JPH04346801A - Agent, device and method for separating lymphocyte subclass - Google Patents

Abstract

PURPOSE:To efficiently separate lymphocyte subclass by bringing a liquid to be tested into contact with a separating agent for lyphocyte subclass prepared by coupling oxirane-acrylic beads with an oxyalkylamine so that the B- lymphocyte and nonlymphocyte luekocyte are selectively adsorbed to the separating agent. CONSTITUTION:The separating agent for lymphocyte subclass is prepared by coupling an oxyalkylamine with glycidyl group of oxirane-acrylic beads as an insoluble carrier. The liquid to be tested is brought into contact with this separating agent so that the B-lymphocyte and nonlymphocyte luekocyte are selectively adsorbed to the separating agent. The oxirane-acrylic beads are polymers obtd. from the mixture of unsatd. radical-polymerizable monomers having oxirane groups and cross-linking monomers. Thereby, lymphocyte subclass cells which act an important roll for immunocyte in tissue or humor such as blood, especially, T-lymphocyte can be separated and corrected without damaging the function of the cell.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention is useful for removing B lymphocytes and T cells in immunological research, clinical tests, and immunological disease treatment.
This invention relates to a lymphocyte subclass separation agent used to purify and collect or import lymphocytes, a separation device using the same, and a separation method.

0002

[Prior art] Pluripotent stem cells, which are derived from blood cells, are
lymphocytes, monocytes (macrophages), granulocytes (neutrophils,
The cells differentiate into eosinophils, basophils), platelets, and red blood cells.

[0003] Lymphocytes, which play an important role in the immune surveillance mechanism, can be roughly divided into T lymphocytes, which mature under the influence of the thymus gland, and T lymphocytes, which mature under the influence of the thymus gland, and T lymphocytes, which mature under the influence of the thymus. B lymphocytes are differentiated into B lymphocytes, which mature under the influence of B lymphocytes. T lymphocytes are responsible for cell-mediated immunity, and B lymphocytes are responsible for humoral immunity.

[0004] T lymphocytes can be divided into several subsets from a functional standpoint. For example, helper T cells, T cells involved in delayed hypersensitivity reactions, and suppressor T cells.
These include cells and cytotoxic killer T cells. B lymphocytes differentiate into plasma cells and produce antibodies based on instructions from T lymphocytes.

[0005] In recent years, attempts have been made to separate and fractionate lymphocytes, which have such important functions, and use them in basic immunological research and various diagnoses and treatments, but each method has the following drawbacks. It was something that I had.

[0006] Namely, [1] a method based on velocity sedimentation that utilizes differences in cell size, [2] a method that uses specific gravity centrifugation that utilizes differences in specific gravity of cells, and [3] a method that uses differences in cell surface charge. The cell electrophoresis method used has no significant difference in physical properties such as specific gravity and density between T lymphocytes and B lymphocytes, so the yield or purity of the separated and collected cells may be affected. High accuracy was not required. In particular, in the specific gravity centrifugation method [2], the price of the medium is expensive, or even if it is cheap, it requires skill to operate, and in the cell electrophoresis method [3] They also have drawbacks such as the mobility differs depending on the cell, the influence of electric fields on cell functions has not been elucidated, and large-scale processing is difficult.

[4] A method using affinity chromatography that utilizes the specific binding ability of cells; [5]
] A rosette sedimentation method using cell rosette formation as an indicator; [6] Fluorescence-activated cell sorter (Fluorescence-A) that utilizes a reaction between a specific antigen on the cell membrane surface, immunoglobulin, and a labeled antibody.
activated Cell Sorting, F
In all methods using the ACS method, the treated cells are subject to strong biologically specific bonds and stimulation, making it difficult to recover the target cells in an intact state and not suitable for large-scale processing. Furthermore, the affinity chromatography in [4] requires expensive antibodies, and the FACS method in [6] requires complicated pretreatments such as labeling cells, and requires expensive antibodies and equipment. It had the following drawbacks.

[7] Field-Flow Fractionation is a method for multi-stage separation that does not use a support or a fixed layer.
ation, FFF) method (see, for example, Junichi Yada, Michio Fujiwara, eds., New Lymphocyte Function Search Method, 1978, Chugai Igakusha, etc.), but the In addition to the difficulties associated with the inflow and outflow equipment, the materials of the rotor-shaped separation cells and flow channels cause adhesion of cells, so mutual separation of cells has not yet been put to practical use.

On the other hand, [8] a method using a column filled with nylon wool, glass wool, etc., which utilizes the difference in non-specific adhesion activity of cells, has little risk of damaging the function of treated cells, and Although it is suitable in terms of price, operability, and large-scale throughput, it is insufficient in terms of selectivity and recovery rate for target cells. There was also the major issue of requiring experiential and technical proficiency in the case.

[0010] As separation agents and separation methods that utilize such non-specific adhesion properties, hydrophobic materials (Japanese Patent Publication No.
No. 17387, No. 59-36963, No. 62-452
06, JP-A No. 57-204454), materials with acidic functional groups (JP-B No. 59-36961, JP-A-56-1998)
No. 140886, No. 56-152740), polymers containing basic functional groups (Japanese Unexamined Patent Publication No. 59-216584,
60-105490), hydroxyapatite fiber (Japanese Patent Application Laid-Open No. 63-284), specific polymers (Japanese Patent Application Laid-open No. 63-284),
1-221123 and 64-34285), but even these methods have not been sufficient in terms of selectivity and recovery rate for target cells.

[0011]

[Problems to be Solved by the Invention] The present invention solves the problem that, in such conventional techniques, high accuracy is not required for the yield or purity of separated and collected cells in cell separation of test fluid containing lymphocytes. This was done in consideration of problems such as the need for proficiency in operation, and the fact that cells are easily stimulated and damaged. Therefore, the present invention provides a lymphocyte subclass separation agent that can efficiently separate lymphocyte subclasses of immunocompetent cells and recover them without impairing cell function, and a separation device and separation method using the same. The purpose is to

The present invention further provides a lymphocyte subclass separation agent that is easy to operate, does not require special equipment, and can efficiently separate lymphocyte subclass cells, and a separation device and separation method using the same. The purpose is to

[0013]

[Means for Solving the Problems] Such objects are achieved by the following inventions 1 to 7. (1) Oxirane - a lymphocyte subclass separating agent characterized by being formed by bonding oxyalkylamine to acrylic beads. (2) The lymphocyte subclass separating agent according to item 1, wherein the oxirane-acrylic beads are a polymer obtained from a mixture of an unsaturated radically polymerizable monomer having an oxirane group and a crosslinkable monomer. (3) The lymphocyte subclass separating agent according to item 1 or 2, wherein the oxirane group content of the oxirane-acrylic beads is 0.1 to 10 mmol per 1 g of dry weight. (4) The radically polymerizable monomer is 5 to 60% by weight
, The lymphocyte subclass separating agent according to item 2 or 3, wherein the crosslinking monomer is 5% by weight or more. (5) The lymphocyte subclass separating agent according to any one of items 1 to 4, wherein the oxyalkylamine is selected from the primary alkanolamine group of monooxymonoamines. (6) A lymphocyte subclass separation device comprising a container filled with the lymphocyte subclass separation agent according to any one of items 1 to 5. (7) Bringing a test liquid containing lymphocytes into contact with the lymphocyte subclass separating agent according to any one of items 1 to 5, and separating B lymphocytes and non-lymphocyte leukocytes in the test liquid into the lymphocyte subclasses. A method for separating lymphocyte subclasses, which comprises purifying T lymphocytes in a test fluid by selectively adsorbing them to an agent.

The lymphocyte subclass separating agent according to the present invention is characterized by being formed by bonding oxyalkylamine to oxirane-acrylic beads, and selectively adsorbs B lymphocytes and non-lymphoid leukocytes. It is possible to separate lymphocyte subclass cells, particularly T lymphocytes, from body fluids such as blood or tissues with high yield.

[0015] As mentioned above, lymphocyte subclass cells play an important role as immunocompetent cells. Therefore, as mentioned above, if B lymphocytes and non-lymphoid leukocytes can be selectively adsorbed and removed and T lymphocytes can be recovered, it will not only be useful for immunological research, clinical tests, and treatment of immune diseases, but also for the prevention of cancer diseases. Adoptive immunotherapy may also be applicable.

The lymphocyte subclass separating agent of the present invention uses oxirane-acrylic beads as an insoluble carrier, and has an oxyalkylamine bonded to the glycidyl group. Therefore, the insoluble carrier, the oxirane alkylamine, and the bonding method thereof will be explained respectively.

The oxirane-acrylic beads used as insoluble carriers are obtained by copolymerization of glycidyl methacrylate or allyl glycidyl ether, methacrylamide, and methylene-bis-methacrylamide. Its manufacturing method is disclosed by Röhm Pharma (UK Patent No. 1329062, German Patent Publication No. 22373).
16, German Patent No. 2263289)
. Among these oxirane-acrylic beads, those that are particularly preferably used as the insoluble carrier in the present invention are:
The proportion of crosslinking monomer is 5% by weight or more, and the proportion of radically polymerizable monomer having an oxirane group is 5 to 60% by weight.
It was obtained from a monomer mixture of A specific example of such particularly preferred oxirane-acrylic beads is Eupergit C, a trade name manufactured by Rehm Pharma.

[0018] The shape of the oxirane-acrylic beads is preferably spherical so that they do not damage cells and are difficult to break or chip. The average particle size is in the range of 0.05 to 5 mm, taking into account the flow rate and circulation pressure of the test liquid.
Preferably, a thickness in the range of 0.1 to 0.2 mm is used.

Oxirane-acrylic beads have oxirane groups on their surfaces. As described below, this oxirane group reacts with the oxyalkylamine to form a covalent bond.

The content of oxirane groups in oxirane-acrylic beads was determined by the thiosulfate method (R.Axe
n, as quoted by L. Sundb
erg and J. Parath in C
chromatography, 90, 89 (1974)
), 0.1 to 10 mmol/g-dr
The range of y is preferably 0.6 to 1.0 m, more preferably 0.6 to 1.0 m.
mol/g-dry.

Next, the oxyalkylamine bonded to the oxirane-acrylic beads will be explained.

Oxyalkylamines are aliphatic compounds having a hydroxyl group and an amino group in their molecules, and are also called alkanolamines or aminoalcohols.

Among oxyalkylamines, compounds belonging to the primary alkanolamine group of monooxymonoamines represented by the following molecular formula (I) have a simple structure and are resistant to interactions between oxyalkylamine molecules and non-specific adsorption. This is particularly suitable because it does not cause HO(CH2)nNH2 n is an integer, preferably n=1 to 10 (Formula I) The above oxyalkylamine is bonded to the oxirane group present on the surface of the oxirane-acrylic beads. The formation of this bond involves
With the opening of the oxirane group, the oxygen atoms forming the oxirane group become hydroxyl groups and bond together. The functional group of the oxyalkylamine involved in this bond is an amino group, but a hydroxyl group may also be involved in some cases.

Next, the lymphocyte subclass separating agent of the present invention obtained as described above will be explained.

One feature of the lymphocyte subclass separating agent of the present invention is that the oxirane-acrylic beads used as the insoluble carrier and the oxyalkylamine are firmly bonded via covalent bonds as described above. Methods for immobilizing organic compounds on the surface of insoluble carriers include, in addition to covalent bonding, carrier binding methods such as ionic bonding, physical adsorption, hydrophobic bonding, and biochemical specific bonding, as well as crosslinking methods, entrapment methods, and composite methods. However, when immobilization is performed by a method other than covalent bonding, the oxyalkylamine is likely to be desorbed in the test solution, resulting in a lack of stability.

The most important feature of the lymphocyte subclass separating agent of the present invention is that it has high efficiency and high selectivity for B lymphocytes and non-lymphoid leukocytes, as shown in the results of the examples described below. The point is that it exhibits excellent adsorption capacity. This characteristic is obtained not only by the contribution of the oxyalkylamine but also by the combination of the contribution by the oxirane-acrylic beads used as a carrier.

Generally, lipids are easily adsorbed to separation agents composed only of hydrophobic compounds, that is, separation agents with strong hydrophobic bonds, and therefore cells to which cell membrane lipids are firmly adsorbed are damaged. On the other hand, separation agents composed of compounds that have many charged groups with strong electrostatic binding properties tend to adsorb proteins containing many basic or acidic amino acids, and therefore cells with strongly adsorbed cell membrane proteins are damaged. receive. Therefore, in order to separate lymphocyte subclasses in a test fluid, a separation agent that has an appropriate combination of various interactions is suitable. From this viewpoint, the lymphocyte subclass separating agent of the present invention is considered as follows.

First, the hydroxyl group formed by ring opening of the oxirane group and the hydroxyl group of the oxyalkylamine exhibit hydrophilicity. Furthermore, the imino group formed by bonding with the oxirane group has hydrogen bonding properties. Next, acrylic beads have a methyl group exhibiting hydrophobicity and an acid amide group exhibiting hydrogen bonding and electrostatic bonding properties on the surface of the polymeric carbon chain.

Due to these factors, the separation agent of the present invention has an interaction with the cell membrane surface substances of B lymphocytes and non-lymphoid leukocytes that has a suitable combination of hydrophobic bonds, electrostatic bonds, and hydrogen bonds. It is presumed to have excellent lymphocyte separation ability.

Next, the lymphocyte subclass separation device of the present invention will be explained with reference to FIG.

FIG. 1 is a diagram showing the structure of a preferred embodiment of the lymphocyte subclass separation device of the present invention.

The lymphocyte subclass separation device 1 of the present invention includes a column container 4 having an inlet 2 and an outlet 3 for body fluid.
The above-mentioned lymphocyte subclass separating agent 5 is filled and held inside.

As the material for the column container 4, glass, stainless steel, polyethylene, polypropylene, polycarbonate, polystyrene, polymethyl methacrylate, etc. can be used, but polypropylene and polycarbonate are particularly preferred because they can be sterilized in an autoclave and are easy to handle. Moreover, it is preferable to provide mesh filters 6a and 6b between the body fluid inlet 2 and outlet 3 and the separating agent 5 layer, through which the body fluid passes but not the separating agent. The material for the filters 6a and 6b is not particularly limited as long as it is physiologically inert and has a certain degree of strength, but polyester and polyamide are preferably used.

That is, the body fluid introduced from the inlet 2 of the column container 4 passes through the lymphocyte subclass separation agent layer held by the filters 6a and 6b, is subjected to adsorption treatment, and is led out from the outlet 3. It is something.

[0035]

[Operation] There are two methods for performing extracorporeal circulation body fluid purification therapy using the lymphocyte subclass separation device of the present invention.

[0036] The first method is to centrifuge the blood taken out from the body to collect the buffy coat (white blood cell layer).
After this buffy coat is treated with the lymphocyte subclass separation device of the present invention, it is returned to the body together with components other than the buffy coat. Alternatively, blood taken from the body can be subjected to density gradient centrifugation to form mononuclear cells (PBM).
C: Peripheral Blood Mononu
This is a method in which mononuclear cells are collected, treated with the lymphocyte separation device of the present invention, and then returned to the body together with the remaining components.

[0037] The second method is to process blood taken from the body by directly passing it through the device.

The second method will now be explained with reference to FIG.

FIG. 2 is a diagram showing a circuit configuration used in extracorporeal circulation body fluid therapy according to the second method.

That is, blood is introduced into the circuit from the blood sampling port 7, passes through the chamber 9a equipped with the blood pump 8 and the pressure gauge 10a, and passes through the inlet in the lymphocyte subclass separation device 1 of the present invention at a constant flow rate. introduced in 2,
It comes into contact with the lymphocyte subclass separating agent 5 of the present invention housed in the column container 4 .

[0041] The blood thus treated in the lymphocyte subclass separation device 1 was led out from the outlet 3 of the column container 4, passed through a chamber 9b equipped with a pressure gauge 10b, and was heated in a constant temperature bath 11. Thereafter, the blood is returned to the patient's body through the blood return port 12.

[0042] The method of circulating body fluids may be carried out continuously or intermittently depending on clinical needs and equipment conditions.

[0043] The lymphocyte subclass separation device of the present invention includes:
Preferably, the areas that come into contact with blood are sterilized before use. Sterilization methods include moist heat sterilization (autoclave sterilization), gas sterilization (eg, ethylene oxide sterilization), and radiation sterilization. The lymphocyte subclass separation device of the present invention does not show any decrease in performance even after sterilization treatment such as moist heat sterilization, gas sterilization, or radiation sterilization, and exhibits stable lymphocyte separation ability.

Although the lymphocyte subclass separation agent and separation device of the present invention have been described above, the present invention is not limited thereto. For example, instead of a column container,
The configuration can be changed in various ways, such as using a syringe outer cylinder.

Next, the present invention will be explained in more detail by showing examples.

[0046]

[Example] [Example 1] 0.2M carbonate buffer (pH 10
) to monoethanolamine (molecular formula HOCH2CH2N
H2) was dissolved at a concentration of 0.5M. In this solution, oxirane-acrylic beads (manufactured by Röhm Pharma, trade name: Eupergit C, the proportion of polymerizable monomers is 20% by weight, the proportion of crosslinkable monomers is 30% by weight, and the average particle size is 0.
．． 15 mm, oxirane group content is 0.8 mmol/g
-dry) at a rate of 0.55 g/ml and degassed. After heating this in a water bath at 80°C for 3 hours, it was heated using a slow rotating mixer (manufactured by Taiyo Kagaku Kogyo Co., Ltd.: trade name Rotator II).
) and stirred overnight. After that, reverse osmosis water (RO water), 0.2M carbonate buffer (pH 10), 0.1M acetate buffer (pH 4), 0.1M phosphate buffer (pH 7.4)
Washed sequentially with Then, using an autoclave, heat with 0.1M acetate buffer (pH 4) and then 0.1M phosphate buffer (pH 7.4) (120℃, 20mi
After n), it was thoroughly washed with RO water.

The obtained separation agent was suspended in phosphate buffered saline (PBS) at pH 7.2, and 1 ml was filled into a 2.5 ml disposable syringe (manufactured by Terumo Corporation) containing a polyester nonwoven fabric inside the tip. did. Then, a polyester nonwoven fabric was placed on top of the filled separation agent to hold the separation agent layer, and a 25G needle and a cell storage container were attached to the tip of the syringe to produce a lymphocyte subclass separation device.

Using this separation device, the following lymphocyte subclass separation experiment was conducted.

Human peripheral blood mononuclear cell suspension (PBMC) was obtained from the upper end opening of the syringe constituting the separation device using a mononuclear cell separation tube (manufactured by Becton Dickinson, trade name: Leucoprep). 200 μm (white blood cell count 200
0x104 cells) and 10% fetal calf serum (FCS).
After submerging the cells in the separating agent layer with 100 μl of RPMI1640 culture solution containing % of the culture solution, the cells were allowed to stand at room temperature for 30 minutes. Next, 2m
The separation agent was rinsed with 10% FCS-RPMI1640, and the cells that had flowed out were collected. After measuring the number of cells in this collected solution using a multi-item automatic blood separation device (manufactured by Toa Medical Electronics Co., Ltd., trade name: Sysmex NE-6000), a phycoerythrin (PE)-labeled anti-Leu-12 antibody (Becton, Dickinson Co., Ltd.) and a flow cytometer (JASCO Co., Ltd.: trade name: Cyto A).
ce-150), the percentages of B lymphocytes and lymphocytes were determined. Then, the number of cells, B lymphocytes, and the percentage of lymphocytes in the suspension before contact with the separation agent were determined in the same way, and by comparing these, the adhesion rate of each cell to the separation agent was calculated. . The results are shown in Table 1.

[Example 2] Instead of monoethanolamine solution, 6-amino-1-hexanol (molecular formula HO
(CH2)6NH2) solution was used, otherwise Example 1
A separating agent was prepared in exactly the same manner. Then, in the same manner as in Example 1, the adhesion rate of each cell to the separation agent was measured. The results are shown in Table 1.

[Comparative Example 1] A separating agent was prepared in the same manner as in Example 1 except that β-alanine (molecular formula: HOOCCH2CH2NH2), which is an amino acid, was used instead of the monoethanolamine solution. Then, in the same manner as in Example 1, the adhesion rate of each cell to the separation agent was measured. The results are shown in Table 1.

[Comparative Example 2] 0.12 g of nylon wool (Bi
A separation device was produced in the same manner as in Example 1, except that a disposable syringe (manufactured by Otest) was filled in a volume of 1 ml instead of the separation agent used in Example 1. Then, in the same manner as in Example 1, the adhesion rate of each cell to the separation agent was measured. The results are shown in Table 1.

[0053]

[Table 1]

From Table 1, it was confirmed that the lymphocyte subclass separating agents according to Examples 1 and 2 had a high value of E and a low value of F, so that B cells were selectively adsorbed. Furthermore, D was also high, indicating that non-lymphoid leukocytes were also selectively adsorbed. On the other hand, the lymphocyte subclass separating agents according to Comparative Examples 1 and 2 have a lower value of E than Examples 1 and 2 and a higher value of F than Examples 1 and 2, so they have poor selectivity for B cells. It was confirmed that it was inferior.

[0055]

[Effects of the Invention] As detailed above, the lymphocyte subclass separating agent of the present invention is characterized by being formed by bonding oxyalkylamine to oxirane-acrylic beads having an oxirane group. Lymphocyte subclasses can be selectively, efficiently and conveniently separated in large quantities. Furthermore, since a suitable oxyalkylamine is a low-molecular synthetic organic compound, sterilization can be performed easily and reliably. Due to these characteristics, the lymphocyte subclass separating agent of the present invention is effective for basic research such as functional analysis of lymphocytes related to immune function, and further for diagnosis and treatment of various immune diseases.

Furthermore, since the lymphocyte subclass separation device of the present invention is characterized by containing the above-mentioned lymphocyte subclass separation agent, it is possible to combine the lymphocyte subclass separation agent with excellent characteristics with body fluids. Contact with cell suspensions can be made more efficiently, for example, when applied to extracorporeal circulation therapy for immune diseases, specific lymphocytes or subclasses of lymphocytes can be removed from the patient's blood in a short time and efficiently. As a result, more efficient treatment or processing operations can be expected.

Furthermore, when the lymphocyte subclass separation device of the present invention is applied to extracorporeal circulation therapy as described above, the separation agent supplemented with specific lymphocytes is treated with a cytokine, a cell differentiation factor, or an immunomodulator while acting on the separation agent. After culturing the cells, it is possible to collect the lymphokines and antibodies produced by the lymphocytes, or to detach the lymphocytes with an adsorption antagonist and return them to the patient's body, making it possible to expect a wider variety of treatments. .

[0058] The lymphocyte subclass separation method of the present invention includes:
By bringing a test solution containing lymphocytes into contact with a lymphocyte subclass separating agent and adsorbing B lymphocytes and non-lymphoid leukocytes in the test solution to the lymphocyte subclass separating agent,
Since it is characterized by purifying T lymphocytes in the sample fluid, it is effective in research related to immune function and in the diagnosis and treatment of various immune diseases.

Furthermore, the lymphocyte subclass separation device of the present invention exhibits stable lymphocyte separation ability without any deterioration in performance even after sterilization treatment such as moist heat sterilization, gas sterilization, or radiation sterilization. Accurate diagnosis and safe treatment will become possible.

[Brief explanation of drawings]

FIG. 1 is a cross-sectional view schematically showing the structure of one embodiment of the lymphocyte subclass separation device of the present invention.

FIG. 2 is a drawing showing the configuration of an extracorporeal circulation therapy circuit incorporating the lymphocyte subclass separation device of the present invention.

[Explanation of symbols]

1 Lymphocyte subclass separation device 2 Body fluid inlet 3 Body fluid outlet 4 Column container 5 Lymphocyte subclass separation agent 6 Filter 7　Blood sampling port 8 Blood pump 9a, 9b Chamber 10a, 10b pressure gauge 11　Thermostatic chamber 12 Blood return port

Claims (7)

[Claims] 1. A lymphocyte subclass separating agent comprising oxirane-acrylic beads bound to an oxyalkylamine. 2. The oxirane-acrylic beads include:
The lymphocyte subclass separating agent according to claim 1, which is a polymer obtained from a mixture of an unsaturated radically polymerizable monomer having an oxirane group and a crosslinkable monomer. 3. The oxirane group content of the oxirane-acrylic beads is 0.1 to 10 per gram of dry weight.
The lymphocyte subclass separating agent according to claim 1 or 2, which is mmol. 4. The radically polymerizable monomer contains 5 to 6
The lymphocyte subclass separating agent according to claim 2 or 3, characterized in that the amount of the crosslinking monomer is 5% or more by weight. 5. The lymphocyte subclass separating agent according to claim 1, wherein the oxyalkylamine is selected from the primary alkanolamine group of monooxymonoamines. 6. A lymphocyte subclass separation device comprising a container filled with the lymphocyte subclass separation agent according to any one of claims 1 to 5. 7. A test solution containing lymphocytes is brought into contact with the lymphocyte subclass separating agent according to any one of claims 1 to 5, and B lymphocytes and non-lymphoid leukocytes in the test solution are separated from the lymphocytes. A lymphocyte subclass separation method characterized by purifying T lymphocytes in a test fluid by selectively adsorbing them to a cell subclass separation agent.