JPH03287067A - Separating agent, separating device and separating method for lymphocyte - Google Patents

Abstract

PURPOSE:To separate and thicken lymphocytes at a high yield and high purity and to speed up a sepn. operation by immobilizing lectin having affinity to the sub-class of the specific lymphocyte or the specific sub-set thereof to a water-soluble solid material. CONSTITUTION:While a three-way cock 4 is held closed, a specimen liquid contg. cells desired to be recovered is injected into an effective diameter part 6 from the top end of a column 1 and is held under prescribed conditions. The specimen liquid permeates a filter member 3 and comes into contact with the separating agent 2. The specific lymphocytes in the specimen liquid are thereby adsorbed at a high adhesion rate on the separating agent 2. The separating agent formed by immobilizing the lectin to the water-soluble solid material is used for the separating agent 2. The cock 4 is thereafter opened to discharge the specimen liquid in the effective diameter part 6 from an outflow port 5 via a reduced diameter part 7. This liquid is recovered in a test tube 8. The cells which are not adsorbed on the separating agent 2 are included in the recovered liquid and particularly the desired cells are held thickened. The desired cells are thickened with the high purity in this way without impairing the function of the cells.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a lymphocyte separation M agent, separation H apparatus, and separation method. The present invention relates to a separation agent that can be selectively (preferentially) captured, a separation device having a container filled with the separation agent, and a method for separating lymphocytes using a separation agent.

<Conventional technology> The main immunocompetent cells are lymphocytes that are equipped with antigen-specific receptors and have the ability to recognize antigens and differentiate into effector cells that produce specific humoral and cellular antibodies. , immunocompetent cells
These cells are called nt cells. However, it is impossible to carry out immune responses in vivo using lymphocytes alone; dendritic cells of the macrophage subgroup are required for the ascending leg of the immune response, which processes antigens and presents them to lymphocytes. In addition, foreign body disposal, which is the final effect of the antigen-antibody reaction, and inflammatory reactions centered on the canal reaction are the descending legs of the immune response, and macrophages (monocytes), granulocytes, mast cells, etc. are involved. There is.

Diverse immunocompetent cells are characterized by the ability to quickly respond to the demands of complex immune responses by local recruitment and proliferation, and proliferation and differentiation in response to stimuli are closely linked to the regulation of functions in other cell systems in the body. It has a corresponding role.

Nonva cells can be broadly divided into T lymphocytes, which mature under the influence of the thymus gland, and B lymphocytes, which mature under the influence of the capsule of Fabricius, or the equivalent organs in mammals (hematopoietic organs such as the liver and bone marrow). However, in the case of B lymphocytes, after receiving antigen stimulation, they show a unidirectional change of differentiation and proliferation into antibody-producing cells and production of antibodies, whereas that of T lymphocytes Not just a single antigen stimulation, but several types of T lymphocytes with various functions undergo changes, with some becoming effector cells and others functioning as regulatory cells (promoting or suppressing differentiation and proliferation).

That is, it is thought that T lymphocytes exist in functional subsets.

So-called helper/inducer T lymphocytes (T,
/l), suppressor/cytotoxic T lymphocytes (
T, /c), T lymphocytes involved in delayed-type allergies (
T, TH), etc. The existence of these subsets is shown by the correlation between classification of T lymphocytes using antibodies (monoclonal antibodies) against surface antigens that serve as markers for these subsets and their functions.

In addition, B lymphocytes also have immunoglobulin classes (IgG, IgM, IgA, Ig
It is thought that there is a subset classified by D, IgE).

In recent years, lymphocytes with such important functions have been isolated,
Attempts have been made to fractionate the lymphocytes and use them in basic immunological research or in various diagnoses and treatments, and various methods as described below are conventionally known as methods for separating lymphocytes.

Namely, N) methods such as velocity pouring that utilize differences in cell size, [2] methods such as specific gravity centrifugation that utilizes differences in specific gravity of cells, and [3] methods that utilize differences in cell surface charge. Methods such as cell electrophoresis r removal, [4] Removal of monocytes by carbanyl iron using oligophagic activity, [5] Filling with nylon wool, glass wool, etc. using differences in non-specific adhesion activity of cells. [6] Affinity chromatography that utilizes the specific binding ability of cells; [7] Rosette sedimentation method that uses cell rosette formation as an indicator; [8] ] Fluorescence activating cell sorter that utilizes the reaction between a specific antigen on the cell membrane surface, immunoglobulin, and a labeled antibody.
Activating Ce11 Sorting.

FAC3) method (for example, see Jun Yada-1 Michio Fujiwara (ed.), New Lymphocyte Function Search Method, 1987, Chugai Medical Pillar, etc.). In addition, as a means of multistage separation without using a support or stationary phase, [9] Field-Flow Fraction
nation, FFF) method is being considered.

However, physical cell separation methods such as those in [1] to [3] have a specific gravity between T lymphocytes and B lymphocytes.
Since there is no significant difference in physical properties such as density, high precision cannot be required for the yield or purity of separated and recovered cells.In particular, in the specific gravity centrifugation method of [2], The price is expensive, or even if it is cheap, it requires skill to operate, and in the cell electrophoresis method [3], the mobility differs depending on the maturity of the cell, and the influence of the electric field on cell function. It also had disadvantages, such as the fact that it was not fully understood and that it was difficult to process in large quantities.

In addition, cell separation methods that use specific antigens, receptors, immunoglobulins, etc. on the surface of cell membranes as indicators, such as those in [61-[8], all require that the treated cells have strong biologically specific bonds and stimulation. In particular, affinity chromatography in [6] requires expensive antibodies, and it is difficult to collect target cells in an intact state due to the high sensitivity of the target cells, and is not suitable for large-scale processing. The FACS method has disadvantages in that pretreatment such as labeling of cells is complicated and requires expensive antibodies and equipment.

In addition, the FFF method of [9] has the equipment difficulty of continuously inflowing and outflowing the eluent via the rotating shaft, and the materials of the rotor-shaped separation cell and the channel cause cell adhesion. We have not reached the point where mutual separation of cells can be put to practical use.

On the other hand, the method [5], which utilizes the non-specific adhesion properties of cells, is less likely to damage the functions of treated cells and is suitable in terms of cost, operability, and mass processing. However, it is insufficient in terms of selectivity and recovery rate for target cells, and in addition, the column method mentioned above requires experience and technical proficiency when performing fractional H operations, which is a major problem. also existed.

As separation agents and separation methods that utilize such non-specific adhesion properties, hydrophobic materials (Japanese Patent Publication No. 59-17387
No. 59-36963, No. 62-45206, JP-A-57-204454), materials with acidic functional groups (JP-A-59-36961, JP-A-56-14088)
6, No. 56-152740), polymers containing basic functional groups (JP-A-59-216584, No. 60-1)
05490), hydroxyapatite fiber (Japanese Patent Application Laid-open No. 6
No. 3-284) Specific polymers (JP-A No. 61-2211)
23, No. 64-34.285), but even these methods have not yet been sufficient in terms of selectivity and recovery rate for target cells.

<Problems to be Solved by the Invention> Therefore, the present invention provides a lymphocyte separation agent and separation agent that can efficiently separate a specific lymphocyte subclass or a specific subset thereof and recover target cells without impairing cell function. The object of the present invention is to provide an apparatus and a separation method.

<Means for Solving the Problems> Such objects are achieved by the present invention described in (1) to (11) below.

(1) A lymphocyte separation agent characterized in that a lectin having an affinity for a specific lymphocyte subclass or a specific subset thereof is immobilized on a water-insoluble solid substance.

(2) The lymphocyte separation agent according to (1) above, wherein the water-insoluble solid substance is in the form of particles.

(3) The lymphocyte separation agent according to (2) above, wherein the water-insoluble solid substance has a particle size of 0.05 to 5 mm.

(4) A lymphocyte separation device comprising a container filled with the separation agent according to any one of (1) to (3) above.

(5) The lymphocyte separation device according to (4) above, wherein the inside of the container is sterilized.

(6) A test solution containing lymphocytes is brought into contact with the separation agent according to any one of (1) to (3) above, and the separation agent is applied to a specific lymphocyte subclass or a specific subset thereof in the test solution. A method for isolating lymphocytes, which comprises adsorbing cells and collecting cells not adsorbed to a separation agent.

(7) A test solution containing lymphocytes is brought into contact with the separation agent according to any one of (1) to (3) above, and the separation agent is applied to a specific lymphocyte subclass or a specific subset thereof in the test solution. 1. A method for separating lymphocytes, which comprises adsorbing cells to a separating agent, and then separating the cells from the separating agent and collecting them.

(8) The method for separating lymphocytes according to (7) above, wherein a specific lymphocyte subclass or a specific subset thereof is selectively detached from among the cells adsorbed to the separation agent and recovered.

(9) Detachment of the cells from the separation agent is carried out by bringing the detachment agent into contact with the separation agent (7) or (8) above.
Lymphocyte isolation method described in .

(10) The method for separating lymphocytes according to (9) above, wherein the withdrawal agent is a carbohydrate.

(11) The carbohydrate is a monomer or oligosaccharide (
10) The method for isolating lymphocytes described in 10).

Effects> The lymphocyte separation agent according to the present invention can be used to isolate specific lymphocyte subclasses (e.g., B lymphocytes or T lymphocytes) or specific subsets thereof (e.g., helper/influenza subclasses of T lymphocytes). This is because a lectin that has an affinity for T-inducing lymphocytes or suppressor/cytotoxic lymphocytes (hereinafter collectively referred to as specific lymphocytes) is immobilized on a water-insoluble solid substance. By utilizing the high affinity of lectins for specific lymphocyte membrane surface antigen receptors, specific lymphocytes can be separated or concentrated from other cells with high selectivity and high yield.

To explain this effect in more detail, cell membrane surface antigen receptors are composed of glycoproteins, glycolipids, proteins, and lipids, so specific lectins recognize the structure of the cell membrane surface of specific lymphocytes and selectively It is thought that significant adsorption will occur. A physicochemical analysis of these specific interaction forces reveals that the lectin that has an affinity for a specific lymphocyte and the cell membrane surface of a specific lymphocyte are , dielectric effect and dispersion effect), the intermolecular forces of hydrogen bond, ionic bond (Coulomb force), ion-dipole opening force, coordinate bond and hydrophobic bond are combined. It is speculated that the spatial structure formed by the surface molecules is sterically compatible, like a key and a keyhole.

As mentioned above, lymphocytes are closely involved in various immune diseases as immunocompetent cells. Therefore, if specific lymphocytes could be selectively and efficiently separated and concentrated as described above, it would be useful for various diagnoses and treatments.

<Configuration of the Invention> Hereinafter, the lymphocyte separation agent and separation device of the present invention will be explained in detail based on the accompanying drawings.

The lymphocyte separating agent of the present invention is characterized in that a lectin (hereinafter also simply referred to as lectin) having an affinity for specific lymphocytes is immobilized on a water-insoluble solid substance.

The lectin used in the lymphocyte separation agent of the present invention is a protein or glycoprotein that interacts with sugars, aggregates cells, or precipitates polysaccharides and complex carbohydrates, but is not an immunological product. say.

Representative examples of such lectins include the following.

1) L-fucose binding lectin: Lotus tetragonolobus, Lllexeuropaeus-I, rabbit serum, etc.

2) D-galactose, N-acetyl-D-galactosamine binding lectin: Arachis hypogae
a (Beanatsu), Glycine wax (
Ricinus communis (soybean)
, Bauhinia purpurea (Mokwanju), Phaseolus vulgaris (
kidney beans).

3) D-mannose binding lectin: CanavalL
aensiformis (jack bean: concanavalin A), Lens culinaris (lentil), Pisumsativum (enthusiast bean), Vicia faba (fava bean)
.

4) Di-N-acetylchitobiose-binding lectin: T
riticum vulgaris (wheat germ; wheat germ agglutinin), Phytolacca american
ana (American pokeweed, PWM lectin),
Solanum tuberosum (potato)
.

5) Sialic acid binding lectin: Limulus polyphemus (horseshoe crab).

In addition, the molecular weight of the lectin is not particularly limited, but io,
000 to 50,000, especially 20,000 to 40,
A value of about 000 is preferable.

Such lectins have high affinity for specific lymphocytes depending on their type.

For example, the above Arachis hypogaea, R
icinus communis has a high affinity for helper/inducer T lymphocytes, a subset of T lymphocytes.

Note that the amount of such lectin supported on the water-insoluble solid substance is preferably 3 to 70011 mol/g,
More preferably, it is 50 to 30011 mot/g.

In the lymphocyte separation agent of the present invention, the water-insoluble solid substance serving as a carrier for immobilizing such lectin includes:
It may be hydrophilic or hydrophobic, and specifically includes polysaccharides such as agarose-based, dextran-based, chitosan-based, cellulose-based, polyacrylamide-based, polyvinyl alcohol-based, Polyvinylpyrrolidone type, polyacrylonitrile type, styrene-divinylbenzene copolymer, Bostyrene type, acrylic acid ester type, methacnolic acid ester type, polyethylene type, polypropylene type, poly4-fluorinated ethylene type, ethylene-vinyl acetate copolymer type , organic synthetic polymers such as polyamide, polycarbonate, polyvinylidene fluoride, polyvinyl formal, polyarylate, and polyethersulfone, natural organic polymers derived from living organisms such as collagen and chitosan, glass, and titanium. , activated carbon, or inorganic materials such as various ceramics such as alumina, silica, and hydroxyapatite. However, known carriers that are normally used in immobilized enzyme methods, affinity chromatography, etc. may be used without any particular limitations. I can do it. In particular, among these (
1. It is preferable that the cell adhesion property is not too high, that is, the cell-substrate interaction is relatively mild.

The form of such a water-insoluble solid substance is not particularly limited, and any known shape such as particulate, fibrous, hollow fiber, membrane, or flat plate may be used; From the viewpoint of liquid permeability for liquids and cleaning liquids, ease of handling when preparing the separation agent, etc., particulate forms are preferable, and in particular, particulate forms with an average particle size of 0.05 to 5 mm have a large effective surface area and liquid permeability. It is preferable from the viewpoint of gender, etc.

That is, if the average particle diameter is less than 0.05 mm, the liquid permeability will decrease, and if it exceeds 5 mm, it will be difficult to ensure a sufficient effective surface area.

In addition, the above average particle size is calculated as the weight average of the particle size of each station, which is the intermediate value between the upper limit particle size and the lower limit particle size of each station after classification using a sieve specified in JIS-Z-8801. be able to.

Further, particle shapes (particularly spherical particles) are preferable because they are less likely to cause physical damage to cells, are less likely to cause flaking or chipping, and are easier to obtain uniform particles.

Further, such a carrier may be porous or dense.

As the porous particles, for example, acrylic porous beads made of a terpolymer of divinylbenzene monomer, glycidyl methacrylate monomer and 2-hydroxyethyl acrylate oligomer are preferably used.

In the lymphocyte separation agent of the present invention, methods for immobilizing the lectin on the surface of a water-insoluble solid substance include carrier binding methods such as covalent bonding, ionic bonding, physical adsorption, hydrophobic bonding, and biochemical specific bonding; Alternatively, any known method such as a cross-linking method, entrapping method, or composite method can be used, but from the viewpoint of stability in body fluids or cell suspensions, it is preferable to immobilize by covalent bond or hydrophobic bond.

Various methods are used to covalently bond lectins to the surface of water-insoluble solid substances, depending on the functional groups of both. For example, cyanogen bromide activation method, condensation reagent method using carbodiimide reagent, acid azide derivative method, diazo method, alkylation method, carrier crosslinking method using dialdehyde, bisepoxide, diisocyanate, etc., γ-glyndoxyprovir A silane coupling agent activation method using trimethoxysilane, β-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, etc. is used.

Furthermore, if necessary, a spacer molecule of any length can be introduced between the lectin and the surface of the water-insoluble solid substance. This spacer molecule is
Preferred examples include diamines such as hexamethylene diamine, and diols such as hexamethylene glycol.

In addition, methods for hydrophobically bonding lectin to the surface of a water-insoluble solid substance include, for example, a method in which it is directly bonded to a hydrophobic polymer such as polystyrene or polypropylene; There is a method in which hydrophobic bonding is performed after bonding a ring agent. As a coupling agent having a hydrophobic group in the molecule,
Octadecyldimethyl [3-(1-rimethoxysilyl)]
Examples include propyl] ammonium chloride, octadecyltrichlorosilane, and phenyltrichlorosilane.

In order to perform lymphocyte separation using the lymphocyte separation agent of the present invention, which is obtained by immobilizing the lectin on a water-insoluble solid substance, these 11IIi agents are added to a test liquid (body fluid, cell suspension, etc.). ) It is also possible to adsorb and collect lymphocytes batchwise by bringing them into contact with a test solution, such as by immersing the lymphocytes in a sample liquid (for example, by immersing the lymphocytes in a sample solution), but it is important to obtain the absolute amount of lectin that affects cell adsorption and to obtain sufficient space for cell adsorption. Therefore, it is preferable to make the contact using a separation device having a container (column) filled with a lymphocyte separation agent.

That is, the lymphocyte separation device of the present invention is characterized by having a container filled with a separation agent in which the lectin is immobilized on the water-insoluble solid substance.

FIG. 1 is a cross-sectional view schematically showing a configuration example of a lymphocyte separation device of the present invention. The lymphocyte separation device shown in the figure has a column (container) 1 composed of a full-diameter section 6 and a reduced-diameter section 7 on the lower end side in the figure.

The actual diameter portion 6 of the column 1 has a cylindrical shape, and inside thereof,
It is filled with the lymphocyte separation agent 2 described above. That is, a pair of filter members 3.3 having a mesh or pores through which various cells in the test liquid can pass through but not through which the separating agent 2 can pass are installed in the actual diameter portion 6, and a portion of both filters Separating agent 2 is held between 3.3 and 3.3.

As this filter member 3, for example, mesh, non-woven fabric, etc. are suitably used, and its constituent material may be any material as long as it is physiologically inert and has high strength.
Particularly preferred is one made of polyester or polyamide.

Note that the filling amount of the separating agent 2 is not particularly limited, but is 0.
It is preferably about 1 to 100 g, particularly about 0.5 to 50 g.

Further, the method of holding the separation agent 2 is not limited to the method using the filter member 3 as shown.

The upper end of the actual diameter portion 6 in the figure may be open or shielded, and a stopper (not shown) made of an elastic material into which a needle or the like can be inserted may be inserted into the open opening. It may have a similar structure.

The reduced diameter portion 7 of the column 1 has a shape in which the inner diameter gradually decreases toward the bottom in the figure, and a liquid outlet 5 is formed at its lower end.

Further, a three-way stopcock 4 is installed in the middle of the reduced diameter portion 7, so that the flow path within the reduced diameter portion 7 can be opened and closed.

A test tube 8 is installed below the column 1 to collect the liquid flowing out from the outlet 5.

Column I can be made of synthetic resins such as polyethylene, polypropylene, polycarbonate, polystyrene, and polymethyl methacrylate, metals such as glass and stainless steel, and various ceramics, which can be sterilized in an autoclave and are easy to handle. Particularly preferred are polypropylene and polycarbonate.

In a lymphocyte separation device with such a configuration, the inside of the column 1 (the part that comes into contact with the sample liquid) is sterilized by heat sterilization (autoclave sterilization), gas sterilization (e.g. EOG sterilization), or radiation sterilization before use. It is preferable that the sterilization process is performed using the sterilization method described above.

The container of the lymphocyte separation device of the present invention is not limited to a column as shown in the figure, but may be, for example, an outer cylinder of a syringe.

Next, a method for separating lymphocytes according to the present invention will be explained by taking as an example the case where the separation apparatus shown in FIG. 1 is used.

The method of separating lymphocytes of the present invention includes a method of collecting cells that are not adsorbed to separation agent 2 (first method), and a method of collecting cells that are adsorbed to separation agent 2 (second method).
There is. The explanation will be given below.

[First method] With the three-way stopcock 4 closed, a test solution containing cells to be collected (hereinafter referred to as target cells) is injected into the actual diameter section 6 from the upper end of the column 1, and a predetermined amount is conditions (e.g. 4~
Hold at 37°C for 0.1-120 minutes).

As a result, the test liquid passes through the filter member 3 and comes into contact with the separation agent 2, and specific lymphocytes in the test liquid are adsorbed to the separation agent 2 with a high adhesion rate.

Thereafter, the three-way stopcock 4 is opened, and the test liquid in the full-diameter portion 6 is allowed to flow out from the outlet 5 through the reduced-diameter portion 7 and collected into the test tube 8 .

Furthermore, the separation agent 2 may be washed by injecting a washing liquid from the upper end of the column 1 using various pumps, pipettes, etc., and this washing liquid may be collected into the test tube 8 in the same manner.

In the recovered liquid collected in the test tube 8 in this way,
It contains cells that were not adsorbed to the fraction M agent 2, and the target cells are particularly concentrated.

Examples of the test liquid include suspensions containing various cells, body fluids such as plasma and lymph, and diluted solutions of these body fluids. Furthermore, in the present invention, whole blood (particularly peripheral blood) or a diluted solution thereof can be used as the test liquid, and even in this case, excellent effects can be obtained.

Furthermore, the cells in the test liquid may be derived from humans or non-human animals.

[Second method] After collecting the liquid in the test tube 8 in the same manner as in the first method, transfer this test tube 8 to another location, and then place another unused test tube 8 at the bottom of the outlet 5. to be installed.

Next, the three-way stopcock 4 is closed, and the detachment liquid containing detachment 1j1 (adsorption antagonist) is injected into the column 1 from the upper end of the column 1, and the minutes). As a result, the separation agent 2 and the detachment agent come into contact with each other, and the cells adsorbed to the separation agent 2 detach from the separation agent 2 due to the action of the detachment agent and float in the detachment liquid.

This release agent is a substance with a structure that is more strongly recognized than the sugar chain structure on the cell membrane surface recognized by the lectin, or the various activities of the lectin (such as aggregation of animal and plant cells, precipitation of polysaccharides and complex carbohydrates). It is a substance that inhibits the property of The detachment effect caused by the detachment agent is thought to be caused mainly by specific binding between the lectin and the adsorbed cells.

As a specific example of the withdrawal agent, carbohydrates are preferred, and among these, monosaccharides and oligosaccharides are particularly preferred.

The concentration of the withdrawal agent in the withdrawal solution is preferably about 1 to 500 mM, and in addition to the withdrawal agent, the withdrawal solution also contains electrolytes,
Additives such as proteins, lipids, antioxidants, and antibiotics may also be included.

Depending on the type and molecular structure of these detachment agents, some detachment agents indiscriminately detach all types of cells that are adsorbed to the separation agent 2, but some detachment agents indiscriminately detach all types of cells that are adsorbed to the separation agent 2, but some detachment agents indiscriminately detach all types of cells that are adsorbed to the separation agent 2. Furthermore, one that selectively (preferentially) detaches target cells among them is preferable.

In this way, the detachment liquid containing the cells detached from the separation agent 2 flows out from the outlet 5 by opening the three-way stopcock 4 and is collected into the test tube 8.

The separated liquid collected in the test tube 8 contains a high concentration of cells that had been adsorbed to the separation agent 2, particularly target cells.

In addition, in the lymphocyte isolation method of the present invention, the first method or the second method may be repeated multiple times, or the first method and the second method may be used together to perform two methods. The above target cells may be collected simultaneously and separately.

<Example> Hereinafter, specific examples of the present invention will be described.

(Example 1) The raw material composition of the carrier (water-insoluble solid substance), divinylbenzene monomer, glycidyl methacrylate monomer, and 2-hydroxyethyl acrylate oligomer (molecular weight 230), was 22.8:4.6.2: 31. ○
Granules of terpolymer (manufactured by Fujikura Kasei, MR1)
104, average particle size 0.15 mm), as a lectin,
The method of Tomita et al. (M, Tomi
ta, et al., ,Experientia, 28
．． 84 (1972))
(Ricinus communisagglutin
in, castor bean lectin, molecular position: approx. 120.000
) was immobilized.

First of all, 0.2M (7) carbonic acid loose i! 1(rl(pt(10
) to the above RCA,. The above carrier was added at a ratio of 0.5 g (wet)/mj to a solution in which 0.815 wt% of
BM-101) and stirred at room temperature for about 48 hours.

Next, after washing the reaction product with distilled water, 1M monoethanolamine (pH 8) was added to remove unreacted oxirane groups.
It was immersed in the solution and stirred for about 20 hours.

Thereafter, the reaction mixture was mixed with distilled water, 0.2M carbonate buffer (pH
10), distilled water, O, LM acetate buffer (pH 4), distilled water, O, LM phosphate buffer (pH 7, 4), and distilled water were washed in this order to obtain a separation agent.

The prepared separation agent was dispersed in phosphate buffered balanced salt solution (PBS) with a pH of 7.2, and an optical device was placed in a wet state in a polypropylene column with an inner diameter of 8 mm and a length of 50 mm.
The oj was packed as close as possible and used as a separation device.

Using this separation device, the following cell separation experiment was conducted.

The test solution is PBMC (peripheral blood) containing lymphocyte fraction collected from blood collected from a normal person and supplemented with CPD (citrate phosphate glucose solution).
mononuclear cells)! e turbid liquid 0.2
After gently injecting 5mj into the column and bringing it into contact with the separation agent, PBS (pH 7,2) was further added to submerge all the cells in the separation agent, and the cells were left at room temperature for 30 minutes.

Thereafter, the inside of the column was washed with 2mj of PBS ([))17.2), and the cells that had flowed out were collected.

After measuring the number of cells in this collected solution with a multi-item automatic blood analyzer (manufactured by Toa Medical Electronics Co., Ltd., Sysmex NE-6000), FITC-labeled anti-Leu2a antibody (equivalent to CDS)
and PE-labeled anti-Leu3a antibody (equivalent to CDJ) (both manufactured by Becton Dickinson), and analyzed with an automatic cell analyzer (Cyto ACE-100, manufactured by JASCO Corporation) to analyze suppressor/cytotoxic child cells and helper/ The percentage of inducer T cells was determined for each. The adhesion rate of each cell was then calculated by comparing it with the proportion of T lymphocyte subsets before contact with the separation agent, and the separation ability of the separation agent was evaluated.

The results are shown in Table 1.

(Example 2) The lectin used as a separation agent was added to RCA-120 (Hima Bean Lectin 120, manufactured by Seikagaku Corporation), the concentration when immobilizing the lectin was set to 0.5%, and the stirring time during immobilization was A cell separation experiment was conducted in the same manner as in Example 1, except that the time period was changed to approximately 120 hours.

The results are shown in Table 1.

(Example 3) Using the same separation device as in Example 1, as a test liquid, CPD-added blood collected from a normal person was gently injected into the column and allowed to flow down naturally. The red blood cells in the collected cells were 0.8
Hemolysis was performed using a 26% ammonium chloride solution (pH 7.4), and the collected cells were evaluated in the same manner as in Example 1.

The results are shown in Table 1.

(Comparative Example) A cell separation experiment was conducted in the same manner as in Example 1, except that the MR1104 itself was used as the separation agent.

The results are shown in Table 1.

Table 1 Ratio of Leu3a''/Leu2a'' in recovered cells B: Ratio of A after passing through the column/A before passing through the column (%) C: Adhesion rate of leukocytes (%) D Adhesion rate of Nirimba cells ( %) E: Adhesion rate of non-lymphocytes (%) F: Suppressor/cytotoxic child cells (Leu2
a') Adhesion rate (%) G: Helper/inducer T cell (Leu3a'') adhesion rate (%) As is clear from the results shown in 1 above, Examples 1 to 3
All of the lymphocyte separation agents of the present invention according to the present invention selectively adsorb helper/inducer T lymphocytes compared to comparative lymphocyte separation agents that do not contain lectin. We were able to concentrate the cells.

In particular, regarding Example 3 in which whole blood was used as the test liquid,
Excellent results were obtained.

As an additional experiment, for each of the separation apparatuses of Examples 1 to 3 above (adsorbed by the separation agent), separation liquids A and B containing monosaccharides and oligosaccharides, respectively, were injected into each column and held for a predetermined time. After that, these detached solutions were collected, and the number of cells contained in the detached solutions was determined in the same manner as described above.

As a result, the presence ratio of helper/inducer T lymphocytes in both withdrawal fluids A and B was high.

<Effects of the Invention> As described above, according to the present invention, target cells, particularly lymphocyte subclasses or subsets thereof, can be separated and concentrated with high yield and purity without impairing cell functions. Moreover, the separation operation is extremely simple and quick.

Therefore, for example, basic research such as functional analysis of lymphocytes related to immune function, various clinical tests, diagnosis and treatment of immune diseases, and especially adoptive immunotherapy (specific cells are activated outside the body and then returned to the body). When applied to medical therapy, more reliable results, more effective treatments, or more efficient processing operations can be expected.

[Brief explanation of drawings]

FIG. 1 is a partially cutaway sectional front view showing an example of the structure of the lymphocyte separation device of the present invention. Explanation of symbols 1...Column 2...Separation agent 3...Filter member 4...Three-way stopcock 5...Outlet port 6...Actual diameter section 7...Reduced diameter section 8...・Test tube application Representative of Hitoshi Terumo Co., Ltd.

Claims (11)

[Claims] (1) A lymphocyte separation agent characterized in that a lectin having an affinity for a specific lymphocyte subclass or a specific subset thereof is immobilized on a water-insoluble solid substance. (2) The lymphocyte separation agent according to claim 1, wherein the water-insoluble solid substance is in the form of particles. (3) The lymphocyte separation agent according to claim 2, wherein the water-insoluble solid substance has a particle size of 0.05 to 5 mm. (4) A lymphocyte separation device comprising a container filled with the separation agent according to any one of claims 1 to 3. (5) The lymphocyte separation device according to claim 4, wherein the inside of the container is sterilized. (6) A test solution containing lymphocytes is brought into contact with the separation agent according to any one of claims 1 to 3, so that the separation agent adsorbs a specific lymphocyte subclass or a specific subset thereof in the test solution. , a lymphocyte separation method characterized by collecting cells that are not adsorbed to a separation agent. (7) A test solution containing lymphocytes is brought into contact with the separation agent according to any one of claims 1 to 3, so that the separation agent adsorbs a specific lymphocyte subclass or a specific subset thereof in the test solution. . A method for separating lymphocytes, which comprises: then separating the cells adsorbed to the separation agent from the separation agent and collecting them. (8) The method for separating lymphocytes according to claim 7, wherein a specific lymphocyte subclass or a specific subset thereof is selectively detached from among the cells adsorbed to the separation agent and recovered. (9) The method for separating lymphocytes according to claim 7 or 8, wherein the detachment of the cells from the separation agent is performed by bringing the detachment agent into contact with the separation agent. (10) The method for separating lymphocytes according to claim 9, wherein the withdrawal agent is a carbohydrate. (11) The method for separating lymphocytes according to claim 10, wherein the carbohydrate is a monosaccharide or an oligosaccharide.