JP5047702B2 - Red blood cell type determination method - Google Patents
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Description
本発明は、赤血球の型判定方法に関する。 The present invention relates to an erythrocyte type determination method.
一般的な血液型判定では、抗血清試薬と検体である赤血球を試験管内或いは皿内で混合し、凝集の有無を観察する。また、ガラスビーズ、セファデックスゲル粒子のような不溶性担体をマイクロカラムに充填し、これに抗血清試薬と赤血球を添加して遠心する方法もある。この方法では、凝集塊をガラスビーズ、セファデックスゲル粒子表面に捕捉させることにより、判定を容易にしている（例えば、特許文献１及び２）。
In general blood type determination, an antiserum reagent and a red blood cell as a sample are mixed in a test tube or a dish, and the presence or absence of aggregation is observed. There is also a method in which an insoluble carrier such as glass beads or Sephadex gel particles is packed in a microcolumn, and an antiserum reagent and erythrocytes are added thereto and centrifuged. In this method, the determination is facilitated by capturing the aggregate on the surface of glass beads or Sephadex gel particles (for example,
多数の検体を試験する場合には、マイクロプレートが用いられることもある。反応液をマイクロプレート中に自然放置し、凝集塊を沈殿させる方法（例えば、特許文献３及び４）や、マイクロプレートを遠心する方法がある。 When testing a large number of specimens, a microplate may be used. There are a method of allowing a reaction solution to stand naturally in a microplate and precipitating an aggregate (for example, Patent Documents 3 and 4) and a method of centrifuging the microplate.
しかしながら、凝集塊を自然沈降させる方法では、判定に時間がかかるという問題があった。遠心機を使用する方法では、自動化する場合に装置が大型化するという問題があり、また、遠心条件の均一化が難しいために判定結果の精度が低下する恐れがあった。また遠心操作に時間がかかるという問題もあった。 However, the method of spontaneously sedimenting aggregates has a problem that it takes time for the determination. In the method using a centrifuge, there is a problem that the size of the apparatus is increased when it is automated, and the accuracy of the determination result may be lowered because it is difficult to make the centrifugal conditions uniform. There is also a problem that it takes time to perform the centrifugation.
さらに、赤血球は天然成分であるために、人為的な加工を施すと赤血球表面の抗原等の反応性が著しく低下したり、失活したりし易い。このため、赤血球を直接加工することにより沈降特性を改変することは非常に困難である。
そこで本発明では、赤血球の型判定をより短時間で簡便に行う方法を提供することを目的とする。 Accordingly, an object of the present invention is to provide a method for easily and easily determining the type of erythrocytes in a shorter time.
上記目的を達成するため、磁性体が封入された担体と、前記担体に固定化され、赤血球に非特異的に結合するリガンドとを含み、抗赤血球抗体とは結合しない磁性粒子と、型を判定すべき赤血球とを、前記抗赤血球抗体が固相化された反応容器内で混合して、前記磁性粒子中の前記リガンドを前記型を判定すべき赤血球に結合させることにより、前記型を判定すべき赤血球を磁性標識する工程と、磁性標識された前記型を判定すべき赤血球を磁力によって沈降させて反応像を形成させる工程とを含み、前記反応像が、前記型を判定すべき赤血球と前記抗赤血球抗体とが結合した場合は陽性像であり、前記型を判定すべき赤血球と前記抗赤血球抗体とが結合しなかった場合は陰性像である赤血球の型判定が提供される。一つの態様において、該方法は、前記型を判定すべき赤血球を磁性標識する工程と前記反応像を形成させる工程との間に、前記反応容器に抗赤血球抗体をさらに加えることを含む。
In order to achieve the above object , the type of the magnetic particle containing a carrier encapsulating a magnetic substance and a ligand immobilized on the carrier and non-specifically binding to erythrocytes and not binding to anti-erythrocyte antibodies is determined. The type is determined by mixing the red blood cells to be mixed in a reaction vessel in which the anti-red blood cell antibody is immobilized and binding the ligand in the magnetic particles to the red blood cells to be determined for the type. A step of magnetically labeling the erythrocytes, and a step of sedimenting the erythrocytes to be magnetically labeled to form a reaction image by magnetic force, wherein the reaction image includes the erythrocytes to be determined for the type and the When the anti-erythrocyte antibody is bound, a positive image is provided, and when the red blood cell whose type is to be determined and the anti-red blood cell antibody are not bound, a type determination of the red blood cell is provided. In one embodiment, the method further comprises adding an anti-erythrocyte antibody to the reaction vessel between the step of magnetically labeling the red blood cells whose type is to be determined and the step of forming the reaction image.
他の側面において、磁性体が封入された担体と、前記担体に固定化され、赤血球に非特異的に結合するリガンドとを含み、抗赤血球抗体とは結合しない磁性粒子と、型を判定すべき赤血球とを混合して、前記磁性粒子中の前記リガンドを前記型を判定すべき赤血球に結合させることにより、前記型を判定すべき赤血球を磁性標識する工程と、磁性標識された前記型を判定すべき赤血球を、前記抗赤血球抗体が固相化された反応容器に加える工程と、前記磁性標識された型を判定すべき赤血球を磁力によって沈降させて反応像を形成させる工程とを含み、前記反応像が、前記型を判定すべき赤血球と前記抗赤血球抗体とが結合した場合は陽性像であり、前記型を判定すべき赤血球と前記抗赤血球抗体とが結合しなかった場合は陰性像である赤血球の型判定方法が提供される。
In another aspect, a magnetic particle containing a carrier encapsulating a magnetic substance , a ligand immobilized on the carrier and binding non-specifically to erythrocytes and not binding to anti-erythrocyte antibodies, and the type should be determined Mixing the ligand in the magnetic particles with the red blood cell whose type is to be determined, and magnetically labeling the red blood cell whose type is to be determined; and determining the magnetically labeled type Adding a red blood cell to be added to the reaction vessel on which the anti-red blood cell antibody is immobilized, and a step of causing the red blood cell to be determined for the magnetically labeled type to settle by magnetic force to form a reaction image, The reaction image is a positive image when the red blood cell to be determined for the type and the anti-red blood cell antibody are bound, and the negative image is when the red blood cell to be determined for the type and the anti-red blood cell antibody is not bound. Red blood Typing method is provided.
前記リガンドは赤血球に非特異的に結合して該赤血球を凝集させるリガンドであることが好ましい。前記磁性体封入担体は、磁性体が封入されたコアセルベートであることが好ましい。かかるコアセルベート粒子としては、ゼラチンアラビアゴムコアセルベートが好ましい。また、前記型を判定すべき赤血球を磁性標識する工程において、0.18〜0.69％の濃度で前記磁性粒子を含む磁性粒子含有溶液と、0.5〜1.2％の濃度で前記型を判定すべき赤血球を含む赤血球含有溶液とが、等しい量で混合されることが好ましい。或いは、前記型を判定すべき赤血球を磁性標識する工程において、0.18〜1.8％の濃度で前記磁性粒子を含む磁性粒子含有溶液と、0.7〜0.9％の濃度で前記型を判定すべき赤血球を含む赤血球含有溶液とが等しい量で混合されることが好ましい。
The ligand is preferably a ligand that non-specifically binds to erythrocytes and aggregates the erythrocytes. The magnetic substance-encapsulating carrier is preferably a coacervate in which a magnetic substance is encapsulated. As such coacervate particles , gelatin gum arabic coacervate is preferable. Further, in the step of magnetically labeling the red blood cells whose types are to be determined, a magnetic particle-containing solution containing the magnetic particles at a concentration of 0.18 to 0.69% and red blood cells whose types are to be determined at a concentration of 0.5 to 1.2% are included. It is preferable that the erythrocyte-containing solution is mixed in an equal amount. Alternatively, in the step of magnetically labeling the red blood cells whose types are to be determined, a magnetic particle-containing solution containing the magnetic particles at a concentration of 0.18 to 1.8% and red blood cells whose types are to be determined at a concentration of 0.7 to 0.9% are included. It is preferable that the erythrocyte-containing solution is mixed in an equal amount.
本発明に従えば、赤血球を磁性標識化することによって、磁力により赤血球の沈降速度を速めることができる。よって、短時間で反応像を形成させることができ、判定時間を短縮することができる。 According to the present invention, the sedimentation rate of red blood cells can be increased by magnetic force by magnetic labeling of red blood cells. Therefore, a reaction image can be formed in a short time, and the determination time can be shortened.
本発明では、赤血球を磁性粒子でコーティングしたり赤血球中に磁性粒子を封入したりすることなく、赤血球と磁性粒子との結合体（凝集塊）を結合反応によって形成することにより、赤血球を直接加工する必要が無く、赤血球の磁性体化を簡便に行うことができる。本発明では、赤血球を磁性標識化するのに適した磁性粒子を用いることによって、赤血球の沈降速度を速めることを可能にした。 In the present invention, erythrocytes are directly processed by forming a conjugate (aggregate) of erythrocytes and magnetic particles by a binding reaction without coating erythrocytes with magnetic particles or encapsulating magnetic particles in erythrocytes. Therefore, it is possible to easily convert the red blood cells into a magnetic substance. In the present invention, it is possible to increase the sedimentation rate of red blood cells by using magnetic particles suitable for magnetic labeling of red blood cells.
磁性粒子として、ナノサイズの粒子を使用する方法は、医用磁性ナノビーズの新しい作成技術（応用物理74巻12；1580-2005）でも述べられている。小さな抗原や抗体を結合させる場合は、比表面積が大きいナノ粒子が好ましいが、赤血球のような大きい（８μｍ）粒子を磁力で迅速に動かすには、小さな粒子では、その表面を全て覆うほどに標識しなければならない可能性もあり、赤血球の抗原がマスクされてしまう恐れがある。 The method of using nano-sized particles as magnetic particles is also described in a new technique for producing medical magnetic nanobeads (Applied Physics 74, 12; 1580-2005). When binding small antigens and antibodies, nanoparticles with a large specific surface area are preferred. However, to quickly move large (8 μm) particles such as erythrocytes by magnetic force, small particles are labeled to cover the entire surface. There is a possibility that the antigen of red blood cells may be masked.
そこで、本発明では、本発明者らが開発した赤血球用の磁性粒子を用いる。該磁性粒子は、ゼラチンアラビアゴムコアセルベートを担体として用い、これに磁性体を封入し、磁性体封入ゼラチンアラビアゴムコアセルベートとした。これにさらに赤血球と結合する赤血球結合物質を固定化して磁性粒子として用いた。ゼラチンアラビアゴムコアセルベートは、ゼラチンと水溶性多糖類であるアラビアゴムを主成分として形成された複合コアセルベートである。これは例えば、「HG Bundenberg de Jong. In Colloid Science 1949 Vol.2, Amsterdam」によって紹介されている。ゼラチンアラビアゴムコアセルベートは天然成分に由来するため容易に分解され、環境に優しいという利点がある。また免疫学的な非特異反応が少ないという利点もある。さらに、簡単な設備があれば容易に安価で製造できるため好適に用いることができる。さらに、ゼラチンアラビアゴムコアセルベートは、任意の粒径を有するように調製することができるため、赤血球に適するように粒径を大きくすることができる。本発明で用いるゼラチンアラビアゴムコアセルベートは、赤血球を標識するために用いるので、その粒径が1〜20μｍであることが好ましく、2〜10μｍであることがより好ましく、3〜6μｍであることが最も好ましい。ゼラチンアラビアゴムコアセルベートの製造方法は、公知であり、例えば、特開2004-157040号公報を参照することができる。 Therefore, in the present invention, magnetic particles for erythrocytes developed by the present inventors are used. For the magnetic particles, gelatin gum arabic coacervate was used as a carrier, and a magnetic material was encapsulated therein to form a magnetic material encapsulated gelatin gum arabic coacervate. Further, an erythrocyte-binding substance that binds to erythrocytes was immobilized and used as magnetic particles. Gelatin gum arabic coacervate is a complex coacervate formed from gelatin and gum arabic, which is a water-soluble polysaccharide, as main components. This is introduced, for example, by “HG Bundenberg de Jong. In Colloid Science 1949 Vol. 2, Amsterdam”. Gelatin gum arabic coacervate has the advantage of being easily decomposed and environmentally friendly because it is derived from natural ingredients. There is also an advantage that there are few immunological non-specific reactions. Furthermore, since simple equipment can be easily manufactured at low cost, it can be suitably used. Furthermore, since gelatin gum arabic coacervate can be prepared to have an arbitrary particle size, the particle size can be increased to suit red blood cells. Since the gelatin gum arabic coacervate used in the present invention is used for labeling red blood cells, the particle size is preferably 1 to 20 μm, more preferably 2 to 10 μm, most preferably 3 to 6 μm. preferable. A method for producing gelatin gum arabic coacervate is known, and for example, JP-A-2004-157040 can be referred to.
ゼラチンアラビアゴムコアセルベートに封入される磁性体は、一個でも良いが複数個であってもよい。ゼラチンアラビアゴムコアセルベートは、粒子内に封入される磁性体の数を容易に調整することができる。従って、この磁性粒子で標識された赤血球の移動速度を磁力によって調整するために、封入される磁性体の数を増減させることも可能である。 The magnetic substance enclosed in the gelatin gum arabic coacervate may be one or more. Gelatin gum arabic coacervate can easily adjust the number of magnetic substances encapsulated in the particles. Therefore, in order to adjust the moving speed of the red blood cells labeled with the magnetic particles by magnetic force, it is possible to increase or decrease the number of magnetic bodies to be enclosed.
ここで述べたように、ゼラチンアラビアゴムコアセルベートは赤血球標識用の磁性粒子に都合よく用いることができるが、現在までにこれを使用して赤血球を磁性標識化した例は報告されていない。 As described herein, gelatin gum arabic coacervate can be conveniently used for magnetic particles for red blood cell labeling, but no examples of magnetic labeling of red blood cells using it have been reported so far.
上記のように製造された磁性体封入ゼラチンアラビアゴムコアセルベートには、赤血球結合物質を担持させる。ここで赤血球結合物質は、赤血球と抗原抗体反応をし、赤血球を凝集させるリガンドであることが好ましい。 The magnetic substance-encapsulated gelatin gum arabic coacervate produced as described above carries an erythrocyte-binding substance. Here, the red blood cell binding substance is preferably a ligand that causes an antigen-antibody reaction with red blood cells to aggregate the red blood cells.
赤血球を凝集させるリガンドは種々のものが知られており、赤血球を非特異的に凝集させるレクチンも数多く市販化されている（例えば、豊年コーポレーションのカタログを参照されたい）。例えば、ウサギ抗グリコフォリン抗体、ニシダレクチン（ＣＳＡ）及びチョウセンアサガオレクチン（ＤＳＡ）などのリガンドが好適に用いられる。
Various ligands for agglutinating erythrocytes are known, and many lectins that agglutinate erythrocytes non - specifically are commercially available (see, for example, the catalog of Toyotomi Corporation). For example, ligands such as rabbit anti-glycophorin antibody, Nishida lectin (CSA) and Datura morning glory lectin (DSA) are preferably used.
リガンドは既知の方法によって磁性体封入ゼラチンアラビアゴムコアセルベートに担持させることができる。その方法は、例えば、特開2004-157040号公報に記載されたＥＤＡＣ／ＮＨＳ法であってよいが、これに限定されない。 The ligand can be supported on the magnetic substance-encapsulated gelatin gum arabic coacervate by a known method. The method may be, for example, the EDAC / NHS method described in JP-A-2004-157040, but is not limited thereto.
本発明では、上記リガンドが結合した磁性体封入ゼラチンアラビアゴムコアセルベートを磁性粒子とし、赤血球の型判定に用いる。以下に、本発明の方法の実施態様を説明する。 In the present invention, the magnetic substance-encapsulated gelatin gum arabic coacervate to which the ligand is bound is used as a magnetic particle and used for erythrocyte type determination. In the following, embodiments of the method of the present invention will be described.
第一の実施態様では、固相担体に抗赤血球抗体を予め固相化しておく。例えば反応容器の内壁に固相化してもよい。次いで、反応容器内に検体である赤血球と、磁性粒子を投入し、容器内で反応させる。これを容器の外側から磁石で吸引することによって迅速に沈降させ、予め固相化されていた抗体によって捕捉させる。陽性の場合は、図１（ａ）に示したように容器中に広がった像が生じる。一方、陰性の場合、赤血球は予め固相化されていた抗体によって捕捉されず、図１（ｂ）に示したように容器底面に沈降した像を生じる。
In the first embodiment, an anti-erythrocyte antibody is immobilized on a solid phase carrier in advance. For example, it may be solid-phased on the inner wall of the reaction vessel. Next, red blood cells, which are specimens, and magnetic particles are put into a reaction container, and reacted in the container . Rapidly precipitated by sucking Re this from the outside of the container with a magnet, it is captured by the antibody which had been previously immobilized. In the case of positive, an image spread in the container is generated as shown in FIG. On the other hand, in the case of negative, the red blood cells are not captured by the previously immobilized antibody, and an image that settles on the bottom surface of the container is generated as shown in FIG.
第二の実施態様では、上記第一の実施態様と同様に抗赤血球抗体を固相化した反応容器に、赤血球、磁性粒子と共に、さらに抗赤血球抗体を同時に又は直後に投入する。これらを反応容器中で混合して反応させ、磁石によって吸引して反応を互いに凝集塊に結びつけるために用いる。この抗体には、誤った反応を防ぐために容器に固相化した抗体と同一の抗体を用いてもよく、非同一の抗体であっても特異性が同じ抗体であれば用いることができる。抗赤血球抗体を追加的に投入することにより、抗体価や赤血球量が患者ごとにばらついても、余すことなく特異的な反応を達成できる。
第三の実施態様では、反応容器とは別の容器において、赤血球と磁性粒子を予め反応させておく。次いで、上記第一の実施態様と同様に抗赤血球抗体を固相化した反応容器にこれを投入し、磁石によって吸引して反応像を形成させる。上記と同様に、陽性であれば凝集塊が固相化された抗体と結合して広がった陽性像を生じる。一方、陰性の場合は、赤血球は予め固相化されていた抗体によって捕捉されず、容器底面に沈降した像を生じる。予め反応させることによって、反応容器上の抗体と反応する前に十分な凝集量を得ることができる。
In the second embodiment, in the same manner as in the first embodiment, together with the red blood cells and magnetic particles, the anti-erythrocyte antibody is added simultaneously or immediately after to the reaction vessel in which the anti-erythrocyte antibody is immobilized. These are mixed and reacted in a reaction vessel, and are used to bind the reactions to agglomerates by suction with a magnet. For this antibody, the same antibody as the antibody immobilized on the container may be used in order to prevent an erroneous reaction, or a non-identical antibody can be used as long as it has the same specificity. By additionally introducing an anti-erythrocyte antibody, even if the antibody titer and the amount of erythrocytes vary from patient to patient, a specific reaction can be achieved without leaving a surplus .
In the third embodiment, red blood cells and magnetic particles are reacted in advance in a container different from the reaction container. Next, in the same manner as in the first embodiment, the anti-erythrocyte antibody is put into a reaction vessel on which a solid phase is formed, and is attracted by a magnet to form a reaction image. Similarly to the above, if positive, the aggregate is bound to the solid-phased antibody to produce a positive image that spreads. On the other hand, in the case of negative, erythrocytes are not captured by the previously immobilized antibody, and an image that settles on the bottom surface of the container is generated. By reacting in advance, a sufficient amount of aggregation can be obtained before reacting with the antibody on the reaction vessel.
以上に説明したように、本発明の方法に従って、赤血球を磁性粒子と結合させることで磁性標識することによって、実質的に自重が増加するだけでなく磁力により強引に移動させることができ、高い反応性を保ちながら赤血球の凝集塊の沈降速度を速めることができる。しかも、赤血球自身を磁性体で加工しないので、表面抗原の反応性が高いまま、高速で、例えばわずか３分間で反応像を判定することができる。凝集塊の自然沈降では、通常30〜60分要するため、本発明の方法によって大幅に判定時間を短縮することができる。また、容器などの固相担体の表面に固定化された抗体により凝集塊を捕捉するので、陽性、陰性の判定が容易である。 As explained above, according to the method of the present invention, by labeling red blood cells with magnetic particles and magnetically labeling, not only the self-weight increases but also it can be forcibly moved by magnetic force, resulting in high reaction. The sedimentation rate of the erythrocyte aggregate can be increased while maintaining the property. Moreover, since the erythrocytes themselves are not processed with a magnetic material, the reaction image can be determined at a high speed, for example, in only 3 minutes while the reactivity of the surface antigen remains high. The natural sedimentation of the agglomerates usually takes 30 to 60 minutes, so the determination time can be greatly shortened by the method of the present invention. In addition, since the aggregate is captured by an antibody immobilized on the surface of a solid phase carrier such as a container, it is easy to make a positive or negative determination.
さらに、他の態様において、赤血球を磁性標識化することによって、Ｂ／Ｆ分離が容易になる。そのため蛍光検出などの測定方法を適用することもできる。例えば、磁性標識化赤血球を抗体と反応させて凝集させ、凝集塊を磁石によって反応容器の壁面（底面や側面）に磁気的に捕捉し、未反応抗体を含む液をノズル等で吸引除去する。次いで、蛍光や化学発光などの光マーカーによって標識した抗体を添加して反応させ、再び洗浄した後検出することもできる。 Furthermore, in other embodiments, B / F separation is facilitated by magnetic labeling of red blood cells. Therefore, a measurement method such as fluorescence detection can be applied. For example, magnetically labeled erythrocytes are allowed to react with antibodies to aggregate them, the aggregates are magnetically captured on the wall surface (bottom surface or side surface) of the reaction vessel by a magnet, and the liquid containing unreacted antibodies is sucked and removed with a nozzle or the like. Subsequently, an antibody labeled with a light marker such as fluorescence or chemiluminescence can be added and reacted, washed again, and then detected.
以下、本発明の各実施態様の実施例を記載する。 Examples of each embodiment of the present invention will be described below.
１．磁性体封入ゼラチンアラビアゴムコアセルベートの作成
特開2002-324015号公報、特開2003-291402号公報に記載された方法に従って、ゼラチンとアラビアゴムのコアセルベートを作成し、磁性体を取り込ませ、粒径2-12μｍの磁性体封入ゼラチンアラビアゴムコアセルベートを製造した。
1. Preparation of magnetic substance-encapsulated gelatin gum arabic coacervate According to the method described in JP-A-2002-324015, JP-A-2003-291402, gelatin and gum arabic coacervate were prepared, the magnetic substance was incorporated, and the particle size 2 A gelatin encapsulated gum arabic coacervate with -12 μm magnetic material was produced.
２．磁性体封入ゼラチンアラビアゴムコアセルベートへのリガンドの結合
特開2002-324015号公報に記載された方法に従って、上記磁性体封入ゼラチンアラビアゴムコアセルベートに、赤血球に対するリガンドであるチョウセンアサガオレクチン（ＤＳＡ）を結合させた。ＤＳＡの濃度は10μｈ／ｍｌとした。結合反応後、ＢＳＡ（ウシアルブミン）でブロッキングを行い、0.1％ BSA/PBS ｐＨ7.2に、５％濃度で懸濁した。
2. Ligand binding to magnetic substance-encapsulated gelatin gum arabic coacervate According to the method described in Japanese Patent Application Laid-Open No. 2002-324015, the magnetic substance-encapsulated gelatin gum arabic coacervate is bound to erythrocyte ligand Datura agaagactin (DSA). It was. The concentration of DSA was 10 μh / ml. After the binding reaction, it was blocked with BSA (bovine albumin) and suspended in 0.1% BSA / PBS pH 7.2 at a concentration of 5%.
３．抗Ｂ抗体のマイクロプレートへの固相化
市販の抗血清には、ＢＳＡ等共存タンパクが存在するので、そのままでは使用できない。そこで、プロテインＡカラム（BioRad製）を使用して、Ortho社のバイオクローン抗Ｂから抗Ｂモノクローナル抗体を単離した。この抗体をNuncMaxisorp Ｕ底プレートにＰＢＳバッファーを用いて固相化した。その後、ＢＳＡでブロッキングを行った。
3. Immobilization of anti-B antibody on microplate Commercially available antiserum contains coexisting proteins such as BSA and cannot be used as it is. Therefore, an anti-B monoclonal antibody was isolated from the Bioclone anti-B of Ortho using a protein A column (manufactured by BioRad). This antibody was immobilized on a NuncMaxisorp U bottom plate using PBS buffer. Thereafter, blocking was performed with BSA.
４．血液型判定
（１）Ｂ型ドナー血球（赤血球）をＰＢＳで0.1〜1.2％に希釈した。また、ＤＳＡ感作磁性体封入ゼラチンアラビアゴムコアセルベート（以下ＤＳＡ粒子と称する）を0.18〜2.75％に希釈した。これらを各25μＬずつマイクロプレートに分注し撹拌した。その後、磁石で３分間吸引した。同様にＡ型ドナー血球（赤血球）についても実施した。
4). Blood type determination (1) Type B donor blood cells (erythrocytes) were diluted to 0.1-1.2% with PBS. Further, DSA-sensitized magnetic substance-encapsulated gelatin gum arabic coacervate (hereinafter referred to as DSA particles) was diluted to 0.18 to 2.75%. 25 μL of each was dispensed into a microplate and stirred. Then, it attracted | sucked with the magnet for 3 minutes. Similarly, it was carried out for type A donor blood cells (erythrocytes).
その結果を図２に示した。Ｂ型赤血球はＤＳＡ粒子と凝集塊を生じ、マイクロプレート内面に固相した抗Ｂ抗体に捕捉されて広がった陽性像を示した。磁石で吸引したために、３分間で判定が可能であった。Ａ型血球は底面に沈降した陰性像を呈し、抗体によって捕捉されなかったことを示した。 The results are shown in FIG. Type B erythrocytes produced DSA particles and aggregates, and showed a positive image that was captured and spread by anti-B antibody solid-phased on the inner surface of the microplate. Since it was attracted by a magnet, it could be judged in 3 minutes. Type A blood cells showed a negative image that settled on the bottom surface, indicating that they were not captured by the antibody.
表１に、血球濃度と磁性粒子の濃度を変化させた結果を示した。表１に示されたように、反応像を形成させるためには、血球濃度と磁性粒子濃度の割合が重要であることがわかった。赤血球の濃度が0.5〜1.2％であり、磁性粒子の濃度が0.18〜0.69％である割合で反応に供することが好ましい。また、赤血球濃度が0.7〜0.9％であり、磁性粒子濃度が0.18〜1.8％、好ましくは0.34〜1.8％の範囲の割合で用いることが好ましい。
（２）Ｂ型ドナー血球をＰＢＳで0.8％に希釈した。また、ＤＳＡ粒子を0.6％に希釈した。これらを各25μＬずつマイクロプレートに分注し、同時に、抗Ｂ抗体を添加した。撹拌後、磁石で３分間吸引した。同様にドナーＡ血球についても実施した。 (2) Type B donor blood cells were diluted to 0.8% with PBS. In addition, DSA particles were diluted to 0.6%. 25 μL of each was dispensed into a microplate, and anti-B antibody was added simultaneously. After stirring, it was sucked with a magnet for 3 minutes. The same procedure was performed for donor A blood cells.
その結果を表１に示した。Ｂ型赤血球はＤＳＡ粒子と凝集塊を生じ、さらに抗Ｂ抗体と凝集を生じ、凝集塊は磁石で吸引することで沈降し、マイクロプレート内面に固相化した抗Ｂ抗体に捕捉されて広がった陽性像を示した。一方、Ａ型赤血球では底面に沈降した陰性像を呈し、捕捉されなかったことを示した。
The results are shown in Table 1 . Type B erythrocytes produce DSA particles and aggregates, and further aggregate with anti-B antibodies. The aggregates are settled by suction with a magnet, and are captured and spread by anti-B antibodies immobilized on the inner surface of the microplate. A positive image was shown. On the other hand, exhibits a negative image of sediment on the bottom in the type A red blood cells, it showed that not captured.
（３）市販の抗Ｂ抗抗体とＡ型或いはＢ型ドナー血球（0.7％）を予め別容器で５分間混合した。該混合物をＤＳＡ粒子（0.69％）と共に抗Ｂ抗体を固相化したマイクロプレートウェルに移して、磁石で３分間吸引した。 (3) A commercially available anti-B anti-antibody and type A or type B donor blood cells (0.7%) were mixed in a separate container in advance for 5 minutes. The mixture was transferred to a microplate well on which anti-B antibody was immobilized together with DSA particles (0.69%), and aspirated with a magnet for 3 minutes.
その結果、Ｂ型ドナー血球では、広がった陽性像を形成せずに崩れてしまった。一方、Ａ型ドナー血球は底面に集まった。結果として、陽性／陰性像の判定はできなかった。これは、凝集塊に磁性粒子が結合できなかったためか、或いは磁性粒子が結合して赤血球の結合サイトが埋まり、固相化抗体と反応できなかったためかと考えられる。 As a result, type B donor blood cells collapsed without forming a widened positive image. On the other hand, type A donor blood cells gathered on the bottom surface. As a result, a positive / negative image could not be determined. This is probably because the magnetic particles could not bind to the aggregate, or because the binding sites of the erythrocytes were buried due to the binding of the magnetic particles and could not react with the immobilized antibody.
（４）Ａ型或いはＢ型ドナー血球（0.7％）とＤＳＡ粒子（0.69％）を予め別容器で５分間混合し、その混合物を抗Ｂ抗体を固相化したマイクロプレートウェルに移して、磁石で３分間吸引した。 (4) A or B donor blood cells (0.7%) and DSA particles (0.69%) are mixed in advance in a separate container for 5 minutes, and the mixture is transferred to a microplate well on which an anti-B antibody is immobilized, and a magnet Aspirate for 3 minutes.
その結果、Ｂ型赤血球はウェルに広がり陽性像を形成した。Ａ型赤血球は底面に沈降し陰性像を形成した。よって、陽性陰性が正しく判定できた。 As a result, type B erythrocytes spread into the well and formed a positive image. Type A red blood cells settled on the bottom surface to form a negative image. Therefore, positive / negative was correctly determined.
（５）Ａ型或いはＢ型ドナー血球（0.7％）とＤＳＡ粒子（0.69％）を予め別容器で５分間混合し、その混合物を、抗Ｂ血清（抗赤血球抗体）25μＬと共に、抗Ｂ抗体（抗赤血球抗体）を固相化したマイクロプレートウェルに移して、磁石で３分間吸引した。 (5) A or B donor blood cells (0.7%) and DSA particles (0.69%) are mixed in advance in a separate container for 5 minutes, and the mixture is mixed with 25 μL of anti-B serum (anti-erythrocyte antibody) together with anti-B antibody ( (Anti-erythrocyte antibody) was transferred to a solid-phased microplate well and aspirated with a magnet for 3 minutes.
その結果、 Ｂ型赤血球はウェルに広がり陽性像を形成した。Ａ型赤血球は底面に沈降し陰性像を形成した。よって、陽性陰性が正しく判定できた。 As a result, type B erythrocytes spread into the well and formed a positive image. Type A red blood cells settled on the bottom surface to form a negative image. Therefore, positive / negative was correctly determined.
Claims (8)
磁性標識された前記型を判定すべき赤血球を磁力によって沈降させて反応像を形成させる工程とを含み、前記反応像が、前記型を判定すべき赤血球と前記抗赤血球抗体とが結合した場合は陽性像であり、前記型を判定すべき赤血球と前記抗赤血球抗体とが結合しなかった場合は陰性像である赤血球の型判定方法。 A carrier to which the magnetic material is encapsulated, is immobilized on the support, and a ligand that bind nonspecifically to erythrocytes, the magnetic particles that do not bind to the anti-red blood cell antibody, and a red blood cell to be determined the type, the A step of magnetically labeling the red blood cells to be judged of the type by mixing them in a reaction vessel in which an anti-erythrocyte antibody is immobilized and binding the ligand in the magnetic particles to the red blood cells to be judged of the type. When,
A step of forming a reaction image by magnetically precipitating erythrocytes to be magnetically labeled for the type, and the reaction image is a combination of the red blood cells to be determined for the type and the anti-red blood cell antibody. A method for determining the type of erythrocytes, which is a positive image and is a negative image when the erythrocyte whose type is to be determined does not bind to the anti-erythrocyte antibody .
磁性標識された前記型を判定すべき赤血球を、前記抗赤血球抗体が固相化された反応容器に加える工程と、
前記磁性標識された型を判定すべき赤血球を磁力によって沈降させて反応像を形成させる工程とを含み、前記反応像が、前記型を判定すべき赤血球と前記抗赤血球抗体とが結合した場合は陽性像であり、前記型を判定すべき赤血球と前記抗赤血球抗体とが結合しなかった場合は陰性像である赤血球の型判定方法。 A magnetic particle- containing carrier, a ligand immobilized on the carrier and non-specifically bound to erythrocytes, and magnetic particles that do not bind to anti-erythrocyte antibodies are mixed with erythrocytes whose type should be determined. Magnetically labeling the red blood cells to be judged of the type by binding the ligand in the magnetic particles to the red blood cells to be judged of the type;
Adding the erythrocytes to be magnetically labeled for the type to a reaction vessel on which the anti-erythrocyte antibody is immobilized;
A step of precipitating the erythrocytes to be magnetically labeled with magnetic force to form a reaction image, and the reaction image is a combination of the erythrocytes to be determined for the type and the anti-erythrocyte antibody. A method for determining the type of erythrocytes, which is a positive image and is a negative image when the erythrocyte whose type is to be determined does not bind to the anti-erythrocyte antibody .
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