉｃｓ社製）を用いて測定することができる。 In the present invention, the contaminant removal material preferably has a specific surface area in the range of 5 to 2500 m 2 / g. If the specific surface area is too small, it may be impossible to sufficiently remove contaminants in the biological sample, or the pretreatment instrument needs to be made larger than necessary. On the other hand, if the specific surface area is too large, the strength of the single fiber may be significantly reduced and the form may not be maintained. The specific surface area means a specific surface area determined by a BET method using a nitrogen gas adsorption isotherm at a liquid nitrogen temperature, and can be measured using, for example, a specific surface area / pore distribution measuring apparatus Gemini 2375 (manufactured by Micromeritics). .
本発明において、夾雑物質の除去材料は、多孔質体を用いる場合、細孔直径が１０Å以下のマイクロポア容積が全マイクロポア容積の９５％以上であることが好ましい。細孔直径が１０Å以下のマイクロポア容積が全マイクロポア容積の９５％未満である場合には、細孔が大きくなりすぎて、夾雑物質が十分に除去されないことがある。なお、細孔直径１０Å以下のマイクロポア容積の割合は、例えば比表面積・細孔分布測定装置Ｇｅｍｉｎｉ２３７５（Ｍｉｃｒｏｍｅｒｉｔｉｃｓ社製）を用いて測定することができる。 In the present invention, when the porous material is used as the contaminant removal material, the micropore volume having a pore diameter of 10 mm or less is preferably 95% or more of the total micropore volume. When the micropore volume having a pore diameter of 10 mm or less is less than 95% of the total micropore volume, the pores become too large, and the contaminants may not be sufficiently removed. The ratio of the micropore volume having a pore diameter of 10 mm or less can be measured using, for example, a specific surface area / pore distribution measuring device Gemini 2375 (manufactured by Micromeritics).
本発明において、夾雑物質の除去材料が繊維形状の場合は、単繊維径が５〜３０μｍであることが好ましい。単繊維径が小さすぎると、夾雑物質を除去するための有効細孔を確保できないことがある。一方、単繊維径が大きすぎると、前処理器具のコンパクト性を損なうことがある。また、夾雑物質の除去材料が粒子状または粉末状である場合は、平均粒子径が１ｎｍ〜１ｍｍであることが好ましい。平均粒子径が小さすぎると、夾雑物質を除去するための有効細孔を確保できないことがある。一方、平均粒子径が大きすぎると、前処理器具のコンパクト性を損なうことがある。 In the present invention, when the contaminant removal material is in a fiber shape, the single fiber diameter is preferably 5 to 30 μm. If the single fiber diameter is too small, effective pores for removing contaminants may not be ensured. On the other hand, if the single fiber diameter is too large, the compactness of the pretreatment instrument may be impaired. When the contaminant removal material is in the form of particles or powder, the average particle diameter is preferably 1 nm to 1 mm. If the average particle size is too small, effective pores for removing contaminants may not be ensured. On the other hand, if the average particle diameter is too large, the compactness of the pretreatment instrument may be impaired.
本発明において、前記した材料を用いて構成された前処理器具がヘモグロビンは除去しないが、ビリルビンや乳ビ等の夾雑物質は除去するという選択性が発揮される理由は実は明らかでない。しかし、後述するようにヘモグロビンを溶解した溶液を前処理器具で処理しても処理前後で吸光度はほぼ低下しないが、ビリルビンを溶解した溶液を前処理器具で処理すると吸光度が大きく低下することからもビリルビンが選択的に除去されることは明らかである。 In the present invention, the pretreatment instrument configured using the above-described material does not remove hemoglobin, but it is not clear why the selectivity of removing contaminants such as bilirubin and milk powder is exhibited. However, as described later, even if the hemoglobin-dissolved solution is treated with a pretreatment device, the absorbance does not substantially decrease before and after the treatment, but if the bilirubin-dissolved solution is treated with the pretreatment device, the absorbance greatly decreases. It is clear that bilirubin is selectively removed.
実施形態１において、例えばシリンジに希釈液で希釈された生体試料を必要量吸上げた後、シリンジの先端に本発明の前処理器具を装着し、シリンジのプランジャーを押し込んで生体試料または希釈された生体試料を前処理器具の試料出口より押し出すことにより、容易にビリルビン等の測定阻害物質を除去することができる。 In the first embodiment, for example, after sucking a necessary amount of a biological sample diluted with a diluent into a syringe, the pretreatment instrument of the present invention is attached to the tip of the syringe, and the plunger of the syringe is pushed into the biological sample or diluted. By extruding the biological sample from the sample outlet of the pretreatment instrument, measurement-inhibiting substances such as bilirubin can be easily removed.
［実施形態２］
実施形態１は、前処理器具が試料採取器具と別体となっているのに対して、実施形態２においては、試料採取器具が前処理器具と一体となっている点で異なる（図２参照）。
[Embodiment 2]
The first embodiment is different from the first embodiment in that the pretreatment instrument is separate from the sampling instrument, but the second embodiment is different in that the sampling instrument is integrated with the pretreatment instrument (see FIG. 2). ).
（比表面積の測定）
夾雑物質除去材料を約３０ｍｇ採取し、１２０℃で１２時間真空乾燥して秤量し、比表面積・細孔分布測定装置Ｇｅｍｉｎｉ２３７５（Ｍｉｃｒｏｍｅｒｉｔｉｃｓ社製）を使用して測定した。液体窒素の沸点（−１９５．８℃）における窒素ガスの吸着量を相対圧が０．０２〜０．９５の範囲で測定し、前記材料の吸着等温線を作成した。相対圧０．０２〜０．１５の範囲での結果をもとに、ＢＥＴ法により重量あたりの比表面積（単位：ｍ２／ｇ）を求めた。
(Measurement of specific surface area)
About 30 mg of the contaminant removing material was collected, vacuum-dried at 120 ° C. for 12 hours, weighed, and measured using a specific surface area / pore distribution measuring device Gemini 2375 (manufactured by Micromeritics). The adsorption amount of nitrogen gas at the boiling point of liquid nitrogen (-195.8 ° C.) was measured in the range of relative pressure of 0.02 to 0.95, and the adsorption isotherm of the material was prepared. Based on the results in the relative pressure range of 0.02 to 0.15, the specific surface area per unit weight (unit: m 2 / g) was determined by the BET method.
（全細孔容積の測定）
夾雑物質除去材料を約３０ｍｇ採取し、１２０℃で１２時間真空乾燥して秤量し、比表面積・細孔分布測定装置Ｇｅｍｉｎｉ２３７５（Ｍｉｃｒｏｍｅｒｉｔｉｃｓ社製）を使用して測定した。液体窒素の沸点（−１９５．８℃）における窒素ガスの吸着量を相対圧が０．０２〜０．９５の範囲で測定し、前記材料の吸着等温線を作成した。相対圧０．９５での結果より全細孔容積（単位：ｃｃ／ｇ）を算出した。
(Measurement of total pore volume)
About 30 mg of the contaminant removing material was collected, vacuum-dried at 120 ° C. for 12 hours, weighed, and measured using a specific surface area / pore distribution measuring device Gemini 2375 (manufactured by Micromeritics). The adsorption amount of nitrogen gas at the boiling point of liquid nitrogen (-195.8 ° C.) was measured in the range of relative pressure of 0.02 to 0.95, and the adsorption isotherm of the material was prepared. The total pore volume (unit: cc / g) was calculated from the result at a relative pressure of 0.95.
（全マイクロポア容積（Ａ））
夾雑物質除去材料を約３０ｍｇ採取し、１２０℃で１２時間真空乾燥して秤量し、比表面積・細孔分布測定装置Ｇｅｍｉｎｉ２３７５（Ｍｉｃｒｏｍｅｒｉｔｉｃｓ社製）を使用して測定した。液体窒素の沸点（−１９５．８℃）における窒素ガスの吸着量を相対圧が０．０２〜０．９５の範囲で測定し、前記材料の吸着等温線を作成した。この結果をＭＰ法によって解析範囲０〜２０Å、ｔ決定式Ｈ．Ｊの条件で解析し、吸着時のマイクロポア径分布数表の結果より全マイクロポア容積（Ａ）（単位：ｃｃ／ｇ）を算出した。
(Total micropore volume (A))
About 30 mg of the contaminant removing material was collected, vacuum-dried at 120 ° C. for 12 hours, weighed, and measured using a specific surface area / pore distribution measuring device Gemini 2375 (manufactured by Micromeritics). The adsorption amount of nitrogen gas at the boiling point of liquid nitrogen (-195.8 ° C.) was measured in the range of relative pressure of 0.02 to 0.95, and the adsorption isotherm of the material was prepared. This result was analyzed by the MP method with an analysis range of 0 to 20 cm and a t-determining formula H.264. Analysis was performed under the conditions of J, and the total micropore volume (A) (unit: cc / g) was calculated from the results of the micropore diameter distribution number table at the time of adsorption.
（細孔直径１０Å以下のマイクロポア容積（Ｂ））
夾雑物質除去材料を約３０ｍｇ採取し、１２０℃で１２時間真空乾燥して秤量し、比表面積・細孔分布測定装置Ｇｅｍｉｎｉ２３７５（Ｍｉｃｒｏｍｅｒｉｔｉｃｓ社製）を使用して測定した。液体窒素の沸点（−１９５．８℃）における窒素ガスの吸着量を相対圧が０．０２〜０．９５の範囲で測定し、前記材料の吸着等温線を作成した。この結果をＭＰ法によって解析範囲０〜２ｎｍ、ｔ決定式Ｈ．Ｊの条件で解析し、吸着時のマイクロポア径分布数表の結果より全マイクロポア容積（Ａ）から細孔直径１０．０３Å以上のマイクロポア容積を引いて、細孔直径１０Å以下のマイクロポア容積Ｂ（単位：ｃｃ／ｇ）を算出した。
(Micropore volume (B) with a pore diameter of 10 mm or less)
About 30 mg of the contaminant removing material was collected, vacuum-dried at 120 ° C. for 12 hours, weighed, and measured using a specific surface area / pore distribution measuring device Gemini 2375 (manufactured by Micromeritics). The adsorption amount of nitrogen gas at the boiling point of liquid nitrogen (-195.8 ° C.) was measured in the range of relative pressure of 0.02 to 0.95, and the adsorption isotherm of the material was prepared. This result was analyzed by MP method with an analysis range of 0 to 2 nm and a t-determining formula H. Analyzing under the condition of J, subtracting the micropore volume with a pore diameter of 10.03 mm or more from the total micropore volume (A) from the results of the micropore diameter distribution number table at the time of adsorption, and obtaining Volume B (unit: cc / g) was calculated.
（吸光度の測定）
各測定用試料について、分光光度計ＵＶ−２４５０（島津製作所社製）を用いて、４２０ｎｍの吸光度を測定した。なお、測定用セルは、１ｍＬ容ディスポーザブルセルを用い、光路長１０ｍｍで測定した。
(Measurement of absorbance)
About each sample for a measurement, the light absorbency of 420 nm was measured using spectrophotometer UV-2450 (made by Shimadzu Corp.). The measurement cell was a 1 mL disposable cell and was measured with an optical path length of 10 mm.
（前処理冶具１の作製）
図１に示されるような入口および出口を有するポリプロピレン製のフィルターホルダーにフェノール繊維を炭化、賦活化して得られた比表面積９７３ｍ２／ｇ、全細孔容積０．４２ｃｃ／ｇの活性炭素繊維を３４ｍｇ充填し、前処理器具１を作製した。
(Preparation of pretreatment jig 1)
An activated carbon fiber having a specific surface area of 973 m 2 / g and a total pore volume of 0.42 cc / g obtained by carbonizing and activating phenol fibers in a polypropylene filter holder having an inlet and an outlet as shown in FIG. Filled with 34 mg, the pretreatment device 1 was prepared.
（前処理冶具２の作製）
フェノール繊維を炭化、賦活化して得られた比表面積１９６２ｍ２／ｇ、全細孔容積０．８５ｃｃ／ｇの活性炭素繊維３４ｍｇを充填した以外は、前処理器具１と同様にして前処理器具２を作製した。
(Preparation of pretreatment jig 2)
Pretreatment device 2 in the same manner as Pretreatment device 1 except that 34 mg of activated carbon fiber having a specific surface area of 1962 m 2 / g and a total pore volume of 0.85 cc / g obtained by carbonizing and activating phenol fibers was filled. Was made.
（前処理冶具３の作製）
ピッチ系繊維を炭化、賦活化して得られた比表面積８５０ｍ２／ｇ、全細孔容積０．３５ｃｃ／ｇの活性炭素繊維３４ｍｇを充填した以外は、前処理器具１と同様にして前処理器具３を作製した。
(Preparation of pretreatment jig 3)
Pretreatment instrument as in the pretreatment instrument 1 except that 34 mg of activated carbon fiber having a specific surface area of 850 m 2 / g and a total pore volume of 0.35 cc / g obtained by carbonizing and activating the pitch fiber is filled. 3 was produced.
（前処理冶具４の作製）
フェノール繊維を炭化、賦活化して得られた比表面積１５２０ｍ２／ｇ、全細孔容積０．６５ｃｃ／ｇの活性炭素繊維３４ｍｇを充填した以外は、前処理器具１と同様にして前処理器具４を作製した。
(Preparation of pretreatment jig 4)
The pretreatment device 4 is the same as the pretreatment device 1 except that 34 mg of activated carbon fibers having a specific surface area of 1520 m 2 / g and a total pore volume of 0.65 cc / g obtained by carbonizing and activating phenol fibers are filled. Was made.
（前処理冶具５の作製）
ピッチ繊維を炭化、賦活化して得られた比表面積１７００ｍ２／ｇ、全細孔容積０．８０ｃｃ／ｇの活性炭素繊維３４ｍｇを充填した以外は、前処理器具１と同様にして前処理器具５を作製した。
(Preparation of pretreatment jig 5)
The pretreatment device 5 is the same as the pretreatment device 1 except that 34 mg of activated carbon fibers having a specific surface area of 1700 m 2 / g and a total pore volume of 0.80 cc / g obtained by carbonizing and activating the pitch fibers are filled. Was made.
（前処理冶具６の作製）
粒状活性炭（ユーイーエス社製、ＫＤ−ＧＷ−Ａ、比表面積１３２９ｍ２／ｇ、全細孔容積０．４４ｃｃ／ｇ）３４ｍｇを充填した以外は、前処理器具１と同様にして前処理器具６を作製した。
(Preparation of pretreatment jig 6)
Pretreatment device 6 in the same manner as Pretreatment device 1 except that 34 mg of granular activated carbon (made by UES, KD-GW-A, specific surface area 1329 m 2 / g, total pore volume 0.44 cc / g) was filled. Was made.
［参考例１］
干渉チェックＡプラス（シスメックス社）の溶血ヘモグロビンを蒸留水で１３５ｇ／Ｌになるように溶解した後、希釈液（１％Ｔｒｉｔｏｎ Ｘ−１００、１％Ｔｗｅｅｎ−２０、５０ｍＭ ＰＢＳ、ｐＨ７．１）で５００倍希釈し測定用試料１を得た。１ｍＬ容のシリンジに前記測定用試料１を１ｍＬ吸い上げた後、シリンジの先に前処理器具１を取付け、前記測定用試料１をセルに押し出した。前記セルを分光光度計にセットし、４２０ｎｍの吸光度を測定した。結果を表１にまとめた。
[Reference Example 1]
After dissolving hemolyzed hemoglobin of interference check A plus (Sysmex) to 135 g / L with distilled water, it was diluted with 1% Triton X-100, 1% Tween-20, 50 mM PBS, pH 7.1. The sample 1 for measurement was obtained by diluting 500 times. After 1 mL of the measurement sample 1 was sucked into a 1 mL syringe, the pretreatment instrument 1 was attached to the tip of the syringe, and the measurement sample 1 was pushed out into the cell. The cell was set in a spectrophotometer and the absorbance at 420 nm was measured. The results are summarized in Table 1.
［参考例２］
干渉チェックＡプラス（シスメックス社）のビリルビンＦを蒸留水で２００ｍｇ／ｄＬになるように溶解した後、参考例１と同様の希釈液で５００倍希釈し測定用試料２を得た。１ｍＬ容のシリンジに前記測定用試料２を１ｍＬ吸い上げた後、シリンジの先の前処理器具１を取付け、前記測定用試料２をセルに押し出した。前記セルを分光光度計にセットし、４２０ｎｍの吸光度を測定した。結果を表１にまとめた。
[Reference Example 2]
After bilirubin F of interference check A plus (Sysmex) was dissolved in distilled water to 200 mg / dL, it was diluted 500 times with the same diluent as in Reference Example 1 to obtain a sample 2 for measurement. After 1 mL of the measurement sample 2 was sucked into a 1 mL syringe, the pretreatment instrument 1 at the tip of the syringe was attached, and the measurement sample 2 was pushed out into the cell. The cell was set in a spectrophotometer and the absorbance at 420 nm was measured. The results are summarized in Table 1.
表１の結果から明らかなように、ヘモグロビンを溶解した溶液を前処理器具に接触させた場合に、吸光度は接触前後でほぼ変化しなかったが、ビリルビンを溶解した溶液を前処理器具に接触させた場合には、吸光度は大きく低下した。これは、本発明の前処理器具を用いると、生体試料中のヘモグロビンは除去されないが、ビリルビンに代表される夾雑物質は除去されることを示している。 As is clear from the results in Table 1, when the solution in which hemoglobin was dissolved was brought into contact with the pretreatment device, the absorbance was almost unchanged before and after the contact, but the solution in which bilirubin was dissolved was brought into contact with the pretreatment device. In the case of the absorption, the absorbance decreased greatly. This indicates that when the pretreatment instrument of the present invention is used, hemoglobin in the biological sample is not removed, but contaminants typified by bilirubin are removed.
［実施例１］
１ｍＬ容のシリンジに前記測定用試料１を１ｍＬ吸い上げた後、シリンジの先に前処理器具１を取付け、測定用試料１をセルに押し出した。前記セルを分光光度計にセットし、４２０ｎｍの吸光度を測定した。また、前記測定用試料２についても同様の処理を施して吸光度を測定した。結果を表２にまとめた。
[Example 1]
After 1 mL of the measurement sample 1 was sucked into a 1 mL syringe, the pretreatment instrument 1 was attached to the tip of the syringe, and the measurement sample 1 was pushed out into the cell. The cell was set in a spectrophotometer and the absorbance at 420 nm was measured. The measurement sample 2 was also subjected to the same treatment and the absorbance was measured. The results are summarized in Table 2.
［実施例２］
１ｍＬ容のシリンジに実施例１と同様の測定用試料を１ｍＬ吸い上げた後、シリンジの先に前処理器具２を取付け、測定用試料をセルに押し出した。前記セルを分光光度計にセットし、４２０ｎｍの吸光度を測定した。結果を表２にまとめた。
[Example 2]
After sucking 1 mL of the same measurement sample as in Example 1 into a 1 mL syringe, the pretreatment instrument 2 was attached to the tip of the syringe, and the measurement sample was pushed out into the cell. The cell was set in a spectrophotometer and the absorbance at 420 nm was measured. The results are summarized in Table 2.
［実施例３］
１ｍＬ容のシリンジに実施例１と同様の測定用試料を１ｍＬ吸い上げた後、シリンジの先に前処理器具３を取付け、測定用試料をセルに押し出した。前記セルを分光光度計にセットし、４２０ｎｍの吸光度を測定した。結果を表２にまとめた。
[Example 3]
After sucking 1 mL of the same measurement sample as in Example 1 into a 1 mL syringe, the pretreatment instrument 3 was attached to the tip of the syringe, and the measurement sample was pushed out into the cell. The cell was set in a spectrophotometer and the absorbance at 420 nm was measured. The results are summarized in Table 2.
［実施例４］
１ｍＬ容のシリンジに実施例１と同様の測定用試料を１ｍＬ吸い上げた後、シリンジの先に前処理器具４を取付け、測定用試料をセルに押し出した。前記セルを分光光度計にセットし、４２０ｎｍの吸光度を測定した。結果を表２にまとめた。
[Example 4]
After sucking 1 mL of the same measurement sample as in Example 1 into a 1 mL syringe, the pretreatment instrument 4 was attached to the tip of the syringe, and the measurement sample was pushed out into the cell. The cell was set in a spectrophotometer and the absorbance at 420 nm was measured. The results are summarized in Table 2.
［実施例５］
１ｍＬ容のシリンジに実施例１と同様の測定用試料を１ｍＬ吸い上げた後、シリンジの先に前処理器具５を取付け、測定用試料をセルに押し出した。前記セルを分光光度計にセットし、４２０ｎｍの吸光度を測定した。結果を表２にまとめた。
[Example 5]
After sucking 1 mL of the same measurement sample as in Example 1 into a 1 mL syringe, the pretreatment instrument 5 was attached to the tip of the syringe, and the measurement sample was pushed out into the cell. The cell was set in a spectrophotometer and the absorbance at 420 nm was measured. The results are summarized in Table 2.
［実施例６］
１ｍＬ容のシリンジに実施例１と同様の測定用試料を１ｍＬ吸い上げた後、シリンジの先に前処理器具６を取付け、測定用試料をセルに押し出した。前記セルを分光光度計にセットし、４２０ｎｍの吸光度を測定した。結果を表２にまとめた。
[Example 6]
After sucking 1 mL of the same measurement sample as in Example 1 into a 1 mL syringe, the pretreatment instrument 6 was attached to the tip of the syringe, and the measurement sample was pushed out into the cell. The cell was set in a spectrophotometer and the absorbance at 420 nm was measured. The results are summarized in Table 2.
［比較例１］
１ｍＬ容のシリンジに実施例１と同様の測定用試料を１ｍＬ吸い上げた後、シリンジの先に前処理器具を取付けずに、測定用試料をセルに押し出した。前記セルを分光光度計にセットし、４２０ｎｍの吸光度を測定した。結果を表２にまとめた。
[Comparative Example 1]
After sucking 1 mL of the same measurement sample as in Example 1 into a 1 mL syringe, the measurement sample was extruded into the cell without attaching a pretreatment instrument to the tip of the syringe. The cell was set in a spectrophotometer and the absorbance at 420 nm was measured. The results are summarized in Table 2.
表２の結果から明らかなように、前処理器具１−６を用いた実施例１−６は、比較例１に比較して、波長４２０ｎｍの吸光度が大きく低下している。ヘモグロビンＡ１ｃ（％）は、ヘモグロビンＡ１ｃ濃度／ヘモグロビン濃度×１００で求められる糖尿病診断の指標であり、ヘモグロビン濃度の測定値に誤差が含まれると正確な診断ができない。本発明によれば、より正確なヘモグロビン濃度を測定することができる。 As is clear from the results in Table 2, the absorbance at a wavelength of 420 nm is greatly reduced in Example 1-6 using the pretreatment instrument 1-6 as compared with Comparative Example 1. Hemoglobin A1c (%) is an index for diagnosing diabetes obtained by hemoglobin A1c concentration / hemoglobin concentration × 100, and accurate measurement cannot be performed if the measured value of hemoglobin concentration includes an error. According to the present invention, a more accurate hemoglobin concentration can be measured.
本発明によれば、夾雑物質の影響を受けずにヘモグロビン濃度を定量的に測定することができる。また、夾雑物質の影響が低減されるので、ヘモグロビンの極大吸収波長域での測定が可能となり、生体試料中のヘモグロビン濃度が低い場合にも高精度に測定することができるので、糖尿病診断において極めて好適である。 According to the present invention, the hemoglobin concentration can be quantitatively measured without being affected by contaminants. In addition, since the influence of contaminants is reduced, measurement in the maximum absorption wavelength region of hemoglobin is possible, and even when the concentration of hemoglobin in a biological sample is low, it can be measured with high accuracy. Is preferred.
１．試料入口
２．夾雑物質の除去材料
３．メンブレンフィルタ
４．試料出口
1. Sample inlet 2. 2. Contaminant removal material 3. Membrane filter Sample outlet
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| CN203941046U (en) * | 2014-06-19 | 2014-11-12 | 深圳市凯特生物医疗电子科技有限公司 | The miniature chromatographic column of a kind of glycolated hemoglobin analysis |
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