JPH1033165A - Selective separation of hematopoietic undifferentiated cell and separator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To carry out the collection of a hematopoietic undifferentiated cell such as leukemia, the removal, etc., of lymphocytes from a cell for bone marrow transplantation by combining two or more monoclonal antibodis or proteins, etc., containing fragments of the antibodies with a hematopoietic undifferentiated cell and recovering the resultant bonded substance. SOLUTION: A hematopoietic undifferentiated cell is separated by bonding two or more monoclonal antibodies capable of recognizing respective different epitopes in the same antigenic molecular species on the hematopoietic undifferentiated cell such as a CD34 molecule or an antiCD34 antibody or proteins or peptides containing fragments of the antibodies as far as the bonding properties to the epitopes are held to the hematopoietic undifferentiated cell and recovering the resultant bonded substance.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method and an apparatus for separating hematopoietic undifferentiated cells using an antibody. According to the method for separating hematopoietic undifferentiated cells of the present invention, hematopoietic undifferentiated cells can be efficiently separated from a blood cell suspension in vitro or from blood by extracorporeal circulation, and the separated cells can be used for treating various diseases. It can be used.

[0002]

2. Description of the Related Art Isolation or concentration of necessary hematopoietic undifferentiated cells from a solution containing various cell types such as blood, or removal of unnecessary cells is effective in enhancing medical effects. Means. Bone marrow transplantation is a method of transplanting normal hematopoietic undifferentiated cells into cancer patients such as leukemia patients with myeloablation and innate immune diseases such as aplastic anemia. Is being established. Recently, a method is used in which hematopoietic undifferentiated cells are obtained not only from bone marrow but also from peripheral blood and cord blood. Allogeneic bone marrow transplantation is a method of injecting a bone marrow fluid collected from a healthy human whose HLA type, which is a leukocyte type, almost coincides with a patient whose bone marrow has been destructed by an anticancer drug or radiation. It causes GVHD (graft-versus-host disease). Therefore, in order to prevent autologous bone marrow transplantation therapy used in the treatment of hematopoietic malignant tumors and cancer in recent years, or graft-versus-host disease (GVHD) caused by allogeneic bone marrow transplantation, isolation of hematopoietic undifferentiated cells has been Demand is growing. Currently, as a method for separating cells, a method for specifically separating only target cells using an antibody is being developed. For example, a fluorescent antibody-labeled cell separation method, a method of immobilizing a ligand having an affinity for a target cell on a water-insoluble carrier and directly or indirectly binding the target cell thereto, separation using an immunoadsorption column, and separation using an immunomagnetic bead. There are laws. In the fluorescent antibody-labeled cell separation method, a cell mixture is first incubated with a fluorescently labeled monoclonal antibody that recognizes the membrane antigen expressed on the target cells, and then the treated cells are irradiated with laser light using a cell sorter or the like. This is a method of separating cells to which fluorescent antibodies are bound by applying that only cells to which antibodies are bound emit fluorescence.

A method in which a monoclonal antibody against an antigen present on the membrane surface of a target cell is directly immobilized on the surface of a separation device, and a cell mixture is first incubated with a monoclonal antibody binding to a membrane antigen on the target cell. hand,
WO 87-04628 describes a method in which a ligand, such as an anti-immunoglobulin antibody, that binds to an antibody on the cell surface is treated with a cell separation device immobilized thereon. Cell separation is a cell separation method performed by directly or indirectly binding the antigen-positive cells to a monoclonal antibody immobilized on a carrier or a device. In these methods, the antibody is separated on a plastic petri dish on which the antibody is immobilized, and the cell mixture is first poured onto the plastic petri dish on which the antibody is immobilized, and the antibody is bound to the membrane antigen on the target cell. To be incubated for. After the incubation, the plastic dish is washed to remove unbound cells and separated. In the immunoadsorption column method, a ligand such as an antibody against a membrane antigen on a target cell is immobilized on the surface of a bead, and this is packed in a column to perform cell separation. A cell-antibody-biotin bond is formed by adding a biotin-labeled antibody against the membrane antigen on the target cell to the cell mixture, and incubating the mixture.The beads in which avidin is immobilized on a porous acrylamide gel are then used. WO 91-16116 describes a method for passing target cells into the column by passing through a packed column and using the strong binding of biotin-avidin to separate the cells. In the immunomagnetic bead method, target cells are labeled with magnetic beads by first incubating a cell mixture with magnetic beads to which antibodies are bound. After labeling, the labeled cells are separated from the unlabeled cells using a magnetic device.

[0004] Although these methods have been studied for use in separating hematopoietic undifferentiated cells, sufficient results have not yet been obtained. That is, it is difficult to efficiently separate hematopoietic undifferentiated cells, which are cells having a low frequency of the antigen on the target cell membrane, for the following reasons. Hematopoietic undifferentiated cells usually have a low frequency of occurrence (purity). Therefore, it is necessary to increase the purity and recovery rate of hematopoietic undifferentiated cells by a cell separation operation. In order to increase the purity of hematopoietic undifferentiated cells, it is necessary to sufficiently wash the carrier to which the cells are bound and to remove non-specifically adsorbed cells as much as possible. On the other hand,
Washing removes the hematopoietic undifferentiated cells that have been weakly bound little by little, and reduces the recovery rate of hematopoietic undifferentiated cells. In particular, the lower the frequency of the surface antigen for the used antibody on the cell membrane, the more remarkable the reduction in the recovery rate. It is desired to increase both the purity and the recovery. Among hematopoietic undifferentiated cells, a separation method particularly using the CD34 molecule is described in Egelland (The Immu).
Nologist, Vol. 2, p. 65, 1994). The CD34 molecule was reported by Civin et al. (USP-4965204, and J. Immunolo).
gy, 133, 157, 1984), and are known as antigen markers for undifferentiated cells that are specifically expressed on blood undifferentiated cells. Attempts to isolate undifferentiated cells using this antigen marker have been reported (S. Saeland et al., Blood, 72, 15:15).
80, 1988). Further, an anti-CD34 antibody,
Medical devices are being developed to separate undifferentiated cells from differentiated cell groups and cancer cells using combinations of biotin, avidin, or magnetic beads (Dreg)
er et al., Expematol, 23, 147,
1995). Incidentally, the epitope in the CD34 molecule has been separated into three types by Sutherland et al. (J. Hematotherapy, Vol. 1, 155).
Page, 1992, The Immunology,
2, 65, 1994). Class 1 recognizes an epitope related to sialic acid of the CD34 molecule. Class 2
Is Glycopro from Pasteurella
It is tease sensitive. Class 3 does not cause epitope inactivation by these enzymes (Egelland,
The Immunology, Vol. 2, p. 65,
1994). For example, it has been reported that the MY10 antibody belonging to class 1 inhibits binding to the ICH3 and 9C5 antibodies belonging to class 2, and it has been considered that two or more antibodies do not bind efficiently (Uchansk)
a-Ziegler et al., In Leucocyte T.
yping IV, Oxford University
yPress, p. 818, 1989, and Lansd.
orp et al., In Leucocyte Typing
IV, Oxford University Pres
s, 826, 1989). Furthermore, the CD34 antigen has a low expression level particularly on the cell surface, and it is difficult for the antibody to bind to the cell firmly. It is a major clinical problem.

[0005]

An object of the present invention is to improve the method for separating undifferentiated hematopoietic cells by using these antibodies to improve the method for separating undifferentiated hematopoietic cells.

[0006]

SUMMARY OF THE INVENTION The present invention comprises two or more monoclonal antibodies or fragments thereof that retain the binding to antigenic species expressed on the cell surface to be isolated. Reacting a protein or peptide with blood, plasma, serum, body fluid, or cell suspension containing the target cells to bind hematopoietic undifferentiated cells and specifically recover the antibody conjugate By
An object of the present invention is to provide a method for selective separation of cells, which comprises recovering desired hematopoietic undifferentiated cells. Further, the present invention provides two or more monoclonal antibodies recognizing different epitopes in the same antigen molecular species on hematopoietic undifferentiated cells, or as long as the antibody retains binding to the epitopes, It is intended to provide an apparatus for separating hematopoietic undifferentiated cells, comprising a protein or peptide containing fragments and a carrier to which the proteins or peptides are bound. The two or more types of monoclonal antibodies in the present invention refer to a plurality of monoclonal antibodies that recognize different epitopes expressed on the cell surface of the same antigen molecular species. Especially CD
Two or more types of antibodies that recognize the 34 antigen can recognize different epitopes, and have the possibility of binding to the same CD34 molecule at the same time. Hematopoietic undifferentiated cells isolated in the present invention are cells that can be proliferated by a specific stimulus, and cells that can differentiate one or more types of mature cell groups such as erythrocytes, monocytes, granulocytes, lymphocytes, and macrophages. Is shown. In particular, when cultured in a semi-solid medium in the presence of a suitable growth factor, undifferentiated blood cells that form colonies,
Includes hematopoietic stem cells that have the ability to differentiate into all lineages and have the ability to self-proliferate. The cells are abundant in bone marrow and cord blood. The cells are also contained in peripheral blood, but can be induced in peripheral blood by administering a cytokine such as granulocyte colony forming factor (G-CSF).
In addition, a technique for culturing these cells to grow hematopoietic undifferentiated cells in vitro has been reported. The cell suspension containing these hematopoietic undifferentiated cells can be a material for separating hematopoietic undifferentiated cells according to the present invention. An epitope is a structure on an antigen molecule that determines the specificity of an antigen-antibody reaction, and is a structural unit on an antigen molecule that reacts with an antigen-binding site of an antibody. Recognition of the same antigen molecular species by two or more antibodies does not necessarily recognize different epitopes of the same molecular species simultaneously, and may recognize the same epitope of the same molecular species.

In the present invention, it is effective to use an antibody that specifically recognizes a CD34 molecule expressed on the surface of hematopoietic undifferentiated cells. Furthermore, the antigenic species according to the present invention include c-kit (Y
arden et al. 6, Volume 3341, 1
987), the receptor FLK-2 (Mathews
Cell, 65, 1143, 1991).
Interleukin-3 receptor α chain (Kitamur
a, et al., Cell, 66, 1165, 1991).
And the like. They also have very low expression frequency and are unsuitable for cell separation, but by combining two or more monoclonal antibodies against these molecules, these expressing cells can be isolated. The anti-CD34 antibody, one of the antibodies used in the present invention, includes 1984 Civ
in (USP-4965204, and J. Immuno
logy, 133, 157, 1984). For example, anti-HP
CA-2 antibody (Becton Dickinson), anti-HPCA
-1 antibody (Becton Dickinson), 4A1 (Nichirei), B1.3C5 (Katz et al., Leuk. Re.
s. Magazine, 9, 191 pages, 1985), 12.8, 1
15.2 (Andrews et al., Blood, 67,
842, 1986), ICH3 (Watt et al., Le).
ukamia, 1, 417, 1987), T
uk3 (Unchanska-Ziegler et al., Ti
sue Antigens, 33, 230, 19
89), QBEND10 (Fina et al., Blood,
75, 2417, 1990), CD34 (Ab-
1) (Oncogene Sciences), Im
mu-133 (Barrande et al., Hybridom)
a, 12, 203 pages, 1993).

As the antibody used in the present invention, an antibody molecule can be used as it is, but Fab, F (ab ') 2 or Fd which is a fragment containing an antigen binding site obtained by various protease treatments is used. Etc. can also be applied. Furthermore, a peptide comprising a variable region, a peptide comprising an amino acid sequence constituting a complementarity determining region among them, and a modified peptide thereof can be used. In addition, when the present invention is applied to extracorporeal circulation of blood in the human body, it is necessary to consider the side effects when the antibody is released into the blood, to consider the antigenicity, etc., and to make the portion other than the variable region a humanized antibody. It is also useful to use chimeric antibodies. Considering the binding strength to cells (marker antigens) and the side effects when released, Fab, F (a
b ′) It is preferable to use a peptide constituting 2 or a variable region. The method for producing these antibodies is not particularly limited, but a highly purified one must be used. The method for producing the monoclonal antibody may be a method in which a hybridoma is usually grown in the abdominal cavity of a mouse to recover ascites, or a method obtained from a culture supernatant in a serum-free medium. In the case of a fragment peptide, it can be obtained by enzymatic treatment of an antibody molecule, but it can also be produced by bacteria, yeast, or the like by genetic engineering techniques. By combining monoclonal antibodies, monoclonal antibody-derived antibody fragments, peptides, and the like obtained by these methods, stronger binding can be achieved and efficient separation can be achieved.

A water-insoluble carrier can be used as one of the antibodies for directly immobilizing two or more kinds of antibodies recognizing the same antigen molecule. In addition, the target hematopoietic undifferentiated cells are reacted with the CD34 antibody, and the bound anti-C
The antibody can be indirectly reacted with a water-insoluble carrier using an antibody against the D34 antibody. For example, binding biotin or magnetic beads to an antibody to be immobilized on cells in advance, and then reacting with a water-insoluble carrier that binds or contains the biotin or magnetic beads, such as avidin or a magnetic substance. This makes it possible to easily recover the water-insoluble carrier to which the cells are bound. Further, by reacting the cells to which the antibody is bound with a water-insoluble carrier to which the anti-immunoglobulin antibody is bound, and washing or recovering the water-insoluble carrier, only the target cells can be selectively separated. If an antibody immobilized on magnetic beads, which is a water-insoluble carrier, is used, the efficiency of separation can be increased by a magnet or the like. Examples of the shape of the container used in the present invention include a column, a flask or a flask-like case filled with a water-insoluble carrier, filled with a water-insoluble carrier. A separator for hematopoietic undifferentiated cells can be prepared by combining these container shapes with a water-insoluble carrier to which these antibodies are directly or indirectly immobilized. In addition, these separators can be made highly usable as a cell separation system by combining with a pump for flowing a cell suspension, physiological saline, or the like.

The water-insoluble carrier in the present invention refers to a carrier which is in a solid form in an aqueous solution at room temperature, and may have any shape. Spherical, cubic, planar,
Examples thereof include chips, fibers, flat membranes, sponges, and hollow fibers. Of these, spherical and granular, from the viewpoint of ease of close packing, relatively easy retention of antibodies on the surface, relatively large effective area, and distribution of cell suspension Is desirable. The material of the water-insoluble carrier can be any inorganic or organic compound as long as it can retain the antibody on the surface, but the amount of eluted material is small when it comes into contact with the cell suspension, and the shape control is easier. Therefore, an organic polymer compound is desirable. Examples of such polymers include polymers and copolymers of vinyl compounds such as polypropylene, polystyrene, polymethacrylate esters, polyacrylate esters, polyacrylic acid, and polyvinyl alcohol; polyamide compounds such as nylon 6 or 66; polyethylene terephthalate; And a plant-derived polysaccharide-based compound such as cellulose. In addition, these can be combined with a magnet to facilitate recovery of these water-insoluble carriers.
The antibody can be covalently bonded to the surface of the water-insoluble carrier by a grafting method using chemical or radiation or electron beams, or can be shared via a functional group on the surface of the water-insoluble carrier by a chemical method. There is a method of joining. Among them, a copolymerization bonding method via a functional group is preferable because there is no danger of elution of the antibody during use. When the water-insoluble carrier has a coating layer, it can be insolubilized on the surface of the coating layer. As an example of a method for obtaining a functional group on the surface of a water-insoluble carrier or its coating layer, a cyanogen halide method,
There are an epichlorohydrin method, a bisepoxide method, a bromoacetyl bromide method, a halogenated acetamide method and the like. Specific examples include an amino group, a carboxyl group, a hydroxyl group, a thiol group, an acid anhydride, a succinylimide group, a substitutable halogen group, an aldehyde group, an epoxy group, and a tresyl group. The Bromcian method and the N-hydrosuccinimide group method are particularly desirable for ease of immobilization of the antibody. Examples of the shape of the container used in the present invention include a column, a flask, or a flask-like case filled with a water-insoluble carrier, which is filled with the water-insoluble carrier.

[0011]

The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

Example 1 Cord blood (27 ml) was adjusted to 48 ml with Hanks balanced saline (Gibco), and 12 ml each was dispensed into four conical tubes into which Ficoll Pack (Pharmacia) was dispensed at 3 ml, and 800 rpm. For 20 minutes, and the cells in the mononuclear cell layer were collected. The collected cells were washed with Hanks' solution. For each 7.0 × 10 ⁶ cells, 5 micrograms of 4A1 antibody (Nichirei) or 5 micrograms of ICH3 antibody (provided by AIS) or a combination of both (2.5 micrograms) The reaction was performed at 1 μl / ml on ice for 30 minutes. After washing them with Hanks' solution, 3 × 10 ⁶ Dynabeads (Dynal) anti-mouse Ig sheep beads were added and reacted on ice for 1 hour. Additional beads were added.
The separation operation of the dynabeads was performed according to the attached manual. The cells bound to the obtained beads were cultured in Dulbecco's MEM medium for 14 hours with 10% fetal calf serum at 37 ° C. in a 5% carbon dioxide atmosphere. After the culture, the cells and beads were completely separated by pipetting, and the beads were removed with a magnet according to the manual attached to Dynabeads.
A part of the collected cells was used to measure the number of cells to determine the number of recovered cells. The remaining cells were reacted with anti-HPCA-2 antibody (Becton) on ice for 30 minutes, washed with cold saline containing 2% fetal calf serum, and collected by centrifugation. The CD34 positive rate of the cells was measured with a Coulter flow cytometer. The CD34 positive rate was defined as the purity of the CD34 cells, and the number of cells obtained by multiplying the CD34 positive rate and the number of recovered cells was defined as the number of recovered CD34 positive cells. As a result, the 4A1 antibody had a purity of 29.9% and a recovery rate of 66.4%, the ICH3 antibody had a purity of 20.9% and a recovery rate of 41.2%, and the combination of the two antibodies had a purity of 40.9% and a recovery rate of 40.9%. 90.8%. Purity and recovery could be improved by combining two types of antibodies, ICH3 antibody and 4A1 antibody.

[0012]

Example 2 In the same manner as in Example 1, cord blood was diluted with Hank's balanced saline (Gibco), and 1 ml of Ficoll Pak (Pharmacia) was dispensed into a circular tube.
The mixture was dispensed in 2 ml increments, separated at 800 rpm for 20 minutes, and the cells in the mononuclear cell layer were collected. The collected cells were washed with Hanks' solution. The 2.9x10 ⁷ cells each in 4
5 micrograms of A1 antibody (Nichirei) or MY10
Antibodies (Becton Dickinson) at 5 micrograms or a combination of both (2.5 micrograms each) were added and reacted at 1 μl / ml on ice for 30 minutes. After washing them with Hanks' solution, 5 × 10 ⁶ Dynabeads (Dynal) anti-mouse Ig sheep beads were added and reacted on ice for 1 hour. The separation operation of the dynabeads was performed according to the attached manual. The cells bound to the obtained beads were placed in Dulbecco's MEM medium for 14 hours.
The cells were cultured with 5% fetal bovine serum at 37 ° C. in a 5% carbon dioxide atmosphere. After the culture, the cells and beads were completely separated by pipetting, and the beads were removed with a magnet according to the manual attached to Dynabeads. A part of the collected cells was used to measure the number of cells to determine the number of recovered cells. The remaining cells were anti-HPCA-2 antibodies (Becton Dickinson)
For 30 minutes on ice, washed with cold saline containing 2% fetal calf serum, and collected with a centrifuge. The cells
The CD34 positive rate was measured with a Coulter flow cytometer. The CD34 positive rate was defined as the purity of the CD34 cells, and the number of cells obtained by multiplying the CD34 positive rate and the number of recovered cells was defined as the number of recovered CD34 positive cells. As a result, the 4A1 antibody had a purity of 64.8% and a recovery of 54.1%, the MY10 antibody had a purity of 63.7% and a recovery of 52.0%, and the combination thereof had a purity of 91.7% and a recovery of 74. 9%.
Purity and recovery could be improved by combining two types of antibodies, MY10 antibody and 4A1 antibody.

[0013]

Example 3 A bone marrow fluid containing heparin was added at 100 × g for 5 minutes.
After centrifugation for a minute, the buffy coat cell layer (nucleated cell layer) with the sediment layer on top is collected. Further, washing with a PSB solution by centrifugation under the same conditions was performed twice. The washed buffy coat cells were suspended in a PBS solution containing 1% BSA and dispersed so that the number of cells became about 5 × 10 ⁷ cells / ml. This cell suspension is placed in an SBA lectin-immobilized flask (Microselector SBA: AIS),
After standing for 1 hour, the cell suspension was recovered. The 1 mmu-133 antibody and the ICH3 antibody were reacted with the cell suspension. The cells were placed in a culture flask (microselector: AIS) on which an anti-mouse Ig antibody was immobilized, and allowed to stand at room temperature for 1 hour. The microselector was used according to the attached manual. After washing the inside of the microselector, the cells immobilized with the antibody were peeled off by applying a shock to the flask by hand, and the cell solution was recovered. The recovery rate of CD34-positive cells was calculated from the results of quantification by fluorescence-labeled flow cytometry. As a result, CD34-positive cells with a recovery rate of 52% were obtained. Thus, it was recognized that separation using an immobilized carrier was also possible by reacting two types of antibodies, Immu-133 antibody and ICH3 antibody.

[0014]

Example 4 An antibody column to which 4Al antibody and MY10 antibody were bound was used to activate CNBr-activated Sepharose 4B.
(Pharmacia). Antibody Sep
The binding method to harose4B beads followed the attached instruction manual. An antibody solution prepared by mixing equal amounts of the 4Al antibody and the MY10 antibody with 4.1 ml of the gel was reacted to prepare an affinity gel with a coupling efficiency of 99.5%. A glass column (2 cm ² × 3 cm) in which a small amount of glass wool was fixed in the lower portion of the column was siliconized, and a gel column for cell separation was prepared by packing this column with 1 ml of the previously prepared gel. To 30 ml of umbilical cord blood, 3 ml of silica solution (Immunological Biology Laboratories) was added, and 1 hour 37
Incubated in degrees. This cell suspension was made up to 48 ml with Hanks balanced saline (Gibco), and 12 ml of this suspension was dispensed into four concentric tubes into which 3 ml of Ficoll Pack (Pharmacia) was dispensed.
The cells were separated under the conditions of rotation / minute and 20 minutes, and the cells in the mononuclear cell layer were collected. The collected cells were washed with Hanks' solution. 1 ml of the cell suspension containing 2.2 × 10 ⁷ cells was placed in the above antibody column, and reacted for 1 hour at 4 ° C. with gentle stirring. The column was washed by flowing Hanks balanced saline containing 10% fetal calf serum, 100 units / ml penicillin, and 100 micrograms / ml streptomycin. This column contains 10% fetal bovine serum, 1
2 ml of a MEM medium (Gibco) containing 00 units / ml penicillin and 100 microgram / ml streptomycin was added, and the mixture was cultured in a 37 ° C. carbon dioxide incubator for 13 hours. By raising and lowering this column, the gel was agitated somewhat strongly, and the medium was recovered from the column. Furthermore,
5 ml of 10% fetal bovine serum, 100 units /
Hanks balanced saline containing 100 ml of penicillin and 100 micrograms / ml of streptomycin was flushed, and the liquid was also collected. The cells in these collected liquids were collected by centrifugation. The number of these cells was counted. The remaining cells were reacted with anti-HPCA-2 antibody (Becton) on ice for 30 minutes, washed with cold saline containing 2% fetal calf serum, and collected by centrifugation. The CD34 positive rate of the cells was measured with a Coulter flow cytometer. The CD34 positive rate is defined as the purity of CD34 cells,
The number of cells obtained by multiplying the CD34 positive rate by the number of recovered cells
The number of D34 positive cells was determined. As a result, the purity was 32.4%,
A result of a recovery of 66.4% was obtained. From this, hematopoietic undifferentiated cells could be separated using a column device packed with gel.

[0015]

According to the present invention, it has become possible to specifically and efficiently separate hematopoietic undifferentiated cells. INDUSTRIAL APPLICABILITY The present invention is effective in fields such as collection of hematopoietic undifferentiated cells for the purpose of medical treatment such as leukemia, and removal of lymphocytes from bone marrow transplantation cells.

Claims (4)

[Claims] Claims: 1. A method for separating hematopoietic undifferentiated cells from a solution containing hematopoietic undifferentiated cells, wherein two or more monoclonal antibodies recognizing different epitopes in the same antigenic molecule species on hematopoietic undifferentiated cells. Or a hematopoietic undifferentiated cell comprising binding a protein or peptide containing a fragment of the antibody to a hematopoietic undifferentiated cell as long as it retains the binding property to the epitope, and collecting the conjugate. Separation method. 2. The method according to claim 2, wherein the antigen species on the undifferentiated hematopoietic cells is CD3.
The method for separating hematopoietic undifferentiated cells according to claim 1, wherein the four molecules and the antibody are anti-CD34 antibodies. 3. Two or more monoclonal antibodies recognizing different epitopes in the same antigen molecular species on hematopoietic undifferentiated cells, or the monoclonal antibody as long as it retains binding to the epitope. An apparatus for separating hematopoietic undifferentiated cells, comprising a protein or peptide containing fragments and a carrier to which they are bound. 4. The method according to claim 1, wherein the antigen species on the hematopoietic undifferentiated cells is CD3.
The apparatus for separating hematopoietic undifferentiated cells according to claim 3, wherein the four molecules and the antibody are anti-CD34 antibodies.