JP2009089612A - Cell information separation method - Google Patents

Abstract

An object of the present invention is to separate and recover cell-derived information with high efficiency using a carrier and a density gradient centrifugation method.
Cell-derived information is bound to a carrier, density gradient centrifugation is performed on the density of the carrier, and specific cell separation / recovery is performed with high efficiency and stability. The carrier α1, the carrier β2, and the carrier γ3 have antibodies bound to the surfaces, and the antigens α4, β5, and γ6 specific to the antibodies are reacted to bind to the surfaces of the carriers α1, β2, and γ3. . The carrier is subjected to density gradient centrifugation. As a result, the carrier α1, the carrier β2, and the carrier γ3 combined with the cell-derived information placed on the density gradient centrifugal separation layer 7 are subjected to centrifugal separation so that they are accurate as in 8 after density gradient centrifugation. It becomes a layer and can be separated. That is, since the separation is intended for the uniformity of the carrier, stable and highly accurate separation is possible.
[Selection] Figure 1

Description

  The present invention relates to a cell information separation method and a collection method using density gradient centrifugation.

Conventionally, this type of cell separation method using density gradient centrifugation is a technique in which a layer of density is formed in a container and centrifuged to collect cells collected at the boundary of the layer. However, this is easy to collect if the cells to be collected have a constant density. However, when there are a plurality of types of cells, the cells are divided into multiple layers, and the distribution may be different within the same layer. Moreover, separation was important and difficult (see, for example, Patent Document 1). In addition, in the step of identifying the cell type after cell separation, it is necessary to identify the cells in the collected layer, such as culture and antibody reaction, in order to determine whether it is the target cell. If stable, it is difficult to confirm accurately. Furthermore, the boundary layer of the density gradient is translucent like the other boundary layers, and a method of visually judging the fluctuation due to the difference in density and collecting the separated cells can be mentioned as a technique. However, the fluctuation due to the difference in density is difficult to judge by visual observation, and there are cases where excessive recovery or excessive recovery of cells recovered in the boundary layer is performed.
JP 2003-319775 A

  Such a conventional cell information separation method using density gradient centrifugation has a problem in that it is difficult to completely separate specific cell information when cells collected in a density boundary layer are collected. Moreover, since it becomes difficult to collect along with the difficulty of separation, there is a problem that accurate separation and collection cannot be performed. Moreover, when distribution is bad, when there are multiple types of specific cell information, there is a problem that separation at a time is difficult. Therefore, there is a need for a method for separating and collecting specific cell information using density gradient centrifugation that can be accurately separated and easily collected.

  The present invention solves such a conventional problem, and is capable of accurate separation by density gradient centrifugation of specific cell information previously bound to a specific carrier, Along with this, it becomes possible to facilitate recovery. Further, by using density gradient centrifugation and a plurality of carriers in combination, it is possible to separate a plurality of types of specific cell information at a time. As a result, an object of the present invention is to provide a method capable of accurately and easily separating specific cell information in a specimen and various kinds of specific cell information at a time.

  In order to achieve the above-mentioned object, the cell information separation method of the present invention uses density gradient centrifugation to achieve accurate separation and recovery. After carrying specific cell information on a carrier, the cell information separation method is adapted to the carrier. It is characterized by performing density gradient centrifugation.

  The cell information separation method of the present invention is characterized in that specific cell information is the cell itself.

  The cell information separation method of the present invention is characterized in that specific cell information is used as a nucleic acid.

  The cell information separation method of the present invention is characterized in that specific cell information is a sugar chain.

  The cell information separation method of the present invention is characterized in that the carrier is a gold colloid, a magnetic colloid, or a silica colloid alone or in combination of at least two kinds.

  The cell information separation method of the present invention is characterized in that the carrier itself is colored.

  In the cell information separation method of the present invention, the size of the carrier is 1 nanometer to 10 millimeters.

  The cell information separation method of the present invention is characterized in that an antibody is bound to the surface of a carrier.

  In addition, the cell information separation method of the present invention is characterized in that one type of specific microorganism is targeted for binding to one type of carrier.

  In addition, the cell information separation method of the present invention is characterized in that only the carrier is targeted for collection after density gradient centrifugation.

  In addition, the cell information separation method of the present invention is characterized in that after the density gradient centrifugation process, the magnetic carrier is separately magnetized and collected.

  According to the present invention, accurate separation is possible by using a specific cell information separation and recovery method in which specific cell information is attached to a carrier and density gradient centrifugation having a density gradient layer suitable for the carrier is used. Accordingly, recovery becomes easier. Moreover, it becomes possible to separate many kinds of specific cell information at a time by using density gradient centrifugation and a plurality of carriers in combination. As a result, specific cell information in the specimen can be accurately and easily separated, and various kinds of specific cell information can be separated at a time, and the separation operation of specific cell information can be facilitated.

  According to the first aspect of the present invention, in the method for separating cell information, specific cell information is attached to a carrier, and density gradient centrifugation having a density gradient layer adapted to the carrier is performed. By performing density gradient centrifugation with a density gradient layer that matches the density gradient layer to the density of the carrier, separation can be performed not for unstable specific cell information density but for the stable density of the carrier. Since it becomes possible, it has the effect | action that specific cell information can be isolate | separated correctly and easily.

  Further, according to the second aspect of the present invention, it is possible to perform separation on the basis of the stable density of the carrier, not the unstable cell density, by performing density gradient centrifugation using the specific cell information as the cell itself. In addition, since the cells themselves can be directly collected, it has the effect of leading to highly efficient separation.

  In addition, according to the invention of claim 3, by performing density gradient centrifugation using specific cell information as a cell-derived nucleic acid, separation not for unstable nucleic acid density but for stable density of the carrier is performed. It is possible to grasp not only the type of cells but also the toxin productivity from a specific nucleic acid.

  In addition, the invention according to claim 4 uses specific cell information as a glycan, and density gradient centrifugation is performed, so that separation targeting a stable density of a carrier, not an unstable glycan density, is possible. This makes it possible to detect cell-derived cell wall constituents and toxins such as lipopolysaccharide and to understand the toxicity of the cells.

  In the invention according to claim 5, the carrier is a gold colloid, a magnetic colloid, or a silica colloid alone or in combination of at least two kinds, and by carrying out density gradient centrifugation, a plurality of density gradient centrifugations can be performed at one time. Separation and collection of specific cell information of species can be facilitated, and different types of cells can be separated and collected by using two or more types of carriers having different densities.

  In the invention of claim 6, the carrier is colored and subjected to density gradient centrifugation, so that the carrier is gathered in the density gradient layer, so that the color of the layer of the carrier can be visually determined. It has the effect of being able to directly grasp existence and recoverability.

  In the invention of claim 7, the carrier size is set to 1 nanometer to 10 millimeters, and density gradient centrifugation is performed, so that the carrier information is larger than the specific cell information to be collected and easy to separate. It has the effect of becoming.

  In the invention according to claim 8, specific cell information can be collected by binding the antibody to the surface of the carrier and performing density gradient centrifugation, and a plurality of specific information obtained depending on the type of antibody. Cell information can be separated and collected.

  The invention according to claim 9 has an effect that a plurality of specific cell information can be separated at a time by performing density gradient centrifugation with at least two carriers.

  The invention according to claim 10 makes it easy to grasp the separation state of the carriers gathered in each layer by coloring each carrier with a different color and performing density gradient centrifugation, and gathered in each layer. It has the effect of facilitating grasping of the carrier layer.

  Further, the invention according to claim 11 collects specific cell information in one carrier in a concentrated manner by performing density gradient centrifugation on one type of carrier for one type of specific cell information. And can be efficiently separated and recovered.

  Further, the invention according to claim 12 is a method for recovering a separated carrier, and after the density gradient centrifugation treatment, only the carrier is targeted for recovery, so that it is stable without taking in an excessive density gradient centrifugation agent. It has the effect that highly efficient separation becomes possible.

  Further, the invention according to claim 13 is a method for recovering a separated carrier, and after the density gradient centrifugation, recovery using a magnetic force can be performed by separately applying magnetism to a carrier provided with magnetism. Only the carrier provided with magnetism can be recovered, and it has the effect that it can be separated efficiently.

  Embodiments of the present invention will be described below.

(Embodiment 1)
Density gradient centrifugation is a separation method for cell density, and is used as a method for separating animal cells, plant cells, intracellular substances, and the like. Density gradient centrifugation uses sucrose, silica gel, cesium chloride, etc., and changes the concentration to give a gradient in density. Differences in density appear in layers, and microorganisms such as animal cells, plant cells, and bacteria, and intracellular substances can be separated and recovered according to the density. However, when the time for moving the density layer is not uniform, resistance of the density layer and the like occur, and the sedimentation speed is affected. Therefore, there are cases where the cells to be collected in the layer cannot be collected accurately. Further, when a plurality of objects are separated at a time, there are a plurality of density layers, which are further difficult to separate. Specific cell information includes all information such as cell-derived information, such as the cell itself, cell-derived nucleic acid, or cell sugar chain, cell itself, cell components, sites, or metabolites. The cell is a cell of any organism such as an animal, a plant, a bacterium, a fungus, or a yeast, and it does not matter whether the cell is alive or dead. It also includes viruses, including virus sugar chains or nucleic acids.

  In the method of separating cell information, in order to achieve accurate separation and collection using density gradient centrifugation, after carrying specific cell information on a carrier and attaching cells that match the specific cell, Density gradient centrifugation is performed with a density gradient layer adapted to the carrier. That is, density gradient centrifugation is performed having a density gradient layer in which the density gradient layer matches the density of the carrier. Thus, by performing density gradient centrifugation conditions for a carrier having a uniform density, stable separation is possible, leading to high-efficiency separation. Specific cell information can be combined through a medium such as avidin and biotin. In addition, after applying biotin to the carrier, it is possible to bind a primary antibody, a secondary nucleic acid treated with avidin by binding a nucleic acid complementary to the target nucleic acid, or a target nucleic acid directly. After the secondary antibody is bound, the target specific cell information is reacted and collected. After the collection, it is separated with a proteolytic enzyme or the like, and only the target specific cell information is collected and analyzed.

  At this time, by setting the cell information as the specific cell itself, it becomes possible to collect cells such as bacteria and sputum whose shape needs to be observed as they are. Further, by directly collecting the cells, it is possible to discriminate the life or death, or the living cells are collected and cultured separately, so that information can be obtained directly from the living bacteria accurately as the next operation step. It is also effective to make the cell information not a cell solid but a specific nucleic acid. By recovering a specific nucleic acid on the surface of the carrier and recovering only the nucleic acid, it can be used for analysis to a gene analysis tool such as polymerase chain reaction. In the case of a nucleic acid, it is difficult to separate the nucleic acid itself by density gradient centrifugation, but it can be easily separated by utilizing the density of the carrier. Nucleic acids complementary to the target nucleic acid, which is specific cell information, are crazed through avidin and biotin. The target nucleic acid which is the collected specific cell information can be recovered by heating at 80 ° C. or higher.

  This makes it possible to easily separate the nucleic acid having the target sequence. The density gradient centrifugation according to the present invention enables separation from a reaction interfering substance contained in a specimen, so that it can be applied to gene analysis tools such as accurate polymerase chain reaction. Cell-derived sugar chains are also subject to the recovery method. An example of a sugar chain is lipopolysaccharide derived from Gram-negative bacteria. Lipopolysaccharide exhibits cytotoxic activity as an endotoxin. Therefore, it is important for detection to separate them efficiently. In the case of a sugar chain, it is difficult to separate by itself by density gradient centrifugation, but it can be easily separated by utilizing the density of the carrier. For the carrier, after collating gold colloidal particles, magnetic colloidal particles, silica colloidal particles, etc. with bovine serum albumin, etc., when attaching the primary antibody to the carrier surface, the surface is biotinylated and the secondary antibody is avidin. Collect the processed specific cell information. In addition, a material capable of directly collecting an antibody having specific cell information of interest on the surface of the carrier and recovering the cell or cell-derived information is effective.

  In addition, particles capable of attaching cell-derived information indirectly or directly are also effective. By combining these alone or in combination of two or more, information derived from several types of cells can be simultaneously separated. By coloring these carriers and using them, it is possible to easily discriminate the density gradient layer, which was difficult to discriminate in the conventional density gradient centrifugation, and to efficiently recover the target carrier.

  In addition, it is possible to visually observe the state in which the target carrier is collected in the density gradient layer, and it is possible to directly grasp the state of recovery. By setting the size of these carriers to a size of 1 nanometer to 10 millimeters, it becomes possible to easily collect information and cells derived from the target cells. These can be efficiently recovered by using a carrier suitable for cell-derived information of a desired size, and can be separated under suitable separation conditions.

  For example, in the case of bacteria, the size is 0.5 to 3 micrometers, and it is easy to separate by using a carrier larger than this. Antibodies are effective for collecting specific cell information. By using an antibody adapted to information derived from the target cell, the information derived from the target cell can be collected on a carrier. The antibody to be bound to the carrier is not limited to one type, and by combining multiple types of antibodies, it is possible to roughly classify the information derived from a plurality of types of cells of interest on the same carrier. When finely separating and recovering at once, antibodies adapted to cell-derived information use different types of antibodies for each carrier type, and when density gradient centrifugation is performed, various types of cells are derived. Information can be separated at a time. It is effective to match the density of the carrier with the type of information derived from the cells to be separated.

  Moreover, about the collection | recovery method, after density gradient centrifugation conventionally, it separated and combined with the density gradient centrifugation agent. This is because the density gradient layer is not clear, and the target cell-derived information and the density gradient centrifugation agent are combined and separated and collected to try to collect the target cell-derived information together. However, as a result, an extra density gradient centrifuge agent is included, so that the amount of specimen increases and measurement may be difficult. Therefore, separation is performed with high efficiency by a method of selectively recovering only the carrier. As a method for selectively recovering only the carrier, a hollow pipe such as a micropipette, pipette, or capillary tube is brought into contact with the direct density gradient layer of the recovered carrier and sucked, or a mesh of a mesh smaller than the size of the carrier. It is also effective to filter and recover the carrier with a sieve. Moreover, it becomes possible to isolate | separate various support | carriers by making this mesh-like sieve into a multistage type layer. For the recovery of the carrier, it is also effective to use a magnet when a carrier having magnetism is used. Only the target layer can be recovered with high efficiency by the magnet.

  Examples of the present invention will be described below. FIG. 1 shows a conceptual diagram of a cell information separation method according to Example 1 of the present invention. FIG. 1 (a) is a conceptual diagram of carriers and antigens in the reaction vessel, and FIG. 1 (b) is a conceptual diagram before and after the same density gradient centrifugation.

  This is a method for separating specific cell information and specifically recovering specific cell information contained in a specimen. Specific cell information is attached to the carrier, and density gradient centrifugation is performed on the carrier (see FIG. 1). The carrier α1, the carrier β2, and the carrier γ3 are each bound with an antibody specific for cell information, and the specific cell information is mixed and reacted as an antigen. As specific carriers, sepharose beads, colloidal gold particles, and glass particles are used.

  These can be used together because of their different densities. Antigen α4, antigen β5, and antigen γ6 correspond to carrier α1, carrier β2, and carrier γ3, respectively. After the reaction, it is placed on the upper layer of the density gradient centrifugation layer 7 and centrifuged. After the density gradient centrifugation 8, the target carrier is recovered on each layer and can be easily taken out. The density gradient centrifugation can be used in conformity with the density of the carrier, but sucrose and silica gel are used. The density gradient layer is prepared by adjusting these with distilled water so as to be about 50 to 80% and laminating them in a density gradient centrifuge container. After adjusting the density gradient layer, the specimen is dropped on the surface layer and centrifuged at 1000 rpm for 10 minutes to recover the carrier recovered in the target layer.

  As an antigen, the cell itself can be targeted. Antigen α4, antigen β5, and antigen γ6 react with carrier α1, carrier β2, and carrier γ3 in reaction vessel 9, respectively. When targeting bacteria, antibodies that bind to specific bacteria such as Escherichia coli and Staphylococcus aureus are effective. Antibodies against specific bacteria are handled by rabbits and mice, and are effective in production procedures. As other antigens, different species such as Legionella, hepatitis C virus, and Candida can be separated because the density of the carrier is the separation target. As the antibody, an antibody whose detection target is a lipopolysaccharide specific to Legionella can be used. Viral surface polysaccharides are also targeted. It is also effective to bind nucleic acids to the surface of the carrier.

  The nucleic acid sequence is complementary to the nucleic acid sequence derived from the target cell information, so that it can be bound and recovered in the same manner. As for the binding means, it is effective to bind the target specific cell information by using biotin coated on the surface of the carrier and binding of a nucleic acid complementary to the target specific cell information. As the collection conditions, density gradient centrifugation can be used in conformity with the density of the carrier, but sucrose and silica gel are used. The density gradient layer is prepared by adjusting these with distilled water so as to be about 50 to 80% and laminating them in a density gradient centrifuge container. After adjusting the density gradient layer, the specimen is dropped on the surface layer and centrifuged at 1000 rpm for 10 minutes to recover the carrier recovered in the target layer.

  After collection, the target specific cell information can be collected by heating to 80 ° C. or higher and analyzed separately. At this time, even if a sample contains a foreign substance or a measurement interfering substance, it can be separated by density gradient centrifugation. After the separation, analysis can be performed using an analysis tool such as a polymerase chain reaction. In addition, a horseshoe crab-derived blood component can be applied to the surface of the carrier to recover lipopolysaccharide, which is a cell wall constituent derived from Gram-negative bacteria. As the carrier, a gold colloid, a magnetic colloid, or a silica colloid is used alone or in combination of at least two kinds. When used alone, it becomes one kind of separation object, but by combining a plurality of separation objects, two or more kinds of separation objects can be separated at a time. At this time, the separation layer needs to match the number of carriers.

  By coloring the carrier itself, it can be easily recovered from the separation layer when separated by density gradient centrifugation. As the colorant, those that do not decolorize with water and those that can color various colloids are suitable. It is also effective to directly color proteins such as bound biotin and bovine serum albumin applied as a blocking agent with methylene blue. The separation layer is transparent and difficult to see visually, but can be easily discriminated by collecting the carrier for the purpose. Further, by separating the color for each carrier, it is possible to grasp the situation of the carriers gathered in each layer and easily separate them. The size of the carrier is 1 nanometer to 10 millimeters so that highly efficient separation can be performed using a carrier suitable for nucleic acid, lipopolysaccharide, and the cell itself, or a carrier suitable for each amount. it can.

  Specifically, when handling microorganisms such as bacteria, the size is 0.5 to 3 micrometers, so 0.5 micrometers or more, or 3 micrometers or more is effective from the viewpoint of adhesion. is there. At this time, by making the carrier larger than the cell-derived information to be collected, detachment after binding can be reduced, and stable separation becomes possible. In order to incorporate a plurality of information into the carrier at a time, a plurality of types of binding methods can be incorporated into one type of carrier. When there are many separation objects, it is effective as a primary separation method. In addition, when various cell-derived information is bound to each carrier, individual information can be separated at a time.

  As for the recovered carrier, only the target product that does not contain an excessive density gradient centrifuge can be recovered by separating only the carrier. For collection, a method of using a hollow tube 9 such as a micropipette, capillary, or pipette, contacting the density gradient layer 10 directly, and separating by suction (see FIG. 2) is effective. For separation, it is also effective to use a sieve.

  The carrier α1, the carrier β2, and the carrier γ3 are combined with cell-derived information, subjected to density gradient centrifugation, collected, and then screened with the layers of the carrier α1, the carrier β2, and the carrier γ3. The eyes of the sieve α11 are on the carrier α1, the eyes of the sieve β12 are on the carrier β2, and the eyes of the sieve γ13 are slightly finer than the size of the carrier γ3. The agent and adhering foreign matter can be separated and washed, and separation can be performed more efficiently (see FIG. 3). At this time, the hollow tube 17 needs to have a larger inner diameter than the respective carriers in order to recover the carriers. Further, when the magnetic carrier 14 is used, after the density gradient centrifugation, the magnet 16 is brought into direct contact with the density gradient centrifuge container 15 to recover the magnetic carrier 14 efficiently. The carrier can be recovered.

  Further, by separating after density gradient centrifugation, it is possible to separate and wash the density gradient centrifugal separation agent and adhering foreign substances, and more efficient separation is possible (see FIG. 4). As the magnetic particles used for the carrier having magnetism, magnetic particles formed by magnetic bacteria are also effective.

  In the detection after recovery, it is also effective to detect the carrier as it is. In the case of detection as it is, a method of directly magnifying the carrier with a microscope or the like, and a method of in contact with a medium when cultivating like a bacterium are effective.

  In the detection after recovery, it is also effective to separate the cell-derived information component from the carrier. As the separation method, physical treatment or chemical treatment such as ultrasonic treatment, chemical washing, buffer washing, and protease treatment is effective.

  In addition, what is bound to the carrier used is not limited to antibodies, nucleic acids, and horseshoe crab blood-derived components as long as cell-derived components adhere to them. Vaginal blood-derived components such as SLP are also effective.

  It is also possible to directly bond to the carrier to be used.

  In addition, as conditions for density gradient centrifugation, those with a rotational speed of 1000 rpm to 100000 rpm are desirable. When it becomes 100,000 rpm or more, the cell-derived component may be affected. Further, when the rotation speed is 1000 rpm or less, the separation efficiency may be lowered.

  In addition, you may put the magnet used as a method of collect | recovering magnetic carriers directly in a density gradient centrifugation layer.

  When a plurality of carriers are used, separation can be efficiently performed by using one of them as a magnetic carrier. When several types of magnetic carriers are used, it is effective to use them at a distance from the density gradient separation layer.

  When a magnet is used as a method for recovering the magnetic carrier, the container used for density gradient centrifugation must be made of iron.

  It is also effective to insert the hollow tube by the number of density gradient layers at a time.

  By using the specific cell separation / recovery method using the density gradient centrifugation of the present invention, it becomes possible to stably perform the density gradient centrifugation, which has heretofore been difficult to clearly separate, and to accompany it. Recovery after density gradient centrifugation has also been simplified. By using a carrier whose density is known in advance and performing density gradient centrifugation, the separation is performed efficiently. By using the carrier as a recovery target, stable separation is possible. As a result, information derived from specific cells such as microorganisms, nucleic acids, and lipopolysaccharides in various specimens can be specifically and efficiently separated and accurately detected.

Schematic diagram of cell information separation method of Example 1 of the present invention ((a) conceptual diagram of carrier and antigen in the same reaction vessel, (b) conceptual diagram before and after the same density gradient centrifugation) The figure of the collection | recovery method after specific cell information separation using the hollow tube of Example 1 same as the above The figure of the collection | recovery method after specific cell information separation using the sieve of Example 1 same as the above The figure of the collection | recovery method after specific cell information separation using the magnet of Example 1 same as the above

Explanation of symbols

1 Carrier α
2 Carrier β
3 Carrier γ
4 Antigen α
5 Antigen β
6 Antigen γ
7 Density gradient centrifugation layer 8 After density gradient centrifugation 9 Reaction vessel

Claims (13)

A cell information separation method in which specific cell information is attached to a carrier, followed by density gradient centrifugation suitable for the carrier. The cell information separation method according to claim 1, wherein the specific cell information is the cell itself. The cell information separation method according to claim 1, wherein the specific cell information is a cell-derived nucleic acid. The cell information separation method according to claim 1, wherein the specific cell information is a cell sugar chain. The cell information separation method according to claim 1, wherein the carrier is a gold colloid, a magnetic colloid, or a silica colloid alone or in combination of at least two kinds. The cell information separation method according to claim 1, wherein the carrier is colored. The cell information separation method according to claim 1, wherein the carrier has a size of 10 micrometers to 10 millimeters. The cell information separation method according to claim 1, wherein an antibody is bound to the surface of the carrier. The cell information separation method according to claim 1, wherein the number of carriers is at least two or more. The cell information separation method according to claim 9, wherein each carrier is colored with a different color. The cell information separation method according to claim 1, wherein one type of specific cell information is a binding target for one type of carrier. The cell information separation method according to any one of claims 1 to 11, wherein only the carrier is a target to be recovered after density gradient centrifugation. The cell information separation method according to any one of claims 1 to 12, wherein, after the density gradient centrifugation, the magnetism is separately applied to a carrier provided with magnetism to be collected.

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