JPS5917387B2 - Method and device for separating lymphocytes into T cells and B cells - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method and a separator for separating lymphocytes into T cells and B cells using a B lymphocyte adsorbent. An insoluble solid substance, in which a functional group that dissociates into an anion per 17 oml volume is dissolved in a solution with a pH of 6 or more and 8 or less, O, 0
A B lymphocyte adsorbent characterized by having an amount of 5 m equivalents (millichemical equivalents) or more and 5 m equivalents or less and lymphocytes O, ly
/dl or more and less than ly/dl of protein suspended in a solution is brought into solid-liquid contact at a temperature of 20°C or more and 15 or more and 40°C or less to adsorb B cells, thereby separating T cells. , relates to a lymphocyte subfraction separation method and separator for obtaining B cells by detaching B cells.

Here, T cells (thymus-derived lymphocytes) are lymphocytes that form a lip with neuraminidase-treated sheep red blood cells, and B cells (bone marrow-derived lymphocytes) are lymphocytes that have immunoglobulin on their cell surface. , respectively.

H5. Conventionally, two types of separation methods have been known for separating TMB cells and B cells, which are subfractions of lymphocytes.

Specifically, one method is to separate T and B cells by utilizing their biological (immunocytological) differences, that is, to separate T and B cells by their specific antigens, receptors, and immune cells on the 30 surfaces of their membranes. This is a separation method that uses the presence of globulins as an indicator and uses organisms that have specific affinity for them or specific antisera (antibodies). The other is
This is a separation method that takes advantage of the differences in the physicochemical properties of T and B cells35, and uses the differences in adhesive ability, surface charge, size, density, and sensitivity to immunosuppressants, steroid hormones, and X-rays independently. This is for A-woo.

An example of using surface charges is “Medical History” Volume 102,
38-40 (1977), an example of separation based on size and density is ElOcitySedimenta
tiOn method”JournalOfImmUrlOlOg
9, pp. 1617-1620 (1977), and methods that utilize differences in sensitivity to immunosuppressants, steroid hormones, and X-rays include, for example, "Medical Immunology" edited by Hiroshi Kikuchi, Nankodo, 1976. It is stated on page 38 of the publication.

A general explanation for each of the above-mentioned methods is given in Junichi Yada, "T lymphocytes and B lymphocytes", Chugai Igakusha, 1975 edition, pp. 45-49. Methods that take advantage of differences in sample composition require complicated sample cleaving and handling procedures, and the sample and separation equipment are generally expensive.Furthermore, although the sample has a certain activity for living organisms, Production and maintenance of activity become difficult.On the other hand, methods that utilize differences in physicochemical properties result in insufficient separation because the differences are slight if each difference is used alone.For example, Among the methods that take advantage of the differences, a specific method that is relatively commonly used is to isolate T cells by taking advantage of the fact that B cells have a higher adhesion to nylon fibers than T cells. There is a method (Wako Pure Chemical Industries, Ltd. "Nylon fiber for T cell isolation" explanatory materials), but even in this case, many T cells and B cells cannot be separated, and only a few highly pure T cells can be obtained. I can't do it. Furthermore, cell damage is inevitable in separation methods that use drugs or X-rays. The present inventors aimed to isolate both T cells and B cells using a lymphocyte suspension with high purity and recovery rate without damaging the cell membrane and cell functions. As a result of intensive research on cell adsorbents, we have found that a water-insoluble solid substance containing an acidic functional group that dissociates anions per 1 ml volume in a solution with a pH of 6 or more and 8 or more has an amount of 0.05 m equivalent or more. The substance (?
The present inventors have discovered that the adsorbent of the present invention (hereinafter referred to as the "adsorbent of the present invention") adsorbs only B fine particles and sulfuric acid at high temperatures, leading to the completion of the present invention.

That is, by bringing a lymphocyte suspension into contact with the adsorbent of the present invention, the additive effects of the superior adhesiveness of B cells over T cells at room temperature or higher and the negative charge of T cell membranes, which are larger than B cell membranes, are utilized. As a result, at room temperature or above, B cells will be adsorbed, but T cells will not be adsorbed.

After isolating unadsorbed T cells, adsorbed B cells can be detached and isolated by shock, application of external pressure, or by reducing the adhesion of B cells at temperatures below 10°C. can be released. Therefore, a column filled with the adsorbent of the present invention and having a liquid inlet/outlet is prepared, a lymphocyte suspension separated from blood is injected into this column, the column is allowed to stand at a high temperature for a certain period of time, and then washed out with a washing solution. By performing this, a T cell suspension can be selectively obtained.

Thereafter, a B cell suspension can be obtained by washing the cells with a washing solution while applying shock, external pressure, or at low temperature. In the present invention, a water-insoluble solid substance containing an acidic functional group is defined as a spherical, granular, or fibrous water-insoluble solid substance in which an acidic functional group, that is, a strong acidic functional group or a weakly acidic functional group is present. can.

A strong acidic functional group is, for example, a sulfone group (-SO3H)
The weakly acidic functional group is, for example, a carboxyl group (
-COOH). These include a phosphonic group (-PO3H2), a phenol group (-C6H5OH), and the like. Specifically, molecules with each of the above acidic functional groups are solidified into organic substances,
Examples of organic substances include agarose and its derivatives, dextran derivatives, cellulose and its derivatives, polyacrylamide derivatives, and polyamide derivatives, and immobilization methods include the bromcyan method, carbodiimide method,
Examples include a covalent bonding method using an esterification method or a crosslinking method using glutaraldehyde, an ionic bonding method, an entrapping method, a physical adsorption method, and the like.

For example, spherical agarose gels, spherical dextran gels created by reacting soluble dextran with epichlorohydrin in alkaline conditions, and cellulose fibers cross-linked with epichlorohydrin are treated with bromic cyan to produce omega-carboxyalkyl amines. or ω-sulfoalkylamine, ω-aminoalkylamine agarose gel, or ω-aminoalkylamine-dextran derivative gel with water-soluble carbodiimide (1-ethyl-3-(3-dimethylaminopropyl)). Carbodiimide hydrochloride and 1-cyclohexyl-3-(
2-morpholinoethyl)carboimide meso-p-toluene saphonphonate, etc.) reacted with ω-carboxyalkylcarboxylic acid or ω-sulfoalkylcarboxylic acid, and cellulose fibers reacted with succinic anhydride. Polyacrylamide gels prepared by polymerizing acrylamide and NlN'-methylenebisacrylamide in the presence of NlN'-methylenebisacrylamide and a catalyst are treated with hydrazine and reacted with succinic anhydride, and polyacrylamide gels are prepared by polymerizing polyacrylamide gels with ethylenediamine. and reacted with ω-carboxyalkylamine or ω-sulfoalkylamine, and polyamides with carboxyalkyl groups or sulfoalkyl groups in the side chains are gelled with glutaraldehyde. Examples include polyamides containing carboxyalkyl groups, sulfoalkyl groups, etc., wrapped in polyacrylamide.

In addition, a copolymer of styrene and divinylbenzene in which each of the above acidic functional groups is directly bonded, for example, an appropriate amount of divinylbenzene is added to styrene, and a small amount of benzoyl peroxide and water are added to the mixture as a polymerization catalyst. There are also those obtained by performing suspension polymerization by adding a suspending agent such as bentonite or alginic acid, and then treating the resulting polymer with concentrated sulfuric acid or chlorosulfonic acid.

In addition, condensates of phenolsulfonic acid, formaldehyde, and phenol, vinylsulfonic acid polymers, atarylic acid polymers, methacrylic acid polymers, itaconic acid polymers,
Maleic acid polymer, fumaric acid polymer, butadiene 1-carboxylic acid polymer, iminodiacetic acid type styrene polymer, 1,
Examples include condensates of 3,5 resorcinic acid and formaldehyde, diallylphosphonic acid polymers, and condensates of phenol and formaldehyde. Furthermore, the molecules having each of the acidic functional groups are added to an alkylamino glass synthesized by a reaction between glass and a silane derivative such as γ-aminopropyltriethoxysilane, using a carbodiimide method, an esterification method, or a glutaraldehyde method. There are those that are bonded by a covalent bonding method using a crosslinking method using, for example, or an ionic bonding method.

The concentration of acidic functional groups in the present invention is such that the functional groups dissociated into anions do not cause cell damage...0.05 m/ml of water-insoluble solid material in a solution of 6 or more and 8 or less.
equivalent or more and 5 m equivalent or less Very preferably, it is suitable to have 0.1 m equivalent or more to exhibit the performance, and 0.
．． When the amount is less than 0.5 m equivalent, the adsorption rate of T cells to the adsorbent tends to increase.

The opposing ions, which are mobile ions of the opposite sign that are electrically neutralized with the acidic functional group, are H+, Na+, K+, self+, and M.
In other words, a water-insoluble solid substance having an acidic functional group on the surface can be used in either the H form or the salt form, but the salt form makes it easier to adjust the pH of the suspended liquid. Therefore, it is preferable for use. An example of the T and B cell separator used in the present invention will be described with reference to a drawing. The separator includes an inlet 1 for injecting the lymphocyte suspension and washing solution, an outlet for draining the suspension and washing solution,
Filters 1 and 3 having pore diameters large enough to prevent the adsorbent from flowing out at the outlet are attached to a container having 4 and filled with the adsorbent 2 of the present invention as described above.

To separate lymphocytes into T cells and B cells using a T and B cell separator, a suspension of lymphocytes separated from blood by a method such as density centrifugation is placed in a separator. When the suspension has finished penetrating, stop the outflow and let it stand for a certain period of time. Then, run the first washing solution to wash out unadsorbed cells. This is carried out by applying shock by applying pressure or by flowing a washing solution while applying external pressure, or by allowing the separator to stand still at a low temperature for a certain period of time, and then flowing a washing solution to wash out the adsorbed cells.

? By the above method, the first wash solution contained 2 T cells.
The wash solution for the second time contains B cells. As the liquid for suspending lymphocytes and the washing liquid, 0.
A liquid containing protein of 1 y/dl or more and less than 19 y/dl, preferably a culture solution or buffer containing 2 to 20% of autologous serum or animal serum, such as beef tallow serum or calf serum, is used.

When using a solution containing protein of 19/dl or more, for example,
When 100% tallow serum is used, the adsorption rate of B cells to the adsorbent tends to decrease.

When using a solution containing less than 0.1 f1/dl of protein,
The percentage of cells attached to the adsorbent tends to increase.

The temperature during lymphocyte adsorption and first washing operation was as follows:
It is carried out at a temperature of 20°C or more and 40°C or less, preferably at a temperature near body temperature (around 37°C), and the temperature for subsequent operations after the first cleaning operation is 0°C or more and 10°C or less. is preferred.

The adsorption time for lymphocytes is preferably about 1 hour in order to reduce the proportion of B cells flowing out without being adsorbed.

The T and B cells obtained in this way are subjected to immunological function tests such as blastogenesis ability, antibody production regulation effect, cytotoxicity ability, and antibody production ability, and are then injected into a separator. It retained its function to the same extent as the previous lymphocyte function.

As described above, a water-insoluble solid substance containing an acidic functional group, which has 0.05 m equivalent or more and 5 m equivalent or less of a functional group that ionically dissociates per 1 d volume in a solution with a pH of 6 or more and 8 or less, is used at the entrance and exit port of the liquid. The present invention, in which lymphocytes are separated into T cells and B cells using a separator filled in a container having a method, has the following effects compared to conventional T and B cell separation methods. (1) The procedure for separating T cells and B cells from lymphocytes is simple, and both T cells and B cells can be isolated from the same lymphocyte sample. That is, by simply injecting a solution containing lymphocytes suspended in a solution containing 0.1 y/dl or more and less than 1 y/dl into a separator, allowing it to stand still under certain conditions, and then flowing a washing solution, T cells are
Thereafter, each B cell can be isolated by simply desorbing the adsorbed cells. (2) There is no need to treat lymphocytes with oxygen or drugs during separation, and the properties of lymphocytes do not differ from the original after separation, resulting in functional changes and declines in T cells and B cells. does not occur.

Therefore, it can also be used for immunological function tests on T cells or B cells. (3) The preparation of the separator is easier and simpler than the preparation of conventional samples and separation equipment for T cell or B cell separation.

(4) When an organic or inorganic synthetic substance is used as the adsorbent, it is easy to produce an adsorbent with a certain level of activity and maintain the activity, and the sterilization operation is also easy.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 Fabrication of a separator. Sulfopropyl dextran gel Na ion type (structural formula: dextran {)C3H6SO3Na particle size range: 1
00-200μ diameter) 2d in phosphate buffer, then 10% tallow serum monophosphate buffer (protein content 0.43y/Dj)
After thorough washing at [PH=7.44 (24°C)], this resin was applied to a tube with an inner diameter of 10 HIIt having a liquid inlet and outlet with a 80 μm mesh nylon net filter on the bottom.
It was prepared by filling an acrylic plastic column (see drawing) with care to avoid air bubbles.

The ionic dissociation concentration of the acidic functional group of this adsorbent in a solution with pH=7.44 (2'C) was 0.3 m equivalent/7n1. Adsorption and recovery procedure: Heparinized human peripheral blood was mixed with a phosphate buffer solution containing 5% each of fine powder of gum arabic.

37°C, stirring thoroughly 3 to 4 times during the process.
Warm for 1 hour. After heating, the sample was thoroughly stirred, separated by specific gravity centrifugation, and thoroughly washed with phosphate buffer to obtain a lymphocyte fraction of 6 x 106 cells/WLI! A solution suspended in 10% tallow serum monophosphate buffer to a concentration of 0.
5WL1 was gently poured into the inlet of the prepared separator, and when the suspended liquid had sufficiently penetrated into the adsorbent, the outlet of the liquid was closed to stop the outflow. Then, after standing at 37°C for 1 hour, the outlet was opened and 100I) tallow serum monophosphate buffer 6w11 heated to 37°C was gradually poured into the column from the column inlet, and adsorbed to the gel at the flow rate of gravity. Untreated cells were washed out and collected. Thereafter, the outlet of the liquid was closed again, and after being allowed to stand at 4° C. for 1 hour, the outlet was opened and 6 ml of beef tallow serum cooled to 4° C. was gradually poured into the column for second collection. Analysis results: The initial cell recovery rate determined by microscopic observation using an automatic hemocytometer and hemocytometer was 83.5.
%, and the second recovery rate was 12.4%.

Furthermore, T cells were analyzed by rosette reaction with neuraminidase-treated sheep red blood cells, B cells by fluorescent antibody method against surface immunoglobulin, and granule and monocyte groups by peroxidase staining. The respective compositions of the cell group were T = 76.7%, B = 23.4%, and granule/monocyte group = 0.1%, whereas the respective compositions of the first collected cell group were T =89.8%, B=10.
1%, granule/monocyte group = 0%, and each composition of the cell group collected the second time is T = 9.8%, B2 90.1%, granule/monocyte group = 0.1 It was (:fl). Therefore, the T cell recovery rate in the first wash was 97.8% and 2
The B cell recovery rate in the second wash is 47.8%.
Comparative Examples 1, 2, 3, 4, 5 Using a lymphocyte suspension with the same concentration and composition as in Example 1, nylon fibers (with a fiber diameter of 3 denier and 0.19
/d) Comparative Example 1-
, Na ion type of carboxymethyl dextran gel (
Structural formula: Dextran-CH2cOOONal Particle size range:
, 100-200 μ diameter PH = 7.44 (24 ° C.) Ionic dissociation concentration of acidic functional group 0.02 m equivalent/m
l) Comparative Example 2 - Dextran gel (particle size range: 100
-200/t diameter) - Comparative Example 3 - Diethylaminoethyldextran gel, which is a weakly basic anion exchanger (structural formula: dextran-0C2H4N+(C2H5)HC!
-, exchange capacity: 0.5m equivalent/ml, particle size range: 100
~200μ diameter) Comparative Example 4-, diethyl-(2-hydroxypropyl)aminoethyldextran gel (structural formula: dextran-10), which is a strongly basic anion exchanger
C! H4N+(02H5)2CH2(0H)CH3Cl
, Exchange capacity: 0.5m equivalent/d, Particle size range: 100-2
Adsorption and recovery operations similar to those in Example 1 were carried out using Comparative Example 5- (2 ml of adsorbent) as the adsorbent.

The cell recovery rates of the weakly basic anion exchanger - Comparative Example 4- and the strongly basic anion exchanger - Comparative Example 5- were 001) in both the first and second times.

Nylon fiber - Comparative example 1-, Carboxymethyl dextran gel - Comparative example 2-, Dextran gel - Comparative example 3-
The analysis results using the following were as follows. Comparative Example 6 The medium was replaced with 10% beef tallow serum monophosphate buffer,
The same adsorption and first recovery operations as in Example 1 were performed except that 0% phosphate buffer was used.

The initial cell composition was T=76.7%, B=23.
4 (!), granule/monocyte group = 0.1%, whereas the first cell recovery rate was 32.2%, and each composition was T.
= 91.8%, B = 7.1%, and granule/monocyte group = 0%. Comparative Example 7 The medium was replaced with 10% beef tallow serum monophosphate buffer,
The same adsorption and first recovery operations as in Example 1 were carried out, except that 0% tallow serum (protein content: 4Ab41) was used.

The initial cell composition was T=76.7%, B=23.
4%, granule/monocyte group = 0.1%, while 1%
The cell recovery rate for the second time was 99.1%. Example 2 Preparation of separator Gel type sulfonated polystyrene (6%) Na ion type of dibi-nylbenzene resin (structural formula: range: 297-8
It was produced in the same manner as in Example 1 using 2 ml of 40μ diameter).

The ion dissociation concentration of the acidic functional group of this adsorbent in a solution with pH=7.44 (2'C) was 1.6 m equivalent/d. Adsorption and collection operation: The procedure up to the first cell collection was carried out in the same manner as in Example 1.

Then, while tapping the area around the column with a metal rod,
Tallow baby serum 67TL1 cooled to 4° C. was gradually poured into the column for second collection. Analysis Results: The analysis method was the same as in Example 1.

The initial cell composition injected was T-83.5 (Fb, B=
16.5%, granule/monocyte group = 0%,
The first cell recovery rate was 90.5, and T = 9 for each composition.
1.8%, B = 7.9%, granule/monocyte group = 0%,
The cell recovery rate for the second time was 8.8%, and T = 7 for each composition.
．． 0%, B=92.8%, and granule/monocyte group 20%. Comparative Example 8 The medium was replaced with 10% beef tallow serum monophosphate buffer.
The adsorption and first recovery operations were carried out in the same manner as in Example 2, except that 0% phosphate buffer was used.

The initial cell composition was T=83.5%, B216.
5% and granule/monocyte group = 0%, whereas the first cell recovery rate was 10.1%, and each composition was T-92.
1%, B-7.2%, and granule/monocyte group = 0%. Comparative Example 9 The medium was replaced with 10% beef tallow serum monophosphate buffer.
The adsorption and first recovery operations were carried out in the same manner as in Example 2, except that Fb tallow serum was used.

The initial cell composition was T=83.5%, B=16.
The cell recovery rate in the first round was 97.1%, whereas the granule/monocyte group was 0%. Example 3 Preparation of separator: Na ion type of carboxymethyl dextran gel (structural formula: dextran-CH2COONal particle size range: 1
It was produced in the same manner as in Example 1 using 2 ml of 0.00 to 200 μm diameter).

The ionic dissociation concentration of acidic functional groups of this adsorbent in a solution with pH=7.44 (2'C) was 0.5 m equivalent/ml. The adsorption and recovery operations and analysis method were performed in the same manner as in Example 1.

The results showed that the initially injected cell composition was T = 78.3
(Fb.B = 21.0%, granule/monocyte group = 0.2%, whereas the first cell recovery rate was 85.6%, and each composition was T = 88. 6%, B = 11.001), granule/monocyte group = 0.201), the second cell recovery rate was 7.5%, and each composition was T - 8.3%, B = 90.
801), and the granule/monocyte group was 0.1%. Example
4 Preparation of separator゜Na ion type of carboxymethyl agarose gel (structural formula: agarose-CH2COONal particle size range: 100
It was produced in the same manner as in Example 1 using 2 ml of 200 μm diameter).

The ion dissociation concentration of the acidic functional group of this adsorbent in a solution with pH=7.44 (24° C.) was 0.1 m equivalent/d.

The adsorption and recovery operations were performed in the same manner as in Example 1.

result of analysis: The analysis method was the same as in Example 1.

The initial injected cell composition was T-78.3%, B=21
．． 00t), granule/monocyte group = 0.2%, whereas the first cell recovery rate was 83.8%, and each composition was
T-87.8%, B=12.1 (fl), granule/monocyte group -0.1%, the second cell recovery rate was 8.1%, and each composition was T-212. .2%, B=86.8%, granule・
The monocyte group was 0.2%. Example 5 Preparation of separator Gel type carboxylated polystyrene (6%) Na ion type of divinylbenzene resin (structural formula: degree range: 297
It was produced in the same manner as in Example 1 using 2 ml of 840μ diameter).

The ion dissociation concentration of acidic functional groups of this adsorbent in a solution with pH=7.44 (24° C.) was 4.1 m equivalent/ml. The adsorption and recovery operations were performed in the same manner as in Example 2. Analysis Results: The analysis method was the same as in Example 1.

The composition of the initially injected cells was T=91.0%, B28.
8%, granule/monocyte group = 0.1%, whereas 1%
The cell recovery rate for the second time was 92.6%, and each composition was T=9.
7.01:Fb,. B = 2.3%, granule/monocyte group = O%
The second cell recovery rate was 7.3%, and each composition was T = 10.1%, B = 88.4%, and granule/monocyte group = 0.
．． It was 2%. Example 6 Preparation of separator: Example 1 was prepared using Na ion type gel type divinylbenzene acrylate resin (structural formula: QH-CH2-CH-CH2 particle size range: 297 to 840μ diameter) 2d. It was prepared in the same manner as .

The ion dissociation concentration of the acidic functional group of this adsorbent in a solution with pH=7.44 (2'C) was 3.5 m equivalent/ml. The adsorption and recovery operations were performed in the same manner as in Example 2. Analysis Results: The analysis method was the same as in Example 1.

The initial injected cell composition was T = 72.2%, B = 26
．． 9%, granule/monocyte group = 0.3%,
The first cell recovery rate was 78.3%, and each composition was T=
91.3%, B = 8.3%, granule/monocyte group = 0.2%, the second cell recovery rate was 20.1%, and each composition was T = 7.1%, B-92.5%, granule/monocyte group = 0.
It was 4%. Example 7 Preparation of separator: Using 2 ml of gel-type metatallylic acid-divinylbenzene resin of Na ion type (structural formula: 297-840μ diameter),
It was produced in the same manner as in Example 1.

The ion dissociation concentration of the acidic functional group of this adsorbent in a solution with pH=7.44 (24° C.) was 2.5 m equivalent/WLI. The adsorption and recovery operations were performed in the same manner as in Example 2.

result of analysis: The analysis method was the same as in Example 1.

The initial injected cell composition was T = 74.9%, B = 24
．． 5%, granule/monocyte group = 0.2%,
The first cell recovery rate was 81.5 (fl), and each composition was T = 89.8%, B = 9.3%, and condylar monocyte group = 0.2%.
The second cell recovery rate was 18.1%, and each composition was T2 6.7%, B = 93.8%, and granule/monocyte group = 0.
．． It was 3%. Example 8 Preparation of separator: Na ion type of succinylated cellulose fiber (structural formula:
Cellulose-102CC2]-14C02Na Fiber diameter: 1
．． 38 denier) 0.2y was thoroughly loosened, and then placed in a 27n1 column of acrylic plastic with an inner diameter of 101111 mm (see drawing), which had a liquid inlet and outlet with a nylon net filter of 80 μm pore size on the bottom. A phosphate buffer solution, followed by a 10% tallow serum monophosphate buffer solution (PH-7.44 (2'C)) was gradually added dropwise from the inlet. , sufficiently wet the fibers.

The ionic dissociation concentration of the acidic functional group of this adsorbent in a solution with pH = 7.44 (24°C) is 0.2 m equivalent/0.1 y
It was hot. The adsorption and recovery operations were performed in the same manner as in Example 2.

result of analysis: The analysis method was the same as in Example 1.

The initial cell composition injected was T = 73.9%, B = 27
．． 0%, granule/monocyte group = 0.1%,
The first cell recovery rate was 70.0%, and each composition was T-
95.8%, B-3.7%, granule/monocyte group -0%, the second cell recovery rate was 19.1%, and each composition was T =
16.6%, B=83.4%, granule/monocyte group -0.1%
It was hot. Example 9 Preparation of separator: Na ion type of succinylaminopropyl glass particles (
Structural formula: Particle size range: 200 to 500 μm diameter) was prepared in the same manner as in Example 1 using 2 ml.

The ion dissociation concentration of acidic functional groups of this adsorbent in a solution with pH=7.44 (24° C.) was 0.1 m equivalent/ml. The adsorption and recovery operations were performed in the same manner as in Example 2. Analysis Results: The analysis method was the same as in Example 1.

The initial injected cell composition was T-73.9%, B=27
．． 0%, granule/monocyte group -0.1%), whereas the first cell recovery rate was 70.8%, and each composition was
= 98.8%, B = 1.2%, granule/monocyte group = 0%, the second cell recovery rate was 20.3%, each composition was T
= 20.3%, B = 79.8%, granule/monocyte group = 0.2
It was %.

[Brief explanation of drawings]

The drawing is a sectional view showing an example of a T and B cell separator. 1 is a liquid inlet, 2 is an adsorbent, 3 is a filter, and 4 is a liquid outlet.

Claims (1)

[Scope of Claims] 1. In an aqueous solution containing animal serum, having an acidic functional group consisting of a sulfone group or a carboxyl group as a B cell adsorbent, and having a pH of 6 or more and 8 or less, per 1 ml volume, Solid-liquid contact between lymphocytes and a B cell adsorbent made of a water-insoluble solid substance having an acidic functional group that dissociates into anions of 0.05 m equivalent or more and 5 m equivalent or less at a temperature of 20°C or more and 40°C or less A method for separating lymphocytes into T cells and B cells, the method comprising: 2. By applying impact or external pressure to the adsorbent that has adsorbed B cells through solid-liquid contact, or by bringing it into contact with a low-temperature washing solution,
Claim 1 encompassing detachment of B cells
Separation method described in section. 3 Separation of lymphocytes into T cells and B cells by filling a B cell adsorbent made of a water-insoluble solid substance in a container having a liquid inlet and outlet and containing at least one filter. As a B cell adsorbent, 1 m of adsorbent was added in an aqueous solution having an acidic functional group consisting of a sulfone group or a carboxyl group and having a pH of 6 or more and 8 or less.
A separator for separating lymphocytes into T cells and B cells, characterized by using a water-insoluble solid substance having an acidic functional group that dissociates into anions of 0.05 m equivalent or more and 5 m equivalent or less per liter.