JPH0623758B2 - B lymphocyte separation material, separation method and separator - Google Patents

Description

TECHNICAL FIELD The present invention relates to a B lymphocyte separation material, a separation method, and a separator. More specifically, the present invention provides a method for treating B lymphocytes from a B lymphocyte-containing liquid such as a cell suspension or a leukocyte fraction in which a lymphocyte fraction obtained from peripheral blood or biological tissue is suspended. The present invention provides a B lymphocyte separation material, a separation method, and a separator that can be simply and inexpensively separated with high yield and high purity while minimizing functional damage.

Lymphocytes are cells responsible for the immune function, which are differentiated from hematopoietic stem cells, and are divided into several subgroups in terms of function or cell membrane trait. They are B lymphocytes that produce antibodies and are responsible for humoral immunity, T lymphocytes that produce immunoregulatory lymphokines, inflammatory lymphokines and the like and are responsible for cell immunity, and lymphocyte subclasses such as null cells. There are functionally different subsets of T and B lymphocytes. In recent years, a technique for purely separating, purifying, and concentrating such subclasses of T and B lymphocytes having different immunological properties with minimal functional damage has been strongly desired in a wide range of social fields. There is. Eg cytology,
In basic research such as immunology, it is necessary to separate at least T and B lymphocyte groups in the in vitro analysis of the in vivo immune system, and it is necessary to make a diagnosis in clinical medicine.
In treatment or the like, it is necessary to transfer or remove a specific fraction with reduced function or promoted, and in obtaining a physiologically active substance of the lymphatic system, separation and concentration of the producing cell fraction are important.

(Prior Art) As a method for separating T lymphocytes and B lymphocytes, first, lymphocyte fractionation from peripheral blood or biological tissue is performed by specific gravity centrifugation using a high-density isotonic solution such as a sodium metrizoate ficoll mixed solution. It has been general practice to obtain T lymphocytes and subsequently to separate them into T lymphocytes and B lymphocytes.
This subsequent separation treatment method can be roughly divided into three (1) a method using specific antigens, receptors and immunoglobulins on the cell membrane surface as indicators, (2) physical cell separation and (3) adsorption chromatography.

Examples of the method (1) include a rosette formation method, affinity chromatography (with an antibody or an immune complex bound thereto), an antibody, a cytolysis method using complement, removal of mitogen-activated lymphocytes, a fluorescein activated cell sorter ( F.A.C.S.) and the like are known,
In these methods, it is difficult to recover T lymphocytes or B lymphocytes in an intact state because they are strongly bound and stimulated with high biological specificity, and many methods are not suitable for mass treatment. In addition, as the method (2),
Density gradient centrifugation method, cell electrophoresis method, etc. are known, but since T lymphocytes and B lymphocytes have no significant differences in physical properties such as specific gravity and density, they were separated and collected. High accuracy cannot be required for cell yield or purity. Further, as the method (3), it is known to utilize the difference in the non-specific adhesion ability of T lymphocytes and B lymphocytes with respect to foreign substances such as nylon, tetron, and cotton fiber, but it is possible to separate cells with high accuracy. In order to obtain, the adsorption carrier fetal calf serum (FC
It is difficult to set treatment conditions such as S) and the like, and it is difficult to set the execution conditions such as the flow rate, and impurities such as macrophages are often mixed. In addition, these conventional methods also have a big problem that empirical technical proficiency in performing a separation operation is indispensable due to complicated operations or preparations.

(Problems to be Solved by the Invention) Therefore, an object of the present invention is to provide a novel B lymphocyte separation material, a separation method and a separator. The present invention also provides a B lymphocyte separation material, a separation method, and a separator that can be simply and inexpensively separated in high yield and high purity without affecting cell activities. The invention further comprises
An object is to provide a B lymphocyte separation material, a separation method and a separator suitable for large-scale processing.

(Means for Solving the Problems) Further, the above-mentioned various objects have a high affinity with at least one immunoglobulin selected from the group consisting of dipeptides and sulfathiazoles via a spacer on the surface of a water-insoluble solid substance. It is achieved by a B lymphocyte separation material characterized in that an organically synthesized ligand having is immobilized.

The present invention also provides a B lymphocyte separation material in which the spacer has a skeleton of a hydrocarbon chain having at least two carbon atoms or a hydrocarbon chain containing a characteristic group. The present invention further has a structure in which the spacer methylene repeats have a skeleton of a straight-chain alkyl of 4 or more.
It shows a lymphocyte separation material. The present invention also shows a B lymphocyte separation material in which the spacer has an ethylene oxide skeleton having a polymerization degree of 1 to 50. The present invention also shows a B lymphocyte separation material in which the water-insoluble solid substance is a particle body made of a synthetic organic polymer, a natural organic polymer or an inorganic substance. The present invention also provides that the water-insoluble solid substance is a particle body having an average particle size of 0.05 to 5 mm.
It shows a lymphocyte separation material.

The above objects are also to immobilize, through a spacer, an organically synthesized ligand having a high affinity for at least one immunoglobulin selected from the group consisting of dipeptides and sulfatizole on the surface of a water-insoluble solid substance. A method for separating B lymphocytes, which comprises contacting a separated liquid containing B lymphocytes with a liquid containing B lymphocytes to selectively capture B lymphocytes having an immunoglobulin molecule (S-Ig) on the membrane surface. To be achieved.

The invention also provides that the spacer in the separating material is at least 2
It shows a method for separating B lymphocytes having a skeleton of a hydrocarbon chain having at least one carbon atom or a hydrocarbon chain containing a characteristic group. The present invention further shows a method for separating B lymphocytes in which the methylene repeating spacers in the separating material have four or more linear alkyl skeletons. The present invention also shows a method for separating B lymphocytes in which the spacer in the separating material has an ethylene oxide skeleton with a degree of polymerization of 1 to 50. The present invention further shows a method for separating B lymphocytes in which the water-insoluble solid substance is a synthetic organic polymer, a natural organic polymer or an inorganic substance. The present invention also shows a method for separating B lymphocytes, which uses an isotonic buffer as a washing solution.

The above-mentioned various objects further include an organically synthesized ligand having a high affinity with at least one immunoglobulin selected from the group consisting of dipeptides and sulfathiazoles via a spacer on the surface of a particulate water-insoluble solid substance. This is achieved by a B lymphocyte separator having a column packed with a separating material that is fixed.

The invention also shows a B lymphocyte separator in which the column is made of polypropylene or polycarbonate. The present invention further comprises a filter having a network between the inlet and outlet of the column and the separation material layer, which has a network through which cell components in the B lymphocyte-containing liquid can pass but the separation material cannot pass. It shows a separator.
The invention further shows a B lymphocyte separator in which the filter is of polyester or polyamide. The term "ligand" as used herein refers to a substance that specifically binds to a protein.

(Action) As is known, the cell membrane surface of B lymphocytes has different classes such as IgG, IgM, and IgA depending on the degree of differentiation, but immunoglobulins are present in all, whereas the cell membrane of T lymphocytes is present. It is known that substantially no such immunoglobulin is present on the surface.

The B lymphocyte separation material according to the present invention is characterized in that an organically synthesized ligand having a high affinity for immunoglobulin is immobilized on the surface of a water-insoluble solid substance. When the lymphocyte separation material is brought into contact with a B lymphocyte-containing liquid such as a lymphocyte suspension, it shows an extremely high affinity with B lymphocytes having immunoglobulins on the cell membrane surface. Is very likely to be captured. On the other hand, it has no affinity for T lymphocytes that do not have immunoglobulin on the cell membrane surface,
T lymphocytes are not captured by the B lymphocyte separation material. Therefore, B lymphocytes and T lymphocytes can be obtained in high purity and high yield by separating the fraction adsorbing the separating material and the fraction not adsorbing the separating material.

Furthermore, another lymphocyte separation material according to the present invention is characterized in that an organically synthesized ligand having a high affinity for immunoglobulin is immobilized on the surface of a water-insoluble solid substance through a spacer. Therefore, when the B lymphocyte separation material is brought into contact with, for example, a lymphocyte suspension, the B lymphocyte separation material shows an extremely high affinity for B lymphocytes having immunoglobulin on the cell membrane surface, and the B lymphocytes It is captured by the B lymphocyte separation material with a very high probability. On the other hand, the B lymphocyte separation material does not show an affinity for T lymphocytes having no immunoglobulin on the cell membrane surface, and the water-insoluble solid substance surface and the organically synthesized ligand having a high affinity for immunoglobulin. Since the spacer interposed between the cells prevents the cells from approaching the surface of the separation material and interacting with each other due to its fluidity and excluded volume effect, the spacer has a weak interaction force with the B lymphocyte separation material as described above. T lymphocytes are not substantially trapped by the B lymphocyte separator in order to suppress non-specific adhesion of T lymphocytes. Therefore, B lymphocytes and T lymphocytes can be obtained with higher purity and high yield by separating the separation material adsorption fraction and the separation material non-adsorption fraction.

Therefore, for example, a lymphocyte suspension is injected at a constant rate into a column having a liquid inlet / outlet filled with the B lymphocyte separation material, and the non-adsorbed fraction in the column is immediately washed away with a washing liquid such as an isotonic buffer. As a result, T lymphocytes can be obtained easily, rapidly and in large quantities in high purity and high yield. Thus, the present invention applies a kind of adsorption chromatography method, but in the case of the B lymphocyte separation material of the present invention, an antibody or an immune complex is immobilized on a carrier like conventional affinity chromatography. It has a clear affinity for B lymphocytes, but does not give any biologically highly specific stimuli, unlike the case where it is transformed into B lymphocytes. Lymphocytes are those that remain intact. Furthermore, such affinity of the B lymphocyte separation material of the present invention is
It is also confirmed that it does not affect the origin of the animal species, the separation operation is completed in a few minutes, and the composition of the lymphocyte suspension may be simple isotonic salt solution, and concentrated serum derived from different animals such as fetal bovine serum. It also has a number of application advantages, such as no need. Hereinafter, the present invention will be described in more detail based on embodiments.

The B lymphocyte separation material of the present invention is characterized in that an organically synthesized ligand having a high affinity for immunoglobulin is immobilized on the surface of a water-insoluble solid substance.
Such ligands having a high affinity for immunoglobulins include dipeptides, sulfathiazoles, etc. These may be used alone or in combination as a ligand.

In the B lymphocyte separation material of the present invention, the heteroatom of sulfathiazole, which is preferably used as a ligand having a high affinity for immunoglobulin, has a lone electron pair and functions as a proton acceptor. Heterocycle having few dissociative groups represents hydrophobic water. Further, the sulfonamide portion of the sulfa drug, the thiol group or hydroxyl group of 2-thiouracil, the acid amide portion of isonicotinic acid hydrazide, and the carbonyl group and imino group of luminol have hydrogen bonding properties. As described above, in the interaction between the immunoglobulin on the surface of the B lymphocyte cell membrane and the compound, the hydrophobicity of the main part of the compound, the electrostatic property due to the hetero atom, and the hydrogen bonding property in the vicinity of the main part are Exerts an interaction force with the amino acid residue of the adsorption site in each, and in addition,
It is considered that the high affinity of the non-antigen / antibody system was obtained because the three-dimensional structure of the compound was appropriately applied to the immunoglobulin molecular pocket on the cell membrane surface.

Further, the dipeptide used in the present invention is a synthetic compound in which two amino acids are obtained through a peptide bond, and among these dipeptides, known compounds such as protein A having a specific binding property with an immunoglobulin molecule are known. Those which mimic the affinity sites of natural substances are preferable, and in particular, dipeptides of lysine and tyrosine, or compounds such as aspartylphenylalanine alkyl esters such as aspartylphenylalanine methyl ester give extremely good results.

It is considered that these dipeptides also have high affinity for the non-antigen / antibody system due to the interaction with specific hydrophobic sites and charge sites in the immunoglobulin molecular chain.

Further, in the present invention, amino acids can be used in combination with the dipeptide, and these amino acids are a single type of amino acid or two or more types of amino acids used at the same time, and are hydrophobic valine and leucine. , Tyrosine, phenylalanine, isoleucine, tryptophan, and basic lysine, arginine, or acidic proline and aspartic acid and derivatives thereof, alone or in combination, give particularly good results. In addition, it does not matter whether these substances are D- or L-forms.

With regard to these amino acids, B lymphocytes having an immunoglobulin and a high non-antigen / antibody system are also produced by the interaction with specific hydrophobic sites and charge sites in the immunoglobulin molecular chain simultaneously with the above-mentioned dipeptide. It is considered that the affinity was obtained.

Furthermore, the organically synthesized ligands described above are
When used alone, it can exert a sufficient affinity for B lymphocytes, but especially when amino acids such as arginine, aspartic acid, phenylalanine, proline and the heterocyclic compound sulfathiazole are simultaneously used as ligands. , Very good results are obtained. Further, since such ligands listed above have high affinity for immunoglobulins derived from human, bovine and rat, it is possible that the ligands may be of any species of target cells. It will be expensive.

In the B lymphocyte separation material of the present invention, as the water-insoluble solid substance as a carrier for immobilizing such an organically synthesized ligand, agarose-based, dextran-based, cellulose-based, polyacrylamide-based, polyvinyl alcohol-based, polyvinyl-based Pyrrolidone type, polyacrylonitrile type, styrene-divinylbenzene copolymer, polystyrene type, acrylic ester type, methacrylic acid ester type, polyethylene type, polypropylene type, poly 4-fluorinated ethylene type, ethylene-vinyl chain acrylate copolymer type , Organic polymers such as polyamide-based, polycarbonate-based, polyvinylidene fluoride-based, polyvinyl formal-based, polyarylate-based, polyether sulfone-based, etc., glass-based, alumina-based, titanium-based, activated carbon-based, ceramic-based, etc. Is but particularly not too high cell adhesive, i.e., the cell - the interaction between the carrier is a relatively mild one is preferred, particularly preferably used are acrylic acid ester type. Further, collagen, chitosan, etc., which are natural organic polymers derived from living bodies, can also be used. The form of such a water-insoluble solid substance is not particularly limited, and those on a flat plate can be used, but preferably, it is in the form of particles, particularly particles having an average particle size of 0.05 to 5 mm. desirable. The average particle size is JIS-7-8801.
The particle size of each grade was calculated by classifying the median value of the upper limit grain size and the lower limit grain size of each grade after classifying using a sieve defined in 1. Further, the particle shape is preferably a spherical shape because physical damage to cells is unlikely to occur and uniform particles are easily obtained.

In the B lymphocyte separation material of the present invention, as a method of immobilizing the organically synthesized ligand on the surface of the water-insoluble solid substance as described above, covalent bond, ionic bond, physical adsorption,
Although any known method such as embedding or insolubilization by precipitation on the surface of the carrier can be used, it is preferable to use by immobilizing or insolubilizing by covalent bond in view of the elution property of the immobilized ligand. Therefore, generally, immobilized enzyme, known insoluble carrier activation method and ligand immobilization method used in the field of affinity chromatography are
It can be preferably used.

Another B lymphocyte separation material of the present invention is characterized in that an organically synthesized ligand having a high affinity for immunoglobulin is immobilized on the surface of a water-insoluble solid substance through a spacer. . As the ligand having a high affinity for the immunoglobulin used in this B lymphocyte, the same ones as listed above are used,
The action is also the same, and as the water-insoluble solid substance as a carrier for immobilizing such an organically synthesized ligand, the same substances as those mentioned above can be used.

The spacer used in the other B lymphocyte separation material of the present invention is a spacer that has a molecular shape and can be provided with an arbitrary length by interposing between the surface of the water-insoluble solid substance and the ligand. Any skeleton structure may be used as long as it does not sterically hinder the binding between the surface of the water-insoluble solid substance and the ligand. When such a spacer is interposed between the surface of the water-insoluble solid substance and the ligand, the fluidity of the spacer and the excluded volume effect prevent cells from approaching the surface of the separation material and interacting with each other,
As a result, it is considered that the adhesion of T lymphocytes having a weak interaction with the separation material is preferentially suppressed, and the effect of suppressing non-specific adhesion of T lymphocytes is produced.

Such a spacer has a skeleton of a hydrophilic hydrocarbon chain having at least two carbon atoms and having a hydrophobic hydrocarbon chain or molecular chain containing a characteristic group such as a hydroxyl group, a carboxyl group or ether oxygen. A known reactive functional group, such as an epoxy group or an isothiocyanate group, which was present at both ends of the molecular chain, is reacted with the surface of the water-insoluble substance and the amino group or the carboxyl group of the ligand, respectively. On the surface of the water-insoluble substance at one end of
It is covalently attached to the ligand at the other end.

As the hydrophobic hydrocarbon chain as the spacer skeleton,
Alkyl chain consisting of simple methylene repetitions, some of which contains various cyclic substituents such as carbon double bonds and benzene rings, and those with branched molecular chains. However, preferably, the number of repeating methylenes is 4 or more, and more preferably, the number of repeating methylenes is 4.
-10 linear alkyl chains are particularly effective as spacers for suppressing non-specific adhesion of T lymphocytes. Further, as the hydrophilic hydrocarbon chain containing a characteristic group as a spacer skeleton, those having a dissociative functional group such as a hydroxyl group, a carboxyl group, or an amino group in the above alkyl chain, those having an ether oxygen, etc. There is
Similarly, there may be a carbon double bond, a cyclic substituent, or a branch of the molecular chain, but preferably an ethylene oxide skeleton having a degree of polymerization of 1 to 50, more preferably a degree of polymerization of 4 to 30, particularly T lymphocytes. It is highly effective as a spacer for suppressing non-specific adhesion of

Preferred examples of the compound that can be a spacer in the B lymphocyte separation material of the present invention by covalently bonding to the surface of the water-insoluble solid substance and the ligand include those having the following structures, but of course, the present invention is not limited to these. Not something.

[In the formula, R ¹ is a linear alkyl having 2 to 20 carbon atoms, preferably 4 to 10 repeating methylene. ] [In the formula, R ² and R ³ are each hydrogen or a carbon number of 1 to 4;
Is an alkyl group, and n is a number of 0 to 49, more preferably 3 to 29. To obtain the other B lymphocyte separation material of the present invention, for example, it is represented by the above structural formula (I) or (II) based on a known method used in the fields of immobilized enzyme and affinity chromatography. A compound having a reactive functional group at both ends such as a bisepoxide and having a molecular skeleton with an appropriate chain length as a spacer is added to the surface of the water-insoluble solid substance and the amino group or carboxyl group of the ligand, respectively. Just react and covalently bond,
A ligand having a high affinity for immunoglobulin is strongly bound to the surface of the water-insoluble solid substance through a spacer by a covalent bond.

The method for separating B lymphocytes of the present invention comprises a separation material obtained by immobilizing an organically synthesized ligand having a high affinity for immunoglobulin G on the surface of a water-insoluble solid substance as described above, or a water-insoluble solid substance as described above. The B lymphocyte-containing liquid is brought into contact with a separation material obtained by immobilizing an organically synthesized ligand having a high affinity for immunoglobulin on the surface through a spacer, and an immunoglobulin molecule (S-I
By selectively capturing B lymphocytes having g), B lymphocytes are efficiently separated.

The B lymphocyte-containing liquid used as a treatment target in the method for separating B lymphocytes of the present invention includes whole blood, a leukocyte fraction obtained by centrifuging whole blood, and a lymphocyte suspension. Be done. A lymphocyte suspension is obtained by purifying a lymphocyte fraction from other blood cell components using a plasma separator or a centrifuge from a cell suspension containing lymphocytes such as peripheral blood or tissue fluid. It is prepared by suspending the fractions in a suitable solution. The solution for suspending the lymphocyte fraction is a simple isotonic salt solution,
It may also contain autologous serum or the like.

In the method for separating B lymphocytes of the present invention, contact of the B lymphocyte-containing liquid such as a lymphocyte suspension with the B lymphocyte-separating material is performed by applying the B lymphocyte-containing liquid to the flat plate B lymphocyte-separating material. It is possible to carry out contacting, but from the viewpoint of obtaining the absolute amount of the organically synthesized ligand on the surface of the water-insoluble solid substance and sufficient cell adsorption space, the particle-like B lymphocyte separation material is filled. This is preferably carried out by using a B lymphocyte separator having a column comprising the above, and passing a B lymphocyte-containing solution through this.

In the B lymphocyte separator having the column filled with the particulate B lymphocyte separation material, polypropylene, polycarbonate,
Synthetic resins such as polystyrene and polymethylmethacrylate, metals such as glass and stainless steel can be used, but polypropylene and polycarbonate which are autoclave sterilizable and easy to handle are particularly preferable. Further, a filter having a mesh between the inlet / outlet of the column of the B lymphocyte separator and the separation material layer filled with the B lymphocyte separation material, in which the cell component of the B lymphocyte-containing liquid passes but the separation material cannot pass. The material for the filter is preferably physiologically inert and has high strength, and polyester and polyamide are particularly preferable.

FIG. 1 is a schematic view showing a mode of use of one embodiment of the B lymphocyte separator according to the present invention. As shown in FIG. 1, a lymphocyte suspension 1 in which lymphocytes as a B lymphocyte-containing liquid are suspended is passed through an inlet 9 of a B lymphocyte separator to a column 6 filled with a B lymphocyte separation material 5. Injected by pump. The B lymphocyte separation material 5 is a filter 4a,
It is held in the column 6 by 4b. After injecting the lymphocyte suspension 1, the washing solution 8 is injected into the column 6 by the pump 3, the non-captured cells remaining in the column are rinsed, and the treatment liquid 7 flowing out from the outlet 10 is collected. To do. The treatment liquid 7 is a separation material non-adsorbed fraction and contains T lymphocytes with high purity and high yield.

As the washing liquid 8 used for washing off the cells (T lymphocytes) not captured without releasing the cells (B lymphocytes) captured by the B lymphocyte separation material 5,
An isotonic buffer, a cell culture medium and the like are used, preferably a divalent cation-free isotonic buffer, and particularly preferably Hanks balanced salts.
solution: HBSS) or the like is used. The method for passing the lymphocyte suspension 1 may be continuous or discontinuous as required, and further, incubation for a certain period of time may be added.

Further, when collecting the cells captured by the B lymphocyte separation material 5, after washing and removing the non-captured cells with the washing solution 8, the cells captured by the B lymphocyte separation material 5 and the surface of the B lymphocyte separation material 5 are removed. The binding force with the ligand may be weakened by an appropriate means. For example, an isotonic buffer solution containing an amino acid, a saccharide or albumin may be used as an eluent, and the column 6 may be subjected to physical vibration for elution. It suffices to flow the agent and elute and collect the trapped cells.

Further, in the method for separating B lymphocytes of the present invention, in order to reduce the non-specific adhesion of T lymphocytes to the B lymphocyte separation material and reduce the yield, it is known that the separation material is bovine serum albumin. It is also possible to carry out the B lymphocyte separation operation after the treatment by the above method.

(Examples) Hereinafter, the present invention will be described more specifically with reference to Examples.

Example 1 As a water-insoluble solid substance, acrylic beads having an oxirane group (manufactured by Euparkid C (EC), Rohm Pharma [Rhm Pharma]) (average particle size:
15 mm) was used to prepare a B lymphocyte selective capture type separation material on which a heterocyclic compound sulfathiazole (ST) was immobilized as a ligand.

First, sulfathiazole was dissolved in 100 ml of a mixed solution (volume ratio 2: 3) of a 0.2 M carbonate buffer (pH 10) and dimethylformamide to a concentration of 0.1 M. Oxirane-acrylic beads (crosslinking monomer ratio 30% by weight) were added to the solution at a rate of 0.1 to 0.2 mg / ml for deaeration. After heating this in a 80 ° C water bath for 3 hours, a blood mixer (Bigaki Medical Science Co., B
The mixture was stirred overnight at room temperature using M-101). Then
1M (pH 8) to remove unreacted oxirane groups
Of ethanolamine solution was added and stirred overnight. Then 0.0 with distilled water, containing 0.5 M sodium chloride
Sulfathiazole-immobilized acrylic beads (EC-ST) were obtained by sequentially washing with 2 M (pH 4) acetate buffer and 0.2 M (pH 10) carbonate buffer.

The separation material obtained in this way has an inner diameter of 3 mm and an outer diameter of 4 mm.
Was filled in a column composed of a Teflon tube, and terminals having polyester filters (150 mesh) were connected to both ends of the tube to hold the adsorbent in the column. The column filled with this adsorbent was replaced with Hank's buffer (HBSS; pH 7.2, Ca ²⁺ , Mg ²⁺ free) and subjected to the subsequent T, B lymphocyte separation operation.

On the other hand, the lymphocyte suspension was prepared by suspending and diluting the lymphocyte fraction isolated from the mesenteric lymph node of Wistar rat to 800 × 10 ⁴ cells / ml using HBSS, This lymphocyte suspension is stored under ice cooling,
Used within 1 hour after dilution.

1 ml of the lymphocyte suspension prepared as described above was injected into the column filled with the separating material at a flow rate of 1 to 3 ml / min by a disposable syringe and a syringe pump (both manufactured by Terumo Corp.), and HBBS was immediately added. Using 2
The column was similarly injected at a flow rate of ml / min to wash the inside of the column, and the separation operation was completed. The collected processing solution is an automatic blood cell coefficient counter (ORTHO INSTRUME
NTS], ELT-8) was used to measure the cell density, and then only B lymphocytes in the treatment solution were fluorescently stained with FITC (Miles [MILES]) to which an antibody against rat IgG was bound. Then, B lymphocytes in the treatment liquid were counted by a fluorescence microscope, and the T, B lymphocyte ratio in the treatment liquid was calculated. By the above operation, the capture ability (SR) was obtained as an index showing the yield (y) and purity (p) of T lymphocytes in the treatment liquid, and the specificity for B lymphocytes. The results are shown in Table 1.

Example 2 As a water-insoluble solid substance, acrylic beads having an oxirane group (Euperkid C, manufactured by Reem Pharma Co.) were mixed with sulfathiazole, arginine, aspartic acid, phenylalanine and proline in a ratio of 16: 1: 1: 1. Using the mixed solution, in the same manner as in Example 1, sulfathiazole, arginine, aspartic acid, phenylalanine, proline-immobilized acrylic beads (EC-S).
TA).

Using this EC-STA, T and B were obtained in the same manner as in Example 1.
Lymphocyte separation operation was performed. The results obtained for yield (y) and purity (p), and scavenging (SR) are shown in Table 1.

Example 3 B-lymphocyte selective trapping type in which sulfathiazole was immobilized using cross-linked chitosan beads (chitopearl (CH), manufactured by Fuji Spinning Co., Ltd.) (average particle size 0.25 mm) as a water-insoluble solid substance A separation material was created.

First, 5w / v adjusted to pH 7.0 with sodium hydroxide
After immersing chitosan beads in a glutaraldehyde (GA) aqueous solution to degas, the mixture was stirred overnight at room temperature using a blood mixer (BM-101 manufactured by Kagaki Medical Science Co., Ltd.). This reaction product, which had been washed with distilled water, was added to a 0.1 M sulfathiazole solution dissolved in a mixed solution (volume ratio 2: 3) of dimethylformamide and a 10 mM calcium chloride solution (pH 8), and deaerated. After stirring overnight,
The chitosan beads were washed with dimethylformamide and distilled water. Next, in order to block unreacted aldehyde groups, the mixture was degassed in a 1M ethanolamine (pH 8) solution, and then stirred overnight with a blood mixer. This was washed again with distilled water, added to a 10 mM calcium chloride solution (pH 8) containing 1 w / v% sodium borohydride and reacted at 4 ° C., and the aldehyde group of glutaraldehyde and the amino group of chitosan and sulfathiazole were added. The azomethine produced by the reaction with was reduced and stabilized.
Finally, distilled water, 0.5M containing 0.5M sodium chloride.
It was washed several times with 02M acetate buffer (pH 4) and 0.2M carbonate buffer (pH 10) to obtain sulfatizole-immobilized chitosan beads (CH-GAST).

Using this CH-GAST, T and B lymphocytes were separated in the same manner as in Example 1. The results obtained for yield (y) and purity (p), and scavenging (SR) are shown in Table 1.

Comparative Example 1 For comparison, T and B lymphocytes were separated according to the procedure of Example 1 using acrylic beads (EC) to which no ligand was bound. The results are shown in Table 1.

Comparative Example 2 For comparison, using chitosan beads (CH) to which no ligand was bound, T and B lymphocytes were separated according to the procedure of Example 1. The results are shown in Table 1.

As is clear from these results, the ability to capture B lymphocytes was significantly improved by introducing a ligand having a high affinity to immunoglobulin, and the concentration of T lymphocytes in the recovered cell suspension was accordingly increased. Was confirmed to increase.

Example 4 After filling the column with the sulfathiazole-immobilized acrylic beads obtained in the same manner as in Example 1, 0.5% bovine serum albumin was injected into the column and left for 16 hours. Using the sulfathiazole-immobilized acrylic beads (AEC-ST) thus treated with bovine serum albumin from which free albumin was removed, T and B lymphocytes were separated according to the procedure of Example 1.
The results obtained for yield (y), purity (p) and scavenging (SR) are shown in Table 1.

As is clear from the results shown in Table 1, as compared with EC-ST (Example 1) which was not treated with albumin, a yield improvement of 20% or more was observed in the separation efficiency without decreasing the purity. It was It is considered that this is because the adsorbed albumin exerted a sea surface active material effect of suppressing non-specific adhesion of T lymphocytes without decreasing the affinity between B lymphocytes and ligands.

The yield (y), purity (p) and scavenging property (SR) were calculated as follows.

Furthermore, the T and B lymphocyte separation material according to the present invention (Example 1)
4), at least 1.0 × per 1 g of the separating material.
Cells capable of processing about 10 ^{7 to} 6.0 × 10 ⁷ lymphocytes, the processing time required for the processing is about 2 minutes / column, and are also outstanding in terms of viability and adhesion form. There was no damage.

Example 5 5 w / v% prepared with phosphate buffered saline, pH 7.4
Except for using the glutaraldehyde solution, cross-linked chitosan beads were bound with sulfathiazole using glutaraldehyde and solidified in the same manner as in Example 3 to form sulfathiazole-immobilized chitosan beads (CH-GAS).
T) was obtained.

The separation material obtained in this way has an inner diameter of 3 mm and an outer diameter of 4 mm.
Was filled in a column made of Teflon tube, and terminals having polyester filters (150 mesh) were connected to both ends of the tube to hold the separating material in the column. The column filled with this separation material was replaced with Hank's buffer (HBSS; pH 7.2, CA ²⁺ , Mg ²⁺ free) and subjected to the subsequent T, B lymphocyte separation operation.

On the other hand, target lymphocytes were collected from human peripheral blood by specific gravity centrifugation using Ficoll pack (specific gravity 1.077, manufactured by Pharmacia), and 80% using HBBS.
A lymphocyte suspension was prepared by suspending and diluting at 0 × 10 ⁴ cells / ml.

1 ml of the lymphocyte suspension prepared as described above was injected into the column filled with the separation material at a constant flow rate shown in Table 2 by a disposable syringe and a syringe pump (both manufactured by Terumo Corp.), and HBBS was immediately added. The column was similarly injected at a constant flow rate shown in Table 2 to wash the inside of the column, and the separation operation was completed. The number of cells in the recovered lymphocyte suspension was measured by an automatic hemocytometer (ORTHO INSTRUMENTS, ELT-8), and then only B lymphocytes in the recovered solution were tested against human immunoglobulin. Fluorescent staining was performed with an antibody-bound FITC (made by Miles [MILES]), and FITC-positive cells in the recovered solution were counted by a fluorescent microscope.
The ratio of ITC-stained cells (FITC (+)) and FITC-unstained cells (FITC (-)) captured in the column was determined. The results are shown in Table 2.

Example 6 Crosslinked chitosan beads (C
[Alpha] -L-aspartyl phenylalanine methyl ester (AT, manufactured by Ajinomoto Co., Inc.) which is a dipeptide consisting of aspartic acid and phenylalanine as a ligand in H).
A B lymphocyte separation material was prepared by binding and immobilizing the above with glutaraldehyde. This B lymphocyte separation material is C
It was called H-GAAT.

First, chitosan beads were soaked in a 5 w / v% glutaraldehyde solution prepared with phosphate buffered saline (PBS) having a pH of 7.4 to degas, and then a blood mixer (BM-101 manufactured by Kagaki Medical Science Co., Ltd.). ) Was used for stirring at room temperature.
The reaction product washed with distilled water was dissolved in 0.1M α-L-, which was prepared by dissolving α-L-aspartylphenylalanine methyl ester in phosphate buffered saline (PBS), pH 7.4.
The mixture was added to the aspartyl phenylalanine methyl ester solution, degassed, and stirred overnight with a blood mixer.

Next, the chitosan beads were washed, degassed in a 1M ethanolamine (pH 8) solution for blocking unreacted aldehyde groups, and then stirred overnight with a blood mixer. This is washed again with distilled water, 1 w / v%
Of 10 mM calcium chloride solution containing sodium borohydride and reacted at 4 ° C. to give an azomethine group formed by reaction of aldehyde group of glutaraldehyde with amino group of chitosan and α-L-aspartylphenylalanine methyl ester. Reduced and stabilized. Finally, distilled water, 0.02 acetate buffer (pH 4) containing 0.5 M sodium chloride, and 0.2 M carbonate buffer (pH
10) Washed several times with α-L-aspartyl phenylalanine methyl ester-immobilized chitosan beads (C
H-GAAT) was obtained.

Using the separating material CH-GAAT thus obtained, T and B lymphocytes were separated in the same manner as in Example 5. The results are shown in Table 2.

Examples 7 to 8 Crosslinked chitosan beads (C
H) is bound with sulfathiazole (ST) or α-L-aspartyl phenylalanine methyl ester (AT) as a ligand using epichlorohydrin (EH).
An immobilized B lymphocyte separation material was prepared. The B lymphocyte separation material was referred to as CH-EHST (Example 7) and CH-EHAT (Example 8), respectively.

First, 2M NaOH and epichlorohydrin were mixed at a ratio of 4: 1 and chitosan beads were added to a solution diluted 5 times with distilled water, and the mixture was mixed with a blood mixer at 40 to 45 ° C.
And stirred for 2 hours. The reaction product washed with distilled water was added to D
0.1M sulfathiazole solution or PB dissolved in a mixture of MF and 10 mM calcium chloride solution (2: 3)
0.1 M α-L-aspartyl phenylalanine methyl ester solution dissolved in S (pH 7.4)
The mixture was added at a rate of 0.7 g / ml, degassed, and stirred overnight at room temperature using a blood mixer.

Next, an ethanolamine solution having a concentration of 1 M (pH 8) was added to block unreacted oxirane groups,
Stir overnight. Then, distilled water, sodium chloride 0.5
Sulfathiazole-immobilized chitosan beads (CH-EHST) were sequentially washed with a 0.02 M (pH 4) acetate buffer containing M and a 0.2 M (pH 10) carbonate buffer.
And α-L-aspartyl phenylalanine methyl ester solidified chitosan beads (CH-EHAT) were obtained.

Separation materials CH-EHST and C thus obtained
Using H-EHST, T and B lymphocytes were separated in the same manner as in Example 5 except that the lymphocyte suspension injection rate and washing rate were shown in Table 2. The results are shown in Table 2.

Comparative Examples 3 to 4 Crosslinked chitosan beads (CH) were directly used as a separating material for comparison, and the lymphocyte suspension injection rate and washing rate shown in Table 2 were used in the same manner as in Example 5. , B lymphocyte separation operation was performed. The results are shown in Table 2.

As is clear from the results shown in Table 2, B according to the present invention
It was found that the lymphocyte separation material (Examples 5 to 8) was effective as a separation material that preferentially traps B lymphocytes as in the results shown in Table 1. Also, B according to Examples 5 to 8
Also in the lymphocyte separation material, there was no remarkable cell damage in terms of viability and adhesion form.

Example 9 Bisepoxide having a polyethylene oxide chain (E
G22, manufactured by Nagase Kasei Co., Ltd. (ethylene oxide repeatability 22) is used as a spacer, and acrylic beads having oxirane groups (Eupergide C (EC),
(Rem Pharma [Rhm Pharma])
A B lymphocyte separation material was prepared by binding and immobilizing sulfathiazole (ST), which is a heterocyclic compound, as a ligand on (average particle diameter 0.15 mm). This B lymphocyte separation material was called EC-EG22ST.

First, 0.2M of 1, dissolved in carbonate buffer (pH 10)
The 3-propanediamine solution was washed with distilled water in advance E
After degassing with the addition of 30 g of C, the mixture was reacted in a water bath at 80 ° C. for 3 hours. Then, the mixture was stirred overnight at room temperature using a blood mixer (BM-101 manufactured by Kagaki Medical Science Co., Ltd.) and washed with distilled water. Next, 18 g (wet weight) of the EC thus introduced with an amino group was added to a carbonate buffer solution (p
H10) was added to a 10% (w / v) EG22 solution, the total amount was 100 ml, and the mixture was rotatably mixed with a blood mixer overnight, then reacted in an 80 ° C water bath for 1 hour and washed with distilled water. . This was again added to 100 ml of a 0.1 M sulfathiazole solution using a mixed solution of carbonate buffer (pH 10) and dimethylformamide (volume ratio 2: 3),
The reaction was carried out in a water bath at 80 ° C. for 3 hours, and the mixture was rotatably mixed with a blood mixer overnight. Furthermore, in order to block unreacted oxirane groups, 100 ml of ethanolamine having a concentration of 1 M (pH 8) was added, and the mixture was stirred overnight. Then, the concentration of distilled water and sodium chloride 0.5M including 0.02M (p
H4) acetic acid buffer and 0.2 M (pH 10) carbonate buffer were sequentially washed, and acrylic beads (E) having sulfathiazole immobilized through a polyethylene oxide chain (E
C-EG22ST) was obtained.

The separation material obtained in this way has an inner diameter of 3 mm and an outer diameter of 4 mm.
Was filled in a column made of Teflon, and terminals having polyester filters (150 mesh) were connected to both ends of the tube to hold the separating material in the column. The inside of the column filled with this separation material is Hanks buffer solution (HBS
S; pH 7.2, Ca ²⁺ , Mg ²⁺ free) and subjected to the subsequent T, B lymphocyte separation procedure.

On the other hand, target lymphocytes were collected from human peripheral blood by specific gravity centrifugation using Ficoll pack (specific gravity 1.077, manufactured by Pharmacia), and 80% using HBBS.
A lymphocyte suspension was prepared by suspending and diluting at 0 × 10 ⁴ cells / ml.

1 ml of the lymphocyte suspension prepared as described above was injected into the column filled with the separating material at a constant flow rate shown in Table 3 by a disposable syringe and a syringe pump (both Terumo Corporation), and immediately HBBS was used. Then, the column was similarly injected at a constant flow rate shown in Table 3 to wash the inside of the column, and the separation operation was completed. The number of cells in the recovered lymphocyte suspension was measured by an automatic blood cell counter (ORETO INSTRUMENTS, LET-8), and subsequently only B lymphocytes in the recovered solution were tested against human immunoglobulin. Fluorescence staining was performed with an antibody-bound FITC (manufactured by Miles [MILES]), and FITC-positive cells in the recovered solution were counted by a fluorescence microscope to obtain FI.
The ratio of TC-stained cells (FITC (+)) and FITC-unstained cells (FITC (-)) captured in the column was determined. The results are shown in Table 3.

Comparative Example 5 For comparison, acrylic beads having an oxirane group (Eupergit C (EC), manufactured by Laem Pharma Co.)
Was used as a separating material as it was, and T and B lymphocytes were separated according to the procedure of Example 9. The results are shown in the third bale.

Example 10 Bisepoxide (E having a polyethylene oxide chain)
G22, manufactured by Nagase Kasei Kogyo Co., Ltd. is used as a spacer, and crosslinked chitosan beads (chitopearl (C
H), manufactured by Fuji Spinning Co., Ltd. (average particle size: 0.3 mm) was bound and immobilized with sulfathiazole (ST) as a ligand to prepare a B lymphocyte separation material. This B lymphocyte separation material was called CH-EG22ST.

First, 30 g (wet weight) of chitosan beads washed in advance with distilled water was added to a 10% (w / v) EG22 solution prepared using a carbonate buffer (pH 10), and the total amount was 100 ml.
And rotate and mix with blood mixer overnight, then at 80 ℃
The reaction was carried out for 1 hour in the water bath and washed with distilled water. This was again added to 100 ml of a 0.1 M sulfathiazole solution using a mixed solution of carbonate buffer (pH 10) and dimethylformamide (volume ratio 2: 3), and the mixture was reacted in a water bath at 80 ° C for 3 hours, and then added to a blood mixer. Mix by vortexing overnight. Further, 100 ml of ethanolamine having a concentration of 1 M (pH 8) was added to block unreacted oxirane groups, and the mixture was stirred overnight. Then, distilled water, sodium chloride 0.5
Chitosan beads (CH-E) immobilized with sulfathiazole were washed successively with an acetic acid buffer containing 0.02M (pH 4) containing M and a carbonate buffer containing 0.2M (pH 10).
G22ST) was obtained.

Using the separation material CH-EG22ST thus obtained, a T / B lymphocyte separation operation was performed in the same manner as in Example 9. The results are shown in Table 3.

Comparative Example 6 For comparison, cross-linked chitosan beads (chitopea)
l, (CH) Fuji Spinning Co., Ltd.) was used as it was as a separating material, and T and B lymphocytes were separated according to the procedure of Example 9. The results are shown in Table 3.

As is clear from the results shown in Table 3, under each separation condition, the separation material in which sulfathiazole was bound via a spacer (Examples 9 to 10) significantly reduced the adhesiveness of lymphocytes themselves, and The non-specific adhesion of T lymphocytes was suppressed without impairing the high affinity for B lymphocytes, and the T and B lymphocyte selectivity was high. Furthermore, such a highly selective expression was observed regardless of whether the water-insoluble solid substance was acrylic beads (EC) or chitosan beads (CH),
It was confirmed that the T and B lymphocyte selectivity does not depend on the type of the water-insoluble solid substance.

Examples 11 to 12 The ethylene oxyside repeatability is 1 as a spacer.
Of bisepoxide (EG
1, manufactured by Nagase Chemical Industry Co., Ltd. (Example 11) or a bisepoxide having a degree of repetition of 9 (EG9, Nagase Chemical Industry)
Acrylic beads in which sulfathiazole was immobilized via an ethylene oxide chain were prepared in the same manner as in Example 9 except that the product (manufactured by KK) (Example 12) was used. The obtained B lymphocyte separation materials were designated as EC-EG1ST (Example 11) and EC-EG9ST (Example 12), respectively. This separation material EC-EG1ST or EC-EG9S
In the same manner as in Example 9 except that T was used to set the lymphocyte suspension infusion rate and the washing rate to those shown in Table 4,
The T and B lymphocytes were separated. The results are shown in Table 4.

Example 13 EC-EG22ST was prepared in the same manner as in Example 9, and the same procedure as in Example 9 was carried out except that the separating material was used to change the lymphocyte suspension injection rate and the washing rate to those shown in Table 4. Then, the T and B lymphocytes were separated. The results are shown in Table 4.

Examples 14 to 15 1,4-butanediol diglycidyl ether (ME4, Tokyo Chemical Industry Co., Ltd.) having a methylene repeating degree of 4 as a spacer
Co., Ltd.) (Example 14) or the degree of repetition of methylene
1,6-hexanediol diglycidyl ether (ME
6 (manufactured by Tokyo Kasei Co., Ltd.) (Example 15) was used, and 10 (w / v) was prepared using a mixed solution of carbonate buffer (pH 10) and dimethylformamide (volume ratio 9: 1) as the solution.
)% ME4 solution, carbonate buffer (pH 10), dimethylformamide and distilled water (volume ratio 2: 2: 1)
Acrylic beads in which sulfathiazole was immobilized via a polyethylene chain were prepared in the same manner as in Example 9 except that a 10 (w / v)% ME6 solution prepared using was used.
The obtained B lymphocyte separation material was EC-ME4, respectively.
ST (Example 14), EC-ME6ST (Example 15)
Was called.

Using the separation material EC-ME4ST or EC-ME6ST, T and B were treated in the same manner as in Example 9 except that the lymphocyte suspension injection rate and the washing rate were set to the rates shown in Table 4.
Lymphocyte separation operation was performed. The results are shown in Table 4.

As is clear from the results shown in Table 4, the separation material in which sulfathiazole was bound via a spacer suppressed non-specific adhesion of T lymphocytes without impairing its high affinity for B lymphocytes, and It was found to maintain accurate T and B lymphocyte separation ability, and in particular EG9 (Example 12), EG
Those using 22 (Example 13) and ME4 (Example 14) as spacers showed outstanding T, B selectivity.

Example 16 Bis epoxide (EG having ethylene oxides having a repetition degree of ethylene oxide of 1 as a spacer (EG
1. Nagase Chemical Industry Co., Ltd., and 10 (w / v)% EG prepared by using a mixed solution of carbonate buffer (pH 10) and dimethylformamide (volume ratio 9: 1) as the solution.
Chitosan beads in which sulfathiazole was immobilized via an ethylene oxide chain were prepared in the same manner as in Example 10 except that one solution was used. The obtained B lymphocyte separation material was referred to as CH-EG1ST.

Using this separating material CH-EG1ST, T and B lymphocytes were separated in the same manner as in Example 10 except that the lymphocyte suspension injection rate and washing rate were set to the rates shown in Table 5. The results are shown in Table 5.

Example 17 3 having 3 ethylene oxide chains as a spacer
Chitosan beads in which sulfathiazole was immobilized via an ethylene oxide chain were prepared in the same manner as in Example 10 except that a functional triepoxide (EG3, manufactured by Nagase Kasei Co., Ltd.) was used. The obtained B lymphocyte separation material was referred to as CH-EG3ST.

Using this separation material CH-EG3ST, T and B lymphocytes were separated in the same manner as in Example 10 except that the lymphocyte suspension injection rate and the washing rate were the rates shown in Table 5. The results are shown in Table 5.

Example 18 Bisepoxy having a polyethylene oxide chain having a repetition degree of ethylene oxide of 9 as a spacer (EG
9, Example 1 except that Nagase Chemical Industry Co., Ltd. is used
In the same manner as in 0, chitosan beads having sulfathiazole immobilized via a polyethylene oxide chain were prepared. The obtained B lymphocyte separation material is CH-EG9ST.
Was called.

Using this separating material CH-EG9ST, T and B lymphocytes were separated in the same manner as in Example 10 except that the lymphocyte suspension entry rate and washing rate were set to the rates shown in Table 5. The results are shown in Table 5.

Example 19 CH-EG22ST was prepared in the same manner as in Example 10,
Using this separating material, T and B lymphocytes were separated in the same manner as in Example 10 except that the lymphocyte suspension injection rate and the washing rate were those shown in Table 5. The results are shown in Table 5.

Examples 20 to 21 1,4-butanediol diglycidyl ether (ME4, manufactured by Tokyo Kasei Co., Ltd.) having a methylene repeating degree of 4 as a spacer (Example 20) or 1,6-butane having a methylene repeating degree of 6 Hexanediol diglycidyl ether (M
E6, manufactured by Tokyo Kasei Co., Ltd. (Example 21) was used, and a carbonate buffer solution (pH 10) was used as the solution.
Mixture of 0 (w / v)% ME4 solution, carbonate buffer (pH 10), dimethylformamide and distilled water (volume ratio 2:
Chitosan beads in which sulfathiazole was immobilized via a polymethylene chain were prepared in the same manner as in Example 10 except that a 10 (w / v)% ME6 solution prepared using 2: 1) was used. The obtained B lymphocyte separation materials were CH-MH4ST (Example 20) and CH-ME6ST, respectively.
It was designated as (Example 21).

By using this separation material CH-ME4ST or CH-ME6ST, the lymphocyte suspension injection rate and the washing rate were set to 5th.
In the same manner as in Example 10 except that the speeds shown in the table were obtained,
The T and B lymphocytes were separated. The results are shown in Table 5.

As is clear from the results shown in Table 5, the separation material in which sulfathiazole was bound via a spacer was similar to the results shown in Table 5 in that T lymphocytes were not impaired with high affinity for B lymphocytes. It was found that the non-specific adhesion of T. melanin was suppressed and the highly accurate T and B lymphocyte separation ability was maintained. In particular, EG22 (Example 19), ME4 (Example 20) and ME6 (Example 21) were used as spacers. The used ones have a FITC (-) capture rate of less than 10% and FIT.
The C (+) capture rate was 80%, which was an outstanding selectivity.

Examples 22 to 25 In addition to T lymphocytes and B lymphocytes, the lymphocyte fraction obtained from human blood exsanguination by specific gravity centrifugation was mainly
Since there are impurities such as monocytes or null cells,
Contamination of these non-T and B lymphoid cells (N) was evaluated by the peroxidase staining method and immunobeads method.
That is, EC-EG2 obtained in the same manner as in Example 9
2ST, CH-EG2 obtained in the same manner as in Example 10
2ST, CH-ME4 obtained in the same manner as in Example 20
ST or CH-M obtained in the same manner as in Example 21
T and B lymphocytes were separated in the same manner as in Example 9 except that E6ST was used to inject and wash the lymphocyte suspension at the speeds shown in Table 6, and the lymphocytes were separated before and after the separation operation. Non-T, B lymphoid cells (N) in suspension
The rate of contamination with the above was evaluated by the above method, and the yield and purity of T lymphocytes recovered by the separation operation were calculated by the above evaluation method to obtain an accurate separation system. The results are shown in Table 6.

As is clear from the results shown in Table 6, both the T and B lymphocyte separation materials according to the present invention have extremely high T and B lymphocyte selectivity, and the yield and purity of T lymphocytes are 80% each. Remarkable values of ~ 95% and 90-95% were shown, and at the same time, it was confirmed that such separation ability was not affected by the contamination of non-T and B cells.

Examples 26 to 28 First, CH-EG1ST was prepared in the same manner as in Example 16, CH-EG9ST was prepared in the same manner as in Example 18, and CH-EG22ST was prepared in the same manner as in Example 10.

Using these separating materials, a lymphocyte suspension prepared from rat mesenteric lymph nodes and human peripheral cells were prepared according to the procedure in Example 9 at the lymphocyte suspension injection rate and washing rate shown in Table 7. The T and B lymphocytes were separated from the blood lymphocyte suspension. FITC conjugated with an antibody against rat immunoglobulin G was used for fluorescent staining of rat-derived lymphocytes. The results are shown in Table 7.

As is clear from the results shown in Table 7, both the T and B lymphocyte separation materials of the present invention have extremely high B lymphocyte affinity for lymphocytes of rat and human origin. It was From this fact, the T / B lymphocyte separation material of the present invention in which a ligand having an affinity for immune glybulin G is immobilized via a spacer has a B lymphocyte affinity which is not affected by the animal species of the lymphocytes to be separated. It was estimated that

Furthermore, the T and B lymphocyte separation material according to the present invention (Examples 9 to
28), at least 1.0 × per 1 g of the separating material
It is possible to treat about 10 ^{7 to} 6.0 × 10 ⁷ lymphocytes, the treatment time required for the treatment is 10 minutes or less, and the cell damage which is remarkable in terms of viability and adhesion form is also caused. It was nothing at all.

(Effects of the Invention) As described above, the present invention provides an organic compound having a high affinity with at least one immunoglobulin selected from the group consisting of dipeptides and sulfathiazoles via a spacer on the surface of a water-insoluble solid substance. Since it is a B lymphocyte separation material characterized in that a synthesized ligand is immobilized, it is possible to selectively capture and separate only B lymphocytes. For example, from a lymphocyte fraction obtained from peripheral blood or living tissue. , T lymphocytes and B lymphocytes can be separated easily and rapidly with extremely high yield and high purity, and the functional damage to the separated T lymphocytes and B lymphocytes is also small. It can be applied to basic techniques for diagnosing and treating diseases related to functions and obtaining physiologically active substances derived from lymphocytes. Furthermore, in the B lymphocyte separation material of the present invention, the spacer has a skeleton of a carbon-hydrogen chain having at least 2 or more carbon atoms or a hydrocarbon chain containing a characteristic group, and more preferably 4 or more repeating methylene spacers. , Which has a linear alkyl skeleton or an ethylene oxide skeleton having a degree of polymerization of 1 to 50, further suppresses non-specific adsorption of T lymphocytes, resulting in higher separation accuracy. In addition, when the organically synthesized ligand is a combination of arginine, aspartic acid, phenylalanine, proline and sulfathiazole, it is possible to obtain T with higher purity and higher yield.
It becomes possible to obtain lymphocytes and B lymphocytes, and the water-insoluble solid substance has an average particle size of 0.05 to 5
B particles can be separated more stably and efficiently with the particles of mm.

In addition, the present invention immobilizes an organically synthesized ligand having a high affinity with at least one immunoglobulin selected from the group consisting of dipeptides and sulfathiazoles via a spacer on the surface of a water-insoluble solid substance. The method for separating B lymphocytes is characterized in that a lymphocyte-containing liquid is brought into contact with the separation material to selectively capture B lymphocytes having an immunoglobulin molecule (S-Ig) on the membrane surface. B lymphocytes can be easily separated with high accuracy without using concentrated serum derived from different organisms or physiologically active substances in the treatment procedure. In addition, in the method for separating B lymphocytes of the present invention, the spacer in the separating material has a hydrocarbon chain having at least two carbon atoms or a hydrocarbon chain containing a characteristic group as a skeleton, and more preferably a spacer. When the repeating methylene has 4 or more straight chain alkyl skeletons or has an elelene oxide skeleton having a polymerization degree of 1 to 50, more accurate B lymphocyte separation can be expected. Also, when the organically synthesized ligand is a combination of arginine, aspartic acid, phenylalanine, proline and sulfathiazole, for example, it is higher in purity and higher in yield than when it is used for separation of lymphocyte fractions. T lymphocytes and B lymphocytes can be separated with high safety, and if an isotonic buffer is used as a washing solution, T and B lymphocytes can be easily and suitably separated. , A better effect can be expected.

The present invention further immobilizes, on a surface of a particulate water-insoluble solid substance, an organically synthesized ligand having a high affinity with at least one type of immunoglibulin selected from the group consisting of dipeptides and sulfathiazoles via a spacer. Since it is a B lymphocyte separator characterized in that it has a column packed with a separated material, the B lymphocyte separation material and the B lymphocytes such as a lymphocyte suspension having excellent characteristics as described above. The contact with the contained liquid can be performed more efficiently, the B lymphocytes can be separated in a short time and successfully, and the column is made of polypropylene or polycarbonate. A filter having a retina that allows the cell components in the B lymphocyte-containing liquid to pass through but does not allow the separator to pass through is provided between the separator and the separator layer. Furthermore, when the filter is made of polyester or polyamide, sterilization and other operations are easy, and there is no risk of damaging the cell components in the B lymphocyte-containing liquid that passes through, and the lymphocyte fraction can be obtained. When used for separation, it enables separation of T lymphocytes and B lymphocytes with higher purity and higher yield.

[Brief description of drawings]

FIG. 1 is a schematic view showing a mode of use of an embodiment of the B lymphocyte separator of the present invention. 1 ... Lymphocyte suspension, 4a, 4b ... Filter, 5
…… B lymphocyte separation material, 6 …… column, 7 …… treatment liquid, 8 …… washing liquid, 9 …… inlet port, 10 …… outlet port.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP 58-170717 (JP, A) JP 63-9450 (JP, A) JP 60-3367 (JP, B2) JP 59- 36961 (JP, B2)

Claims (16)

[Claims] 1. An organically synthesized ligand having a high affinity with at least one immunoglobulin selected from the group consisting of dipeptides and sulfathiazole is immobilized on the surface of a water-insoluble solid substance through a spacer. B lymphocyte separation material characterized by being present. 2. The B lymphocyte separation material according to claim 1, wherein the spacer has a skeleton of a hydrocarbon chain having at least two carbon atoms or a hydrocarbon chain containing a characteristic group. 3. The B lymphocyte separation material according to claim 2, wherein the repeating methylene of the spacer has at least 4 linear alkyl skeletons. 4. The B lymphocyte separation material according to claim 2, wherein the spacer has an ethylene oxide skeleton having a polymerization degree of 1 to 50. 5. The B lymphocyte separation material according to claim 1, wherein the water-insoluble solid substance is a particle body made of a synthetic organic polymer, a natural organic polymer or an inorganic substance. 6. The water-insoluble solid substance has an average particle size of 0.05 to
The B lymphocyte separation material according to claim 1 or 2, which is a 5 mm particle body. 7. Separation comprising immobilizing an organically synthesized ligand having a high affinity for at least one immunoglobulin selected from the group consisting of dipeptides and sulfathiazoles via a spacer on the surface of a water-insoluble solid substance. A method for separating B lymphocytes, which comprises contacting a material with a B lymphocyte-containing liquid to selectively capture B lymphocytes having an immunoglobulin molecule (S-Ig) on the membrane surface. 8. The spacer in the separating material has at least 2 spacers.
The method for separating B lymphocytes according to claim 7, wherein the skeleton is a hydrocarbon chain having one or more carbon atoms or a hydrocarbon chain containing a characteristic group. 9. The method for separating B lymphocytes according to claim 8, wherein the repeating methylene of the spacer in the separating material has four or more linear alkyl skeletons. 10. The spacer in the separating material has a degree of polymerization of 1 to 1.
The method for separating B lymphocytes according to claim 8, which has 50 ethylene oxide skeletons. 11. A water-insoluble solid substance is a synthetic organic polymer,
The method for separating B lymphocytes according to any one of claims 7 to 10, which comprises a natural organic polymer or an inorganic substance. 12. The method for separating B lymphocytes according to claim 7, wherein an isotonic buffer solution is used as the washing solution. 13. An organically synthesized ligand having a high affinity with at least one immunoglobulin selected from the group consisting of dipeptides and sulfathiazoles is immobilized on the surface of a particulate water-insoluble solid substance through a spacer. A separator for B lymphocytes, which comprises a column packed with a separating material. 14. The separator for B lymphocytes according to claim 13, wherein the column is made of polypropylene or polycarbonate. 15. Between the inlet and outlet of the column and the separation material layer,
The B according to claim 13 or 14, which comprises a filter having a mesh that allows the cell components in the B lymphocyte-containing liquid to pass through but does not allow the separation material to pass therethrough.
Lymphocyte separator. 16. The B lymphocyte separator according to claim 15, wherein the filter is made of polyester or polyamide.