JP2009002686A - Kind determination method for erythrocyte - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for simply performing kind determination on erythrocytes in a shorter time. <P>SOLUTION: This method for determining the kind of erythrocytes is characterized in that erythrocytes are reacted with magnetic particles for turning the erythrocytes into magnetic labels in a reaction vessel with anti-erythrocyte antibodies solidified therein to sediment them by magnetic force, thereby forming a reaction image. In one mode thereof, the erythrocytes, the magnetic particles, and further, the anti-erythrocyte antibodies are reacted with each other in the reaction vessel. As a result, the erythrocytes are turned into the magnetic labels, making it possible to accelerate the sedimentation velocity of the erythrocytes by the magnetic force. Consequently, the reaction image can be formed in a short time, making it possible to shorten determination time. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an erythrocyte type determination method.

  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, Patent Documents 1 and 2).

  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.

  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.

Furthermore, since erythrocytes are natural components, when artificially processed, the reactivity of antigens on the erythrocyte surface is remarkably reduced or inactivated easily. For this reason, it is very difficult to modify the sedimentation characteristics by directly processing erythrocytes.
Japanese Patent Publication No.8-7215 EP 725276 Japanese Patent Publication No.61-44268 Japanese Patent Publication No. 63-60854

  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.

  In order to achieve the above object, a method for determining the type of erythrocytes, in which a reaction vessel in which an anti-erythrocyte antibody is immobilized is reacted with erythrocytes and magnetic particles for magnetic labeling of erythrocytes, and precipitated by magnetic force. To form a reaction image. In one embodiment, the red blood cells, magnetic particles, and anti-red blood cell antibodies are reacted in the reaction vessel.

  In another aspect, a method for determining erythrocyte type, wherein erythrocytes and magnetic particles for magnetically labeling erythrocytes are reacted in advance in a reaction solution, and the reaction solution is solidified with an anti-erythrocyte antibody. A method is provided which is transferred into a reaction vessel and sedimented by magnetic force to form a reaction image.

  The magnetic particles for magnetically labeling erythrocytes preferably have a ligand for agglutinating erythrocytes on the surface, and in particular, coacervate particles to which an anti-erythrocyte antibody is bound are preferred. Such coacervate particles are preferably magnetic substance-encapsulated gelatin gum arabic coacervates. Further, it is preferable that the red blood cells have a concentration of 0.5 to 1.2% and the magnetic particles have a concentration of 0.18 to 0.69%. Furthermore, it is preferable that the red blood cells have a concentration of 0.7 to 0.9% and the magnetic particles have a concentration of 0.18 to 1.8%.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  Various ligands for agglutinating erythrocytes are known, and many lectins that agglutinate erythrocytes non-specifically or 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.

  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.

  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.

  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. If the reaction is positive, red blood cells bind to the magnetic particles and aggregate. This is quickly settled by sucking with a magnet from the outside of the container, and captured by the antibody that has been immobilized in advance. Therefore, 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 aggregated, and as shown in FIG.

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. Form an image. Two or more red blood cells not bound to magnetic particles are added to the antibody. 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 do not aggregate and an image that settles on the bottom 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.

  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.

  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.

  Examples of each embodiment of the present invention will be described below.

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 a magnetic material of -12 μm was produced.

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 above-mentioned magnetic substance-encapsulated gelatin gum arabic coacervate is bound to erythrocyte ligand, datura asagarectin (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. 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). 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).

  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.

Table 1 shows the results of changing the blood cell concentration and the magnetic particle concentration. As shown in Table 1, it was found that the ratio between the blood cell concentration and the magnetic particle concentration is important for forming a reaction image. It is preferable that the red blood cell concentration is 0.5 to 1.2% and the magnetic particle concentration is 0.18 to 0.69%. The red blood cell concentration is 0.7 to 0.9%, and the magnetic particle concentration is preferably 0.18 to 1.8%, preferably 0.34 to 1.8%.

  (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. Similarly, it was performed on donor A blood cells.

  The results are shown in FIG. B erythrocytes produce DSA particles and aggregates, and further aggregate with anti-B antibodies. The aggregates settle by suction with a magnet, and are captured by the anti-B antibody immobilized on the inner surface of the microplate and spread. I showed the statue. On the other hand, A blood cells exhibited a negative image that settled on the bottom surface, indicating that they were not captured.

  (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.

  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) Type A or type 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. Aspirate for 3 minutes.

  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 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.

  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.

The schematic diagram which shows the mode of sedimentation of an aggregate.

Claims (8)

  A method for determining the type of erythrocytes, wherein the erythrocytes are magnetically labeled by reacting the erythrocytes with magnetic particles that do not bind to the anti-erythrocyte antibodies in a reaction vessel in which an anti-erythrocyte antibody is immobilized. And the magnetically labeled red blood cells are precipitated by magnetic force to form a reaction image.   The method according to claim 1, wherein the red blood cells, magnetic particles, and anti-red blood cell antibodies are reacted in the reaction vessel.   A method for determining the type of erythrocytes, wherein erythrocytes and magnetic particles that do not bind to the anti-erythrocyte antibodies are reacted in advance in a reaction solution to magnetically label the erythrocytes. A method characterized in that it is transferred into a phased reaction vessel and precipitated by magnetic force to form a reaction image.   The method according to any one of claims 1 to 3, wherein the magnetic particles for magnetically labeling red blood cells have a ligand for aggregating red blood cells on the surface.   The method according to claim 4, wherein the magnetic particles for magnetically labeling red blood cells are magnetic substance-encapsulated coacervates to which anti-erythrocyte antibodies are bound.   6. The method of claim 5, wherein the coacervate is a gelatin gum arabic coacervate.   The method according to any one of claims 1 to 6, wherein the red blood cells have a concentration of 0.5 to 1.2% and the magnetic particles have a concentration of 0.18 to 0.69%.   The method according to claim 1, wherein the red blood cells have a concentration of 0.7 to 0.9% and the magnetic particles have a concentration of 0.18 to 1.8%.

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