JP2009002685A - Magnetic particle for labeling erythrocyte - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide magnetic particles for turning erythrocytes into magnetic labels. <P>SOLUTION: Magnetic particles for labeling erythrocytes are provided wherein an erythrocyte bonding substance is carried by a carrier containing magnetic bodies. In one mode thereof, the carrier containing the magnetic bodies is gelatin gum-arabic coacervate with the magnetic bodies sealed therein and is selected from among a group comprising an erythrocyte-bonding substance anti-glycophorin antibody, broom lectin (CSA), and thorn apple lectin (DSA). <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a magnetic particle for magnetically labeling red blood cells.

  In the field of immunoassay, a method of binding an antigen or antibody to a magnetic substance and capturing and measuring the target antigen or antibody in a specimen is widely known. Also known is a method of sorting cells and separating B / F by binding various antibodies to a magnetic material and separating them with a magnet (for example, Patent Document 1).

  Therefore, if the erythrocytes can be magnetically labeled, a sedimentation pattern in the erythrocyte agglutination reaction can be rapidly formed by a magnetic force, or a secondary labeled antibody can be used to develop an antibody bound to the erythrocytes. The operation in the immunological test can be made simple and rapid.

However, there is no known method for magnetically labeling red blood cells and using them for immunological examinations. Only DiaGast is magnetically labeled with nanomagnetic particles, but the particles used are not disclosed and the technical content is completely unknown.
JP 2001-91521 A

  Accordingly, an object of the present invention is to provide magnetic particles for magnetically labeling red blood cells.

  According to the present invention, there is provided a magnetic particle for red blood cell labeling characterized in that a red blood cell binding substance is supported on a carrier containing a magnetic substance. In one embodiment, the carrier containing the magnetic material is gelatin gum arabic coacervate in which the magnetic material is encapsulated. The red blood cell binding substance is preferably a red blood cell ligand. In addition, the ligand is preferably selected from the group consisting of an anti-glycophorin antibody, any lectin lectin (CSA) and datura saga orectin (DSA).

  Red blood cells can be magnetically labeled by using the magnetic particles of the present invention. Thereby, operation in an immunoassay can be made simple and rapid.

  Polystyrene particles or the like are used as carriers for general magnetic particles used in immunological tests and the like. In addition, a carrier using a new production technique for medical magnetic nanobeads (see Applied Physics 74, 12; 1580-2005) is also used. Such nano-sized magnetic particle carriers are preferred for binding to small antigens and antibodies. However, in order to move large particles such as erythrocytes (about 8 μm) rapidly by magnetic force, small nanoparticle carriers may have to be bound to cover the entire surface of erythrocytes. There is a risk of being masked.

  Therefore, in the present invention, gelatin gum arabic coacervate was used as a carrier, and a magnetic material was encapsulated therein to be 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.

  Gelatin gum arabic coacervate can easily adjust the amount of magnetic substance enclosed in the particles. Therefore, in order to adjust the moving speed of the red blood cells labeled with the magnetic particles by the magnetic force, the amount of the magnetic substance to be encapsulated can be increased or decreased.

  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.

  Gelatin gum arabic coacervate is manufactured by known methods and can be manufactured to contain the desired amount of magnetic particles. The manufactured magnetic particles carry 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.

  Hereinafter, a method for producing a magnetic substance-encapsulated gelatin gum arabic coacervate will be described. For further details, refer to JP-A-2004-157040. First, at a temperature equal to or higher than the gelling temperature of gelatin (preferably 35 ° C. or higher, for example, about 40 ° C.), 0.01 to 2% by weight of gelatin and 0.01 to 2% by weight of gum arabic, G / A = 0.5 to 1.5, It mixes in 29-65weight% of a water-soluble organic solvent so that it may become 0.5-1.1, More preferably, it is 0.5-1.0, More preferably, it is 0.6-0.9. Here, methanol, ethanol, propanol, acetone or the like can be used as the water-soluble organic solvent, but ethanol is desirable in view of toxicity. On the other hand, when G / A exceeds 1.6, it is difficult to adjust the coacervate diameter. As is known in the art, a surfactant is preferably added to the coacervate preparation solution in order to prevent coacervate particles from aggregating. The type and addition amount of the surfactant can be appropriately set by those skilled in the art within the range where the effect is exhibited.

  The gelatin gum arabic coacervate is then precipitated by the addition of an acid (eg acetic acid, propionic acid, dilute hydrochloric acid, dilute sulfuric acid). Here, the addition amount of the acid is appropriately set according to the coacervate diameter to be produced. Thereafter, it is cooled to a gelation temperature or lower (preferably 35 ° C. or lower, for example, about 10 ° C.), and crosslinked with an aldehyde such as glutaraldehyde or formalin.

Further, in the production of a carrier having a coacervate form, a so-called microcapsule in which a core substance is added and the core substance is wrapped with coacervate may be used. By using a desired core material, it is possible to adjust the specific gravity of the coacervate, to magnetize the coacervate, and to color the coacervate. In that case, it is desirable to add a suitable surfactant (for example, Tween 20, Tween 80, Triton X-100, etc.) to increase the dispersibility. Examples of the core material, SiO _{2 (glass)} can be utilized fine powder of TiO 2, CuO, CoO, metal oxides such as _Fe 2 _{O 3,} carbon, any of various talc. The carrier containing the magnetic material can be subjected to various processing steps such as stirring, washing, and measurement in a liquid in a short time by appropriate magnetism generating means (permanent magnet, electromagnet, etc.). A person skilled in the art can appropriately select the type of the core substance and the amount of the core substance used to make the carrier have the desired properties.

  Since the gelatin gum arabic coacervate of the present invention is used for labeling red blood cells, its particle size is preferably 0.1 to 200 μm, more preferably 1 to 30 μm, most preferably 2 to 12 μm. .

  Next, a known method can be used to support the ligand on the magnetic particles. For example, the EDAC / NHS method described in JP 2004-157040 A can be used. The outline will be briefly described.

  The coacervate is crosslinked with aldehyde, washed with pure water, dyed as necessary, and a known N-hydroxysuccinimide method is applied. First, the previously crosslinked coacervate is suspended in an aqueous solution (MES (2-Morpholinoethanesulfonic acid solution)) in which N-hydroxysuccinimide and carbodiimide are dissolved, and reacted at room temperature for 1 to 3 hours. After the reaction, the coacervate is washed by centrifugation, and the ligand of interest is dissolved in an appropriate buffer (for example, pure water, acetate buffer, MES buffer) at an appropriate concentration, and is added at room temperature or refrigerated (2 to 8 ° C.). React overnight from time. After the reaction, BSA (bovine serum albumin), gelatin, animal serum, etc. are used to block unreacted functional groups (blocking) to produce a coacervate carrying a desired ligand. The prepared coacervate may be used as it is for the intended reaction by substituting the solution for the desired immunological reaction, or it may be dispensed into vials and freeze-dried for long-term storage. Is possible.

  Such an N-hydroxysuccinimide method is a method in which the carboxyl group of the carrier and the amino group of the ligand are combined. In addition to N-hydroxysuccinimide, 1-hydroxybenzotriazole is used as a substance that activates the carboxyl group of the carrier. N-hydroxy compounds containing 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine can be used. Moreover, EDAC; 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, DIPC: diisopropylcarbodiimide, etc. can be used as the carbodiimide that functions as a dehydrating agent during the binding reaction of the ligand to the carrier.

  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).

  However, it has been clarified that some ligands having erythrocyte agglutination ability usually remarkably reduce erythrocyte aggregation ability when immobilized on the magnetic particles of the present invention. For example, when WGA described in Japanese Patent No. 02532670 was immobilized on gelatin and gum arabic coacervate by the method shown in JP-A-2004-157040, red blood cells could not be aggregated.

  Therefore, the present inventor conducted a test for selecting a ligand that exhibits hemagglutination ability when immobilized on the magnetic particles of the present invention. Details are described below.

(1) Preparation of magnetic material-encapsulated gelatin gum arabic coacervate (magnetic particles) Gelatin and gum arabic coacervate were prepared by the method described above, and the magnetic material was taken in to produce magnetic particles having a particle size of 2-12 μm.

(2) Immobilization of ligands on magnetic particles Various ligands shown in Table 1 were immobilized on the magnetic particles prepared in (1) above by the method described above. Reactions were carried out at all material rates of 10 μg / mL. After the reaction, it was blocked with BSA (bovine albumin) and suspended in 0.1% BSA / PBS pH 7.2 at a concentration of 5%.

(3) Measurement of erythrocyte agglutination ability One drop of O, A or B type red blood cell (reagent blood cell Ortho Selectgen, Apharma Gen) to one drop (25 μL) of 5% suspension of magnetic particles to which the ligand is immobilized And stirred on a watch glass. After 3 minutes, hemagglutination was visually observed. The results are shown in Table 1.

  In Table 1, “0”, “+”, “++”, and “++” display indicate aggregation states as shown in FIG. That is, (a) 0 represents a state where magnetic particles and red blood cells do not react. (B) + represents a state in which the magnetic particles and red blood cells are weakly aggregated to cause fine aggregation. (C) ++ represents a state in which the magnetic particles and red blood cells are strongly aggregated to cause relatively large aggregation. (D) ++ represents a state where magnetic particles and red blood cells are strongly aggregated to form a large aggregate.

  As shown in Table 1, the ligands having the ability to aggregate erythrocytes in the state of being immobilized on the magnetic particles of the present invention were anti-glycophorin antibody, Nishida lectin (CSA) and Datura saga orectin (DSA). . In particular, DSA has a strong cohesive force, indicating that it is most suitably used. Although other ligands usually have an aggregating ability, they did not exhibit an aggregating ability when immobilized on the magnetic particles of the present invention.

(4) Measurement of hemagglutination ability in a state in which donor-derived plasma was mixed Using the same method as in (3) above, the hemagglutination power in a state in which serum was mixed in 25% was observed. Type O donor erythrocytes were used. The results are shown in Table 1. It was confirmed that the ligand having an aggregating power in (3) strongly aggregates erythrocytes even when plasma components are mixed.

  From the above test, it was revealed that the ligands that can be used for magnetic particles for labeling erythrocytes are anti-glycophorin antibody, Nishida lectin (CSA) and Datura saga orectin (DSA). Therefore, as the magnetic particles for erythrocyte labeling of the present invention, a ligand selected from anti-glycophorin antibody, Nishida lectin (CSA) and datura saga lectin (DSA) is immobilized on magnetic particles using gelatin gum arabic coacervate as a carrier. What was made is used suitably.

  By using the magnetic particles for red blood cell labeling according to the present invention, red blood cells can be magnetically labeled in an immunological test such as blood type determination. For example, in blood type determination, erythrocytes are allowed to agglutinate in a reaction vessel to be settled, and whether it is positive or negative is determined according to the pattern. This sedimentation usually takes about 30 to 60 minutes. However, when red blood cells are magnetically labeled, the sedimentation rate can be significantly increased by magnetic force, and the sedimentation pattern can be observed in only about 3 minutes.

  Furthermore, B / F separation can be easily performed by labeling red blood cells with the magnetic particles of the present invention. Since unreacted substances can be easily washed, observation with a fluorescent label such as EIA or CLEIA can be performed. In these measurements, the results can be quantified, which is suitable for fully automated immunological tests such as blood type.

The conceptual diagram showing the intensity | strength of the aggregation of the erythrocyte by a magnetic particle.

Claims (4)

  A magnetic particle for erythrocyte labeling, characterized in that an erythrocyte binding substance is supported on a carrier containing a magnetic substance.   The magnetic particle for erythrocyte labeling according to claim 1, wherein the carrier containing the magnetic substance is gelatin gum arabic coacervate in which the magnetic substance is encapsulated.   The magnetic particle for red blood cell labeling according to claim 1 or 2, wherein the red blood cell binding substance is a ligand of red blood cells.   The magnetic particle for erythrocyte labeling according to claim 3, wherein the ligand is selected from the group consisting of an anti-glycophorin antibody, any lectin lectin (CSA) and datura saga orectin (DSA).

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