JPH0332680A - Separating material and separating device for t lymphocyte - Google Patents

Abstract

PURPOSE:To efficiently separate the T lymphocytes of immune charge cells and to recover the cells without impairing the cell function by immobilizing the peptide having affinity to the T lymphocytes to a water insoluble solid material. CONSTITUTION:The T lymphocyte separating material is formed by immobilizing the peptide having the affinity with the T lymphocytes to the water insoluble solid material. The peptide having the affinity with the T lymphocytes refers to an org. compd. in the form in which 2 to 100 pieces of amimo acids are conjugated by a peptide bond (-CO-NH-). Particularly the thymic hormone relating to the maturation of the T lymphocytes and further the bioactive section of the thymic hormone and peptide T among these peptides are more preferable examples. The T lymphocytes in the bodily fluid, such as blood or tissue are separated from the other cells in this way with high selectivity or at a high yield by utilizing the high affinity of the peptide or lipid with the antigen receptor on the membrane surface of the T lymphocyte.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Applications) The present invention relates to a T lymphocyte separation material and a separation device. Specifically, the present invention relates to a separation material capable of selectively capturing T lymphocytes among cells that play an important role in the immune surveillance mechanism, and a separation device having a column filled with the separation material. The present invention also relates to a T lymphocyte separation material and a separation device that can be applied to the diagnosis and treatment of various immune diseases by selectively capturing T lymphocytes that are deeply involved in cellular immunity.

(Prior art) 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 (1mmunocompete
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, the final effects of the antigen-antibody reaction, such as foreign body disposal and inflammatory reactions centered on vascular reactions, are the parallel legs of the immune response, and involve macrophages, granulocytes, and mast cells.

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.

Lymphocytes are broadly divided into T lymphocytes, which mature under the influence of the thymus gland, and B lymphocytes, which mature under the influence of the bursa of Fabricius, or the equivalent organs in mammals (hematopoietic centers such as the liver and bone marrow). However, in the case of B lymphocytes, after antigen stimulation, they show a unidirectional change of differentiation and proliferation into antibody-producing cells and production of antibodies, whereas T lymphocytes show a simple change. Not just one, but one antigen stimulus causes changes in several types of T lymphocytes with various functions, some of which function as effector cells and others that function as control cells (promoting or suppressing differentiation and proliferation). That is, it is thought that T lymphocytes exist in functional subsets. So-called helper lymphocytes (T,, ), killer (cytotoxic, cy
toxic) T lymphocytes (Tc), suppressor T lymphocytes (Ts), T involved in delayed-type allergies
A subset such as lymphocytes (TDTI+). 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 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.

In other words, (1) methods such as velocity sedimentation that take advantage of differences in cell size, (2) methods such as specific gravity centrifugation that take advantage of differences in cell specific gravity, and (3) methods that take advantage of differences in cell surface charge. Methods such as cell electrophoresis, (4) Removal of monocytes with carbanyl iron using negative-negative activity, (5) Filling with nylon wool, glass wool, etc. using differences in non-specific adhesion activity of cells. Methods such as using columns or using petri dishes made of glass, plastic, etc., (6)
Affinity chromatography that utilizes the specific binding ability of cells (7) Rosette sedimentation method that uses cell rosette formation as an indicator (8) Fluorescein activation that utilizes the reaction between specific antigens on the cell membrane surface, immunoglobulins, and labeled antibodies Ted Cell Sorter (Fluorescenc)
e Activating Ce1l Sortlng
, FACS) method, etc. (see, for example, Ya10 Jun-1, edited by Michio Fujiwara, New Lymphocyte Function Exploration Method, 1987, Chugai Igakusha, etc.). In addition, as a means of multistage separation without using a support or stationary phase, (9) field flow fractionation (Peil-Plow
['actionation, F F F) 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 are no significant differences in physical properties such as density, high precision cannot be required for the yield or purity of separated and collected cells. In the cell electrophoresis method (3), the mobility differs depending on the maturity level of the cells.
It also had drawbacks such as the lack of understanding of the effects of electric fields on cell functions and the national difficulty in processing large quantities.

In addition, cell separation methods using specific antigens, receptors, immunoglobulins, etc. on the cell membrane surface as indicators in (6) to (8) all require that the treated cells have strong biologically specific bonds or stimulation. In addition, the affinity chromatography method (6) requires expensive antibodies, and the FAC3 method (8) The disadvantages are that pretreatment such as labeling the cells is complicated and that expensive antibodies and equipment are required.

In addition, in the FFF method (9), the eluent is continuously flowed in and out through the rotating shaft, which is difficult in terms of equipment, and the materials of the rotor-shaped separation cell and the channel cause cell adhesion. We have not yet reached the point where mutual separation of the two is put to practical use.

On the other hand, the method (5) that utilizes the non-specific adhesion properties of cells has less risk of damaging the functions of treated cells, and is also suitable in terms of cost, operability, and mass processing. However, it is insufficient in terms of selectivity and recovery rate for target cells, and there is also a major problem in that the column method mentioned above requires experience and technical proficiency when performing separation operations. It was something that was done.

As separation phases 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 containing 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
3-284), specific polymers (JP-A No. 61-221)
123, No. 64-34285), but even these methods have not yet been sufficient in terms of selectivity and recovery rate for target cells.

(Problem A to be Solved by the Invention) Therefore, an object of the present invention is to provide a novel T lymphocyte separation material and separation device. The present invention also enables efficient separation of T lymphocytes, which are immunocompetent cells,
Another object of the present invention is to provide a T lymphocyte separation material and a separation device that can recover cells without impairing cell function.

A further object of the present invention is to provide a T lymphocyte separation material and a separation device that are easy to operate and can efficiently separate T lymphocytes without requiring special equipment. A further object of the present invention is to provide a T lymphocyte isolation material and isolation method that have wide applicability and can be applied not only to clinical tests but also to immunological disease treatment, cell culture, etc.

(Means for Solving the Problems) The objects mentioned above are achieved by a material for separating T lymphocytes, which is characterized in that a peptide having an affinity for T lymphocytes is immobilized on a water-insoluble solid substance.

The present invention also provides a material for separating T lymphocytes, in which the peptide having affinity for T lymphocytes is a thymus hormone. The present invention also provides a material for isolating T lymphocytes in which the peptide having affinity for T lymphocytes is the biologically active site of thymic hormone. The present invention further provides a T lymphocyte isolation material, wherein the thymic hormone is selected from the group consisting of thymosin fraction 5, thymopoietin, thymohumoral factor, thymostimulin, and thymosin. The present invention also provides a material for separating T lymphocytes, in which the peptide having affinity for T lymphocytes is peptide T, which is a homologous portion of HIV coat protein (gp120) and EBV gene.

The above advantages can also be achieved by a T lymphocyte separation material characterized by immobilizing a lipid having an affinity for T lymphocytes on a water-insoluble solid substance.

The present invention also provides a material for separating T lymphocytes, in which the lipid having affinity for T lymphocytes is a complex lipid. The present invention further provides that the complex lipid is phospholipid or glycolipid.
This shows a material for separating lymphocytes. The present invention further provides a material for separating T lymphocytes in which the phospholipid is an inositol phospholipid.

The above features further include a separation material characterized by immobilizing a peptide having an affinity for T lymphocytes on a water-insoluble solid substance, or a separation material characterized by immobilizing a lipid having an affinity for T lymphocytes on a water-insoluble solid substance. This can also be achieved by a T lymphocyte separation device characterized by having a column filled with a separation material characterized by the following.

The present invention also provides a device for separating T lymphocytes that has been heat sterilized, gas sterilized, or radiation sterilized.

(Function) Therefore, since the T lymphocyte isolation material according to the present invention is characterized by immobilizing a peptide or lipid that has an affinity for T lymphocytes on a water-insoluble solid substance, T lymphocytes can be isolated from T lymphocytes. By utilizing the high affinity of peptides or lipids for bulb surface antigen receptors, T lymphocytes in body fluids such as blood or tissues can be separated from other cells with high selectivity and high yield.

To explain this effect in more detail, as mentioned above, T lymphocytes mature under the influence of the thymus, so they develop receptors or recognition mechanisms on the cell membrane for thymic hormones (thymic humoral factors) produced by epithelial cells within the thymus. It is thought that it is held in

Furthermore, since cell membrane surface antigen receptors are composed of glycoproteins, glycolipids, proteins, and lipids, it is thought that certain peptides or lipids have a strong affinity for the cell membrane surface. A physicochemical analysis of these specific interaction forces shows that the peptides or lipids that have affinity for T lymphocytes and the cell membrane surface are affected by the Van der Waals force (the contents of this force include orientation effects, dielectric Three effects are considered: hydrogen viscosity, ionic viscosity (Coulomb force), and dispersion effect.
It is assumed that the intermolecular forces of dipole opening forces, coordination bonds, and hydrophobic bonds combine to interact, and that the spatial structure formed by surface molecules is sterically compatible, like a lock and a keyhole. It is something that

As mentioned above, T lymphocytes are responsible for cell-mediated immunity as immunocompetent cells, and are closely involved in various immune diseases. Therefore, if T lymphocytes can be selectively and efficiently isolated as described above, it is considered to be useful for diagnosis and treatment of 6-cell.

Hereinafter, the present invention will be explained in more detail based on embodiments.

The first T lymphocyte separation material of the present invention is characterized in that a peptide having affinity for T lymphocytes is immobilized on a water-insoluble solid substance.

T used in the first T lymphocyte separation material of the present invention
Peptides that have rf affinity for lymphocytes are 2 to 100
It is an organic compound in which several amino acids are linked by a peptide bond (-CO-NH-). Among these peptides, thymus hormone, which is involved in the maturation of T lymphocytes,
Furthermore, the biologically active part f of thymus hormone: 1. , or peptide T, etc. can be mentioned as preferable examples.

Here, thymosin fraction 5 (Thymosin fraction 5) is an acidic polypeptide containing more than 30 components, has a molecular weight of 1000 to 15000, and is stable up to about 80°C.
sin Pr-5), the molecular weight is 5562, and the stability is less than Hatake.
, thymopoietin (1"hymopoietin), which has an isoelectric point of 5.2 and contains 49 amino acid residues; and acidic polyester, which has a molecular weight of 3200, has an isoelectric point of 5.7 to 5.9, and contains 31 amino acid residues. The peptide thymic humoral factor (T
hyiic humoral factor, Tl1
l'), has a molecular weight of 857, an isoelectric point of 7.3, and contains 9 amino acid residues.
factor), and molecules! 12,000 or less, and the isoelectric point is 8.30.
Ilulin, TS), etc.

In addition, the biologically active site of thymic hormone is an oligopeptide whose peptide synthesis is relatively easy. -Glu-
Ala-Glu-Asn), 32-36 amino acid residues of thymopoietin I belonging to thymopoietin (specifically Arg-Lys-Asp-Val-
1'yr), and 9 neutral amino acid residues in thymic blood bath factors (specifically, Glu-Ala-Lys
-8cr -Gin -Gly -Gly -3cr
-Asn).

Furthermore, peptide T used as a suitable peptide in the first T lymphocyte isolation of the present invention is human immunodeficiency virus (llua+an 1ma+unodcl).
'1cicney virus, IIIV) coat protein (gp120) and Epstein-Barr virus (
1Epstein-BarrVlrus, a homologous portion of the EBV) gene. In other words, HIV, which is a virus belonging to the genus AIDS (Acquired Immunodeficiency Disease Mosquito Complex), infects T4 cells (herher/inducer T cells);
Infection is established when gp120, an HIV coat protein, binds to the CD4 antigen of T4 cells. Therefore, peptide T, which is a homologous portion between the lymphophilic EBV gene and gp120, is presumed to have an affinity for T lymphocytes. Peptide T 1
The following structure is Ala-8cr-1”hr-Thr-
1”hr −Asn −Tyr−Thr, and 1゛h
It is called peptide T because it has a lot of

In the first T lymphocyte separation phase of the present invention, the water-insoluble solid substance used as a carrier for immobilizing the peptide having an affinity for T lymphocytes may be hydrophilic, such as Alternatively, it may be hydrophobic, such as polysaccharides such as agarose, dextran, chitosan, and cellulose, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylonitrile, and styrene. -Divinylbenzene copolymer, polystyrene, acrylic ester, methacrylic ester, polyethylene, polypropylene, poly(4-fluoroethylene), ethylene-vinyl acetate copolymer, polyamide, polycarbonate, polyfluoride vinylidene type, polyvinyl formal type,
Examples include inorganic materials such as polyarylate-based, polyether sulfone-based, etc., G-organized synthetic leather molecules, glass-based, titanium-based, activated carbon-based, and various ceramic-based materials such as alumina, silica, and hydroxyapatite. Known carriers used in immobilized intoxicant methods, affinity chromatography, etc. can be used without any particular difficulty.

The form of such water-insoluble solid substances is not particularly specified, and any known shape such as particulate, fibrous, hollow fiber, membrane, or plate-like shapes can be used, but body fluids From the viewpoint of liquid permeability of the cell suspension and ease of handling when preparing the separation material, particulates are preferable, and in particular, particulates with a particle size of 0.05 to 5 mm have a large effective surface area and liquid permeability. Desirable from aspects such as gender. Further, the particle shape is preferably spherical because it is less likely to cause physical damage to cells, less likely to cause flaking or chipping, and easier to obtain uniform particles. Further, such a carrier may be porous or dense. In the T lymphocyte separation material of the present invention, methods for immobilizing the above-mentioned peptides having affinity for T lymphocytes on the surface of the water-insoluble solid substance include covalent bonding, ionic bonding, physical adsorption, etc. Any known method can be used, such as carrier binding methods such as hydrophobic bonding, biochemical specific bonding, crosslinking method, entrapping method, and composite method, but from the viewpoint of stability in body fluids or cell suspension fluids. , it is preferable to fix by adhesion or hydrophobic bonding.

In order to co-bond a peptide that has an affinity for T lymphocytes to the surface of a water-insoluble solid substance, a variety of methods can be used depending on the functional groups of the two. For example, cyanogen bromide activation method, condensation wiping method using carbodiimide trial straw, acid azide derivative method, diazo method, alkylation method, carrier crosslinking method using dialdehyde, bisepoxide, diisocyanate, etc., γ-glyside A silane coupling agent activation method using xybrobyltrimethoxysilane, β-(3,4-epoxychlorohexyl)ethyltrimethoxysilane, etc. is used. Furthermore, if necessary, a spacer molecule of any length may be introduced between the peptide having affinity for T lymphocytes and the surface of the water-insoluble solid substance.

Preferred examples of this spacer molecule include diamines such as hexamethylene diamine and diols such as hexamethylene glycol.

In addition, methods for hydrophobically adhering peptides that have an affinity for T lymphocytes to the surface of water-insoluble solid substances include, for example, methods for directly bonding them to hydrophobic polymers such as polystyrene and polypropylene, and methods for attaching them to carriers by the above-mentioned covalent bonding method. There is a method of bonding a coupling agent that binds a hydrophobic group to the molecule and then forming a hydrophobic bond. As a coupling agent for H-hydrophobic groups in the molecule, octadecyldimethyl[3-
(()rimethoxysilyl)propyl]ammonium chloride, octadecyltrichlorosilane, phenyltrichlorosilane, etc.

The second T lymphocyte separation material of the present invention is characterized by immobilizing a lipid that has an affinity for T lymphocytes on a water-insoluble solid substance.

T used in the second T lymphocyte separation material of the present invention
Lipids that have an affinity for lymphocytes include simple lipids, complex lipids, and derived lipids, and among these, complex lipids are particularly preferred, since they exhibit an important distinction in cell-to-cell interaction. Furthermore, among complex lipids, inositol phospholipids such as lipositol, diphosphoinositide, and monophosphoinositide, which are component lipids of glycolipids on the surface of cell membranes, and glycolipids such as cerebroside and ganglioside exert even stronger interaction forces. This is preferable because it is considered to be

In the second T lymphocyte separation material of the present invention, the water-insoluble solid substance as a carrier on which the lipid having an affinity for T lymphocytes is immobilized is used for the first T lymphocyte separation. The same material as that used in the material can be used, and the immobilization method is also the same, so the explanation will be omitted.

The first T lymphocyte separation material of the present invention is obtained by immobilizing a peptide having an affinity for T lymphocytes on a water-insoluble solid substance, or a water-insoluble lipid having an affinity for T lymphocytes. The second aspect of the present invention is immobilized on a solid substance.
To perform T lymphocyte isolation and imaging using T lymphocyte isolation materials, these isolation materials are immersed in body fluids or cell suspensions, and T lymphocytes are adsorbed in a batch manner. It is also possible to collect, but more preferably,
Particulate T lymphocyte separation materials are suitable for obtaining the absolute amount of peptides or lipids that exist on the surface of the T lymphocyte separation materials and act on cell adsorption, as well as sufficient space for cell adsorption. It is desirable to carry out the contact using a separation device having a packed column.

That is, the separation device of the present invention includes the first separation material of the present invention, which is characterized in that a peptide having an affinity for T lymphocytes is immobilized on a water-insoluble solid substance, or a separation device having an affinity for T lymphocytes. It is characterized by having a column filled with the second separation material of the present invention, which is characterized in that lipids are immobilized on a water-insoluble solid substance.

In this T lymphocyte separation device, the column container can be made of synthetic resins such as polyethylene, polypropylene, polycarbonate, polystyrene, polymethyl methacrylate, and metals such as glass and stainless steel. Particularly preferred are polypropylene, polycarbonate, etc., which can be sterilized and are easy to handle.

Furthermore, between the entrance and exit of the column of this T lymphocyte separation device and the separation material layer filled with the T lymphocyte separation material, there is a mesh that allows cell components in the blood cell suspension to pass through but not the separation material. It is preferable to include a filter, and the material constituting this filter may be any physiologically inert and strong material, particularly polyester and polyamide.

FIG. 1 is a sectional view schematically showing the structure of an embodiment of the T lymphocyte separation device of the present invention. In the T lymphocyte separation apparatus 1 in the embodiment shown in FIG. 1, a column container 4 comprising a fluid inlet 2 and a fluid outlet 3
is filled with the T-lymphocyte separation material 5 according to the present invention in the shape of a filter, and this separation material 5 is passed through the column container 4 by filters 6a and 6b provided near the fluid inlet 2 and the fluid outlet 3. held within.

In order to perform extracorporeal circulation therapy on a patient with an immune disease using this T lymphocyte separation device 1, for example, if this T lymphocyte separation device dl is incorporated into a circuit as shown in FIG. good. To briefly explain extracorporeal circulation therapy using the extracorporeal circulation circuit shown in FIG. Chamber 9 with
a, was introduced into the T lymphocyte separation device 1 from the fluid introduction port 2 at a constant flow rate, and was adsorbed by the T lymphocyte separation material 5 according to the present invention housed in the column container 4. Thereafter, the fluid is led out from the fluid outlet 3 to the T lymphocyte separation device 1. In this way, T lymphocyte separation device 1
The treated blood passes through a chamber 9b equipped with a pressure gauge 10b, is heated in a color temperature tank 11, passes through a bubble detector 12, and is returned to the patient's body through a blood outlet 13 of the circuit. It is something.

The T lymphocyte separation device of the present invention having such a configuration is usually sterilized by a sterilization method such as heat sterilization, gas sterilization, or radiation sterilization before use.

(Example) Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 About 1 g of dry weight chloromethylated polystyrene beads (Peptide Kenkyushin, particle size 100-200 mesh, 1% divinylbenzene, Cρ3 amount = 0.68 milliequivalents/g
) using the cesium salt method to add 1 g of Boa -L-Asn -
After coupling the OH and removing the Boe (t-butoxycarbonyl) group with trifluoroacetic acid, Boc-L-Glu
(Ocllcx)-0H was peptide-bonded. Boe-L-Ala
-OH, Boc -L-Glu(OeHcx)-OH
S Boc-L-Glu(Ocllcx)-0H
, Boc-L-Val-OH, and then removing the protecting group by catalytic reduction using trifluoroacetic acid and palladium black.
A T lymphocyte separation material was obtained in which hexapeptides corresponding to 3 to 28 amino acid residues were bound to polystyrene beads.

This separation material was dissolved in a phosphate buffered salt solution (P B
A disposable syringe with a capacity of 1 ml (Terumo ■) containing a polyester nonwoven fabric inside the tip.
0.5 ml was filled into

A 25G needle and a cell collection container were attached to the tip of this syringe to serve as a T lymphocyte separation device.

Cell separation experiments were conducted using this separation device. That is, C from the upper end opening of the syringe that constitutes the separation device.
1 ml of PD-added human peripheral blood was added, and the outflowing treatment curve was made concave. After determining the number of blood cells in this processing capacity l& with an automatic blood cell counting device (ELT-8, manufactured by Orso Instruments), T lymphocytes were determined by FI.
TC-labeled anti-Leu-4 antibody, FITc-labeled anti-Leu-4
2a antibody and PEA-recognized Leu-3a antibody (both manufactured by Becton Dickinson), T cells (mitogen activated), killer /
The percentages of suppressor T cells, helper/inducer T cells, and lymphocytes were determined, respectively.

The adhesion rate of each cell was then calculated by comparing it with the blood cell count and the percentage of lymphocytes, T lymphocytes, and T lymphocyte subsets before contact with (min AI) rA.

The results are shown in Table 1.

Examples 2 to 4 Using the same solid-phase method as in Example 1, a pentapeptide corresponding to amino acid residues 32 to 36 of thymopoietin I (Example 2) and a nonapeptide corresponding to 9 amino acid residues of thymic serum factor (Example 2) were prepared. Example 3) and octapeptide Peptide T (Example 4) were bound to polystyrene beads to create a material for separating T lymphocytes.

Using the thus obtained T lymphocyte separation material, a T lymphocyte separation device was prepared in the same manner as in Example 1, and C
Cell separation experiments were conducted using PD-subjected human peripheral blood. The results are shown in Table 1.

Comparative Example 1 A cell separation experiment was conducted in the same manner as in Example 1, except that chloromethylated polystyrene beads were used as they were as a T lymphocyte separation material. The results are shown in Table 1.

Example 5 2 dissolved in 2-propanol adjusted to pH 4 with acid resistance
% octadecyldimethyl [3-()rimethoxysilyl)
Glass beads (diameter 0.15 mm) were placed in a solution of ammonium chloride (propyl) (manufactured by Toshi Silicone ■, AY43-021) and heated at room temperature for 15 minutes using a blood mixer (manufactured by Kayagaki Irika Kogyo, BM-101). Stir r1! After drying, the glass beads were dried in an oven at 110° C. for 1 hour to obtain glass beads having AY43-021 covalently bonded to the surface. This was added to a 0.5% phosphatidylinositol solution dissolved in chloroform (manufactured by Serdary Research Laboratory [:5erdary Res, Lab, Co., Ltd., Soyam Nyorai), and stirred and pressed overnight with a blood mixer to dissolve phosphatidylinositol into AY43-0. 21
Glass beads hydrophobically bound to were obtained as a material for separating T lymphocytes.

Using the thus obtained T lymphocyte separation material, a T lymphocyte separation device was prepared in the same manner as in Example 1, and C
We conducted cell separation experiments facing the periphery of PD patients. The results are shown in Table 1.

Example 6 Example 5 except that ganglioside (manufactured by Sigma, Ushino Yamaki) was used instead of phosphatidylinositol.
A separated phase for T lymphocytes was prepared in the same manner as in Example 1, and a T lymphocyte separation device was prepared in the same manner as in Example 1.
A cell separation experiment was conducted using D-substituted human peripheral blood. The results are shown in Table 1.

Comparative Example 2 A cell separation experiment was conducted in the same manner as in Example 1, except that glass beads were used as they were as a separation phase for T lymphocytes. The results are shown in Table 1.

As is clear from the results shown in Table 1, polystyrene beads (Examples 1 to 3) bound with an oligopeptide corresponding to the biologically active site of thymic hormone, which is an implementation IFI of the T lymphocyte isolation material of the present invention; Polystyrene beads bound with peptide T (Example 4), glass beads bound with inositol phospholipid (Example 5), and glass beads bound with ganglioside, a glycolipid (Example 6), were all made from polystyrene beads as they were. Compared to the separation material used as a separation material (Comparative Example 1) or the separation material made directly from glass beads (Comparison 2), this product selectively captured T lymphocytes or a subset of T lymphocytes. .

(Effects of the Invention) As described above, the present invention has an affinity for T lymphocytes.
A material for separating T lymphocytes characterized by immobilizing a peptide that acts on a water-insoluble solid substance, and a T lymphocyte separation material characterized by having a lipid having an affinity for T lymphocytes immobilized on a water-insoluble solid substance. Since it is a cell separation material, it is possible to selectively, efficiently and easily separate T lymphocytes, which are deeply involved in cell-mediated immunity, in large quantities, and serves as a basis for functional analysis of T lymphocytes related to immune function. This technology can be effectively applied to the diagnosis and treatment of various immunological diseases.

Furthermore, in the T lymphocyte isolation material of the present invention, the peptide having affinity for T lymphocytes is a thymic hormone or a biologically active site of a thymic hormone, and furthermore, the thymic hormone is thymosin fraction 5, thymopoietin, thymic body a peptide selected from the group consisting of thymostimulin, thymostimulin and thymic serum factor, or a peptide with affinity for T lymphocytes
120) and peptide T, which is a homologous part of the EBVii gene.
In this case, the separation material exhibits higher affinity for the TIJ lymphocyte membrane surface antigen receptor, and an excellent effect can be expected. In addition, in the T lymphocyte separation material of the present invention, when the lipid having affinity for T lymphocytes is a complex lipid, and furthermore, the complex lipid is a phospholipid such as inositol phospholipid or a panicle lipid, the separation material is It shows stronger interaction with lymphocytes and is expected to have superior effects.

The present invention also provides a separation material characterized in that a peptide having an affinity for T lymphocytes is immobilized on a water-insoluble solid substance, or a separation material characterized in that a lipid having an affinity for T lymphocytes is immobilized on a water-insoluble solid substance. Since this is a T lymphocyte separation device characterized by having a column packed with a separation material characterized by For example, when applied to extracorporeal circulation therapy for immune diseases, it is possible to remove specific T lymphocytes or subsets of T lymphocytes from the patient's blood in a short time and in a protective manner. Therefore, more efficient treatment or processing operations can be expected. Furthermore, when the T lymphocyte separation device of the present invention is applied to extracorporeal circulation therapy as described above, lymphokines, monokines, cytotoxic agents, cell differentiation factors, or immunomodulators may be added to the separation material that traps T lymphocytes. After culturing cells while applying
It is also possible to detach T lymphocytes with an adsorption antagonist and return them to the patient's body, which promises a more diverse range of treatments.

Furthermore, the T-lymphocyte separation device of the present invention includes thermal sterilization,
No deterioration in performance is observed even after gas sterilization or radiation sterilization, and since it exhibits stable T lymphocyte separation ability, accurate diagnosis and safe treatment can be performed.

[Brief explanation of drawings]

FIG. 1 is a sectional view schematically showing the structure of an embodiment of the T lymphocyte separation device of the present invention, and FIG. It is a drawing showing the configuration of a circulation therapy circuit. 1... T lymphocyte separation device, 2... fluid inlet,
3...Fluid outlet, 4...Column container, 5...
- T lymphocyte separation material, 6a, 6b... filter 7... blood inlet, 8... blood pump, 9a, 9b... chamber 10a. 10b...Pressure gauge, 1...Thermostat, 12...Bubble detector, 13...Blood outlet. Patent vendor Terumo Corporation

Claims (11)

[Claims] (1) A material for separating T lymphocytes, characterized in that a peptide having an affinity for T lymphocytes is immobilized on a water-insoluble solid substance. (2) The T lymphocyte separation material according to claim 1, wherein the peptide having affinity for T lymphocytes is a thymus hormone. (3) The T lymphocyte isolation material according to claim 1, wherein the peptide having affinity for T lymphocytes is a biologically active site of thymic hormone. (4) The T lymphocyte isolation material according to claim 2 or 3, wherein the thymic hormone is selected from the group consisting of thymosin fraction 5, thymopoietin, thymic humoral factor, thymostimulin, and thymic serum factor. (5) Peptides with affinity for T lymphocytes are linked to human immunodeficiency virus (Human Immunodeficiency virus)
encyVirus, HIV) coat protein (gp1
20) and Epstein-Barr virus (Epstei
The material for separating T lymphocytes according to claim 1, which is peptide T, which is a homologous portion of the (n-BarrVirus, EBV) gene. (6) A material for separating T lymphocytes, characterized in that a lipid having an affinity for T lymphocytes is immobilized on a water-insoluble solid substance. (7) The material for separating T lymphocytes according to claim 6, wherein the lipid having an affinity for T lymphocytes is a complex lipid. (8) Claim 7 wherein the complex lipid is a phospholipid or a glycolipid.
The T lymphocyte isolation material described in . (9) Claim 8 wherein the phospholipid is an inositol phospholipid.
The T lymphocyte isolation material described in . (10) A T lymphocyte separation device comprising a column filled with the separation material according to any one of claims 1 to 10. (11) The T lymphocyte separation device according to claim 10, which has been subjected to thermal sterilization, gas sterilization, or radiation sterilization.