JP2002159292A - Method for separating/collecting target material - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for separating/collecting a target material efficiently and in high purity and speed without lowering its function, and to provide an apparatus for the method. SOLUTION: This method for separating/collecting a target material is characterized by involving utilizing the temperature-dependent base pair formation (hybridization) by the hydrogen bond formation between the complementary strands of a nucleotide and the dissociation (dehybridization) of the base pairs formed above.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and an apparatus for selectively separating and recovering target substances such as cells, cell micro-tissues and proteins from biological tissue fluids such as blood and body fluids. The present invention also relates to a method and an apparatus for separating and removing bacteria, viruses, and the like from a biological tissue fluid. More specifically, by controlling the temperature of the nucleotide by using the complementary binding of the nucleotide, the separation / separation is performed.
The present invention relates to a method and an apparatus for performing recovery or the separation / removal. Further, the present invention relates to a nucleotide used for selective separation and recovery of a target substance from a biological tissue fluid.

[0002]

2. Description of the Related Art Techniques for separating and recovering specific cells are used for preventing autoimmune diseases, AIDS, and acute rejection after transplantation. Separation and collection of hematopoietic stem cells expressing CD34 ⁺ , collection of lymphocytes having an autoreactive antigen receptor for the treatment of autoimmune diseases,
Removal of lymphocytes from cells for bone marrow transplantation is effectively used in the treatment of hematopoietic malignancies and cancer. further,
This is an important technology for cell medicine such as leukemia cell detection and treatment. In recent years, it has been suggested that embryonic stem cells can be differentiated and propagated in all organs, and separation and recovery without damaging cells such as embryonic stem cells, undifferentiated cells, various differentiated growth factors, etc., and intracellular micro-tissues is a regeneration process Medical,
This is an indispensable technology in the field of gene therapy. Currently used methods for separating cells and intracellular microstructures include:
Membrane separation method, centrifugation method, electrical separation method, method of killing cells other than target cells, method of binding target cells to a carrier on which a ligand having affinity for target cells is immobilized,
There is a separation method using magnetic beads, and the like.

[0003]

Each of these techniques has a problem due to the principle used for separating cells. For example, membrane separation or centrifugation is a method of separating cells based on differences in cell size and specific gravity, so that separation is possible when there are large differences in physical properties such as white blood cells, red blood cells, and platelets. However, it cannot be used when the physical difference between target substances is small, such as separation of subpopulations of leukocytes (for example, separation of T and B cells), and has a drawback of low specificity. In addition, the electrical separation method utilizes the difference between the charge characteristics and the dielectric characteristics of cells in an electric field, but similarly has a limitation in cell selectivity. Various methods for killing cells other than the target cells have been studied and developed, but the target cells may be damaged and cause side effects. The magnetic bead method is a method of labeling target cells with magnetic beads by incubating a cell mixture with magnetic beads to which antibodies are bound, and separating labeled cells from unlabeled cells using a magnetic device. It is. In this method, a long incubation time is required to efficiently bind beads and target cells,
As a result, non-specific adsorption of cells other than the target may occur, resulting in a decrease in the purity of the target cells, and it is difficult to recover the target cells adsorbed on the small magnetic beads. In the immunoadsorption column method, a ligand such as an antibody against a membrane antigen of a target cell is immobilized on the surface of a base material such as beads, and this is packed in a column to perform cell separation. As a method utilizing the interaction between ligands such as immunoadsorption, a method utilizing the binding between biotin and avidin is disclosed.However, since the binding force between biotin and avidin is very strong, separation using It is difficult to collect the recovered cells, and has the drawback that the collection efficiency is reduced. These methods have the advantage of high selectivity for target cells because of the use of antibodies, but the use of enzymes such as protease and papain for the recovery of adsorbed cells results in low recovery rates and damage to the cells themselves. Has the disadvantage of being large. JP-A-6-269499 discloses that a nucleotide is bound to an antibody recognizing a target cell, contacted with a cell mixture, and then filled with a water-insoluble carrier on which a nucleotide having a nucleotide sequence complementary to the nucleotide is fixed. A method for separating cells of interest by adsorbing cells to a water-insoluble carrier through a prepared column is disclosed. It should be noted that JP-A-6-2694
The nucleotide used in the example of No. 99 consists of 10 bases. However, the method focuses on separation by adsorption of a target substance, and the recovery of cells after separation has not been specifically studied. Therefore, as for the recovery of the separated cells, the examples merely describe that the gel in the column is manually shaken lightly to the extent that the gel moves, and is detached from the beads. The recovery by such physical vibration has a problem that the recovery efficiency is low, the cells are damaged by collision of the carrier with the cells, and the function thereof is deteriorated. In addition, when one or both ends of the nucleotide are immobilized, steric hindrance significantly lowers the melting temperature of the double helix with the complementary strand. In particular, when the complementary strand is 30 base pairs or less, sufficient hybridization cannot be formed, and the efficiency of cell separation / recovery decreases. Therefore, in order to perform excellent separation / recovery, both the separation step and the recovery step are required to have high efficiency, and the required conditions are very limited. The present invention improves the above-mentioned drawbacks, enables efficient and high-speed separation / recovery of a target substance, and has a high purity of the recovered target substance and does not cause a decrease in function. An object of the present invention is to provide a method and an apparatus for separation and recovery.

[0004]

Means for Solving the Problems As a result of considering and considering the above problems, the present inventors have reached the following invention. That is, by utilizing the temperature-dependent base pair formation (hybridization) and the dissociation of the base pair formation (dehybridization) by hydrogen bonding between complementary strands of nucleotides for separation and recovery of the target substance, I found that the problem could be solved. Specifically, the present invention is achieved by the following contents. (1) the target substance-containing liquid, the complementary chain length is 30 or more,
Mixing the first nucleotide bound to the ligand of the target substance and binding the target substance to the ligand of the first nucleotide via the ligand;
At a temperature, the complementary strand length including the first nucleotide bound to the target substance and the base sequence capable of hybridizing to the first nucleotide is 30 or more, and the second nucleotide immobilized on the carrier Contacting the first nucleotide and the second nucleotide bound to the target substance to separate the target substance, and bringing the second temperature to a second temperature. A method for separating and recovering a target substance, comprising the step of dehybridizing the first nucleotide and the second nucleotide, and recovering the target substance. (2) The ligand of the target substance and the first nucleotide are separated and mixed with the liquid containing the target substance to bind the ligand of the target substance and the first nucleotide. ) A method for separating and collecting a target substance. (3) After the step of binding the target substance to the first nucleotide via the ligand, the method further comprises a step of removing the first nucleotide not bound to the target substance (1) or (2). ) A method for separating and collecting a target substance. (4) The method for separating and recovering a target substance according to (1) to (3), further comprising a step of removing the unhybridized first nucleotide in the step of separating the target substance. (5) the target substance-containing solution, the complementary strand length is 30 or more,
A first nucleotide bound to a ligand of a target substance;
A complementary strand length including a base sequence capable of hybridizing to the first nucleotide is 30 or more, and a second nucleotide bound to a ligand of a target substance is mixed,
A step of binding the target substance to the first nucleotide and the second nucleotide via the ligand, respectively; and a step of binding the first substance bound to the target substance at a first temperature.
A step of contacting the nucleotide with the second nucleotide bound to the target substance and hybridizing to obtain an aggregate of nucleotides;
Dehybridize the aggregate of nucleotides,
A method for separating and collecting a target substance, comprising a step of collecting the target substance. (6) The ligand of the target substance, the first nucleotide and the second nucleotide are separately mixed with the target substance-containing solution, and the ligand of the target substance is mixed with the first nucleotide and the second nucleotide. (5) The method for separating and recovering a target substance according to (5), wherein the target substance is bound to each of the nucleotides. (7) The method for separating and recovering a target substance according to (1) to (6), wherein the second temperature is higher than the first temperature. (8) The method for separating and recovering a target substance according to any one of (1) to (7), wherein the first temperature is a temperature in a range of 1 to 20 ° C., and the second temperature is a temperature in a range of 25 to 40 ° C. . (9) a column having an inlet and an outlet, a complementary nucleotide having a complementary strand length of 30 or more, and a first nucleotide bound to a ligand of a target substance, and a complementary nucleotide containing a base sequence capable of hybridizing to the first nucleotide; A chain length of 30 or more,
An apparatus for separating and recovering a target substance, comprising a second nucleotide immobilized on a carrier packed in the column and a means for controlling the temperature in the column. (10) The complementary strand length is 30 or more, which hybridizes with the first nucleotide bound to the ligand of the target substance at the first temperature and dehybridizes at the second temperature with the complementary nucleotide length of 30 or more. A pair of nucleotides consisting of the second nucleotide of

[0005]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention will be described below in detail. A first aspect of the present invention is a method for separating and recovering a target substance from a target substance-containing solution using temperature-dependent hybridization and dehybridization of a pair of nucleotides having mutually complementary base sequences, Any of the components required to be collected or removed as cells or cell micro-tissues from a liquid containing cells or cell micro-tissues as components required to be recovered or removed such as peripheral blood, lymph fluid, bone marrow fluid, cord blood, etc. The purpose is to separate and collect.

[0006] In the present invention, cells refer to all cells irrespective of animals and plants, and specifically, embryonic stem cells,
Hematopoietic stem cells, platelets, macrophages, dendritic cells, monocytes, neutrophils, vascular endothelial cells, smooth muscle cells, fibroblasts,
Cardiomyocytes, skeletal muscle cells, hepatic parenchymal cells, non-parenchymal liver cells, functional cells such as pancreatic islet cells, cancer cells, tumor cells such as leukemia cells, new cholera, bacteria such as O-157, Legionella, HIV virus, Microorganisms such as hepatitis virus, and viral disease-causing substances. The intracellular micro-tissue refers to a physiologically active substance such as a cell growth factor released into a cell or from a cell to the outside of a cell, and specifically includes liver growth factor (HGF), bone formation factor (BMP), and platelet Derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), nerve cell growth factor (NG
F), epidermal growth factor (EGF), insulin growth factor (IGF), transforming growth factor (TG
F) and each family.

[0007] In the method according to the first aspect of the present invention, there is provided a method comprising the steps of:
One nucleotide of the pair of nucleotides (hereinafter referred to as
It is referred to as "first nucleotide." ) Includes a target cell or an intracellular microstructure (hereinafter, referred to as a “target substance”).
, That is, a substance that specifically binds a target substance. Substances that recognize such target substances include antigen-antibody, enzyme-substrate, ligands based on specific interactions such as various physiologically active substances and their receptors, higher-order structures, electrostatic interactions, hydrophobicity, etc. Supramolecular structures designed and synthesized in consideration of non-specific interactions such as interactions can be mentioned. The substance that recognizes the target substance is not particularly limited as long as it can recognize a specific target substance. For example, when using an antibody, either a monoclonal antibody or a polyclonal antibody may be used. Further, a substance having a hydrophobic group such as a cholesterol group or a long-chain alkyl group, which is known to anchor to a cell membrane, may be used as the cell recognition site. The bond between the nucleotide and the ligand can be achieved by a method using a known chemical reaction, but a bond composed of a spacer or two or more compounds may exist between the two bonds. It is sufficient that a functional group capable of binding a ligand having an affinity for a target substance is present at the terminal or in the chain of the nucleotide, and an amino group,
Carboxyl group, hydroxyl group, thiol group, acid anhydride group, succinylimide group, chlorine atom, aldehyde group,
Examples include an epoxy group and an amide group. For example, a method of condensing an amino group introduced into a nucleotide or a terminal with a carboxyl group in an antibody by a carbodiimide method, using (strept) avidin at a binding portion between a biotinylated antibody and a nucleotide having a terminal or intrachain biotinylated nucleotide Is also good. Here, the nucleotide and the ligand may be bound in advance, or may be separately mixed with a target substance-containing solution and bound in the mixture. In this case, the formation of the bond may be before or after the formation of the bond between the ligand and the target substance. When the nucleotide and the ligand are bound in advance, the formation of the bond between the two is guaranteed, the generation of unbound nucleotide and ligand is small,
Both can be used effectively. On the other hand, when both are separately mixed with the target substance-containing liquid, it is not necessary to provide a separate step of binding the nucleotide and the ligand.

[0008] A pair of nucleotides used for temperature-dependent hybridization and dehybridization has a base sequence capable of hybridizing to each other, such as complementary to each other.
Any one may be used as long as it hybridizes at the above temperature and dehybridizes at the second temperature. It may be a natural nucleotide or an artificial nucleotide.

In natural nucleotides, for deoxyribonucleic acid (DNA), it is known that adenine-thymine or guanine-cytosine is complementary to each other and forms a specific base pair in a double helix structure. I have.
Similarly, in ribonucleic acids (RNA), it is known that adenine-uridine, cytosine-tosine, or cytosine-guanine are complementary to each other and form specific base pairs in a double helix structure. Hybridization is formed between these nucleotides having complementary base pairs. Alternatively, a nucleotide having a base pair forming a triple helix structure such as cytosine-guanine-cytosine or thymine-adenine-thymine may be used.

[0010] Artificial nucleotides known to hybridize similarly to natural nucleotides (DNA, RNA) include DN artificially synthesized by a nucleic acid synthesizer.
A, RNA, poly (A) / poly (U), pol
y (I) • poly (C), poly (G) • poly
Known substances such as (C) and poly (A) / poly (G) can be exemplified. In addition, peptide nucleotides (PN
A), polyvinyl adenine, polyvinyl thymine, polyvinyl guanine, polyvinyl cytosine, polyvinyl uracil, and the like, and artificial nucleotides in which a nucleobase is introduced into a side chain amino group of polylysine or polyethylene imine by an active ester method, and meta (alkyl) Examples include those in which a nucleobase is bonded directly or via a spacer to the main chain of a synthetic polymer such as a skeleton. In particular, when an artificial nucleotide such as a meta (alkyl) skeleton is used, the heat resistance is improved, so that a sterilizable, low-cost separation / recovery device can be manufactured.

In the method of the present invention, the combination of nucleotides is not particularly limited as long as they have base sequences complementary to each other.
Any combination of natural nucleotides vs. natural nucleotides, artificial nucleotides vs. artificial nucleotides may be used. Here, the combination of nucleotides does not need to be completely complementary, and does not form a partially complementary base sequence if hybridization and dehybridization can be appropriately performed in the method of the present invention. It may have a mismatch sequence. When a nucleotide has a palindrome, one nucleotide forms a complementary base sequence as shown below. In addition, the following base sequence is an example of the base sequence of a palindrome, and the nucleotide of the present invention is not limited to this. 5 '… TCC
GGA ... 3'3 '... AGGCCT ... 5' In this case, one pair of nucleotides can be constituted by one nucleotide, which is convenient in the process.

In the present invention, the length of the nucleotide, more specifically, the length of the complementary chain is not particularly limited. When the target substance is a cell, the outermost surface of the cell is formed by using a lipid bilayer as a matrix. 10 by protein and polysaccharide
Since it is covered with a thickness of ２０20 nm, steric hindrance occurs when nucleotides hybridize on the surface of the cell. In addition, when one end or both ends of nucleotides are immobilized, the melting temperature of the double helix formed with the complementary strand is significantly reduced, and sufficient hybridization cannot be achieved with less than 30 base pairs. . Therefore, in order to perform good hybridization, the chain length of the complementary strand is preferably at least 30 base pairs or more. More preferably 5
It has a chain length of 0 base pairs or more. The upper limit is not particularly limited, but is preferably 500 base pairs or less because a very long one is difficult to handle.

In the method of the present invention, the first temperature at which the nucleotides hybridize and the second temperature at which the nucleotides hybridize are not particularly limited.
Is a temperature higher than the first temperature. in this case,
During the target substance separation / recovery step according to the method of the present invention, unintended dehybridization of nucleotides is prevented, and appropriate separation / recovery of the target substance can be performed. In addition,
The first temperature is preferably between 1 ° C and 20 ° C, more preferably between 4 ° C and 10 ° C. On the other hand, the second temperature is preferably between 25 ° C and 40 ° C. More preferably 35
° C to 38 ° C. As described above, the second temperature is preferably higher than the first temperature, and
This is because, if the temperature of the dehybridization exceeds 40 ° C. during collection, the target cells may be damaged, and dissociation preferably occurs near human body temperature.

Here, the first and second temperatures can be controlled by the length of the complementary strand of nucleotides or the base sequence constituting the complementary strand. When the length of the complementary strand increases, the complementary base sequence contained in the strand increases, so that the dissociation temperature generally rises. On the other hand, regarding the base sequence constituting the complementary chain, guanine-cytosine which is a complementary base sequence forms a triple bond, and adenine-cytosine which is not a complementary base sequence forms a hydrogen bond by a double bond, for example, For the same complementary chain length, guanine-
As the content of cytosine increases, the dissociation temperature of the complementary strand increases, and as the content of adenine-cytosine increases, the dissociation temperature of the complementary strand decreases. Therefore, when a nucleotide having a long chain length (100 base pairs or more) and a high dissociation temperature of the complementary strand is used, the content of adenine-cytosine in the base sequence of the complementary strand is increased to form a partially complementary base pair. An unmatched mismatch sequence may be introduced.

As described above, the nucleotide length or the base sequence constituting the nucleotide can be appropriately selected according to the required dissociation temperature, and the efficiency of introduction into the carrier and the compatibility with the ligand of the target substance can be determined. From the viewpoint of sex, the constituent base sequence may be appropriately selected. When a natural nucleotide is used as the nucleotide, in order to obtain a required chain length or base sequence, a restriction enzyme such as endonuclease, exonuclease, DNase, or RNase may be added to cleave the nucleotide. .

In carrying out the method of the first aspect of the present invention,
In one form, the nucleotide binds to the ligand.
The nucleotide on the side that does not match (hereinafter, referred to as “second nucleotid
Do. " ) Is fixed to the carrier. In this case, the carrier
Materials that can be used are cotton, hemp, cellulose,
Natural polymers such as galose, chitin and chitosan, Niro
, Polyester, polyacrylonitrile, polyolefin
, Halogenated polyolefin, polyurethane, poly
Amide, polysulfone, polyethersulfone, poly
(Meth) acrylate, polyvinyl alcohol, police
Tylene, ethylene-polyvinyl alcohol copolymer,
Synthetic polymers such as tadiene-acrylonitrile copolymer
Or mixtures thereof. The shape of the carrier is particularly
Not limited, for example, porous body, fiber, non-woven fabric, particles,
Beads, films, sheets, tubes, hollow fibers, powders, etc.
Any of these may be used. However, porous materials, nonwoven fabrics and
For particles and particles, the maximum or average pore size is 0.1 μm
(0.1 × 10^Threenm) to 800 μm (800 × 10^Three
nm), more preferably 1 μm
(1 × 10^Threenm) to 500 μm (500 × 10 ^Threen
m). On the other hand, the minimum diameter is to prevent clogging,
It is preferable that the diameter be equal to or larger than the diameter of the target substance. The carrier
Immobilization of the second nucleotide on the substrate can be performed by any known method.
Can be used. For example, hydroxyl group, carboxy
A sil group, an amino group, a glycidyl group, an isocyanate group,
Aldehyde group, sulfonic acid group, haloacetyl group, disul
Immobilization using functional groups such as sulfide groups or thiol groups
Do. In addition, the surface of the carrier is
Activated by imidazole, tosyl, tresyl, etc.
It does not matter. Using these functional groups, directly or
Can be bonded using a condensing agent or a crosslinking agent.
The carrier on which the second nucleotide is immobilized is a polyolefin.
, Halogenated polyolefin, polycarbonate, polycarbonate
Urethane, poly (meth) acrylate, polyamide, etc.
Cylindrical, disk with plastic inlet and outlet
And conical columns. However, the column shape
The state is not limited to this. In addition, fixed to the carrier
The amount of the second nucleotide to be converted is
1 to 1000 μg, preferably 5 to 200 μg
is there.

In another form of performing the method of the first aspect of the present invention, the second nucleotide is not immobilized. That is, the second nucleotide is mixed with the target substance-containing solution in the same manner as the first nucleotide. After binding the target substance to the ligand bound to the first nucleotide and the ligand bound to the second nucleotide, respectively, the first nucleotide bound to the target substance and the second nucleotide bound to the target substance By bringing the solution containing the nucleotides to the first temperature, the first nucleotides and the second nucleotides are hybridized to form aggregates of the nucleotides, which are collected by filtration or the like, and then the second temperature is reached. By doing so, the aggregate of the nucleotides is dehybridized, and the target substance is recovered.
In the present embodiment, the timing of performing the step of mixing the second nucleotide into the target substance-containing solution is not particularly limited, and may be performed simultaneously with the mixing of the first nucleotide.
Alternatively, it may be after the ligand of the first nucleotide is bound to the target substance.

Further, in the above embodiment, the nucleotide may be one nucleotide having a palindrome. If the nucleotide is the above, it is possible to bind the ligand of the target substance to all nucleotides,
As a result, all nucleotides can be used for collecting the target substance.

In the method according to the first aspect of the present invention, a target substance ligand and a ligand for the target substance are collected after the step of binding the target substance in order to minimize unnecessary substances present in the system when the target substance is recovered. A step of washing unbound nucleotides may be included. For the same reason, the step of hybridizing nucleotides may include a step of washing unhybridized nucleotides.

A second aspect of the present invention is an apparatus for separating and recovering a target substance from a target substance-containing liquid containing a nucleotide to which a target substance and a ligand recognizing the target substance are bound. The apparatus according to the second aspect of the present invention provides a column having an inlet and an outlet, a first nucleotide fixed to a carrier packed in the column, and a ligand for a target substance, A second nucleotide having a base sequence capable of hybridizing with the above, and a temperature control means for controlling the temperature in the column. Second embodiment of the present invention
In the apparatus according to the aspect, the column, the carrier for immobilizing nucleotides, and the method for immobilization are not particularly limited, and are as described in the method of the first aspect of the present invention.
In the apparatus according to the second aspect of the present invention, the second nucleotide may be subjected to temperature-dependent hybridization and dehydration with the first nucleotide in the column after binding a target substance to a ligand bound to the nucleotide. In order to perform hybridization and thereby separate and collect a target substance, the target substance is preferably contained as a reagent to be added to the target substance-containing solution.

In the apparatus according to the second aspect of the present invention, the means for adjusting the temperature in the column is not particularly limited as long as the temperature in the column can be adjusted to the temperature at which the nucleotide hybridizes and the temperature at which the nucleotide hybridizes. Not done. For example, it may be a means for attaching a heater around the column and adjusting the temperature in the column, or a means for flowing a fluid at a predetermined temperature into the column and thereby adjusting the temperature.

In the apparatus according to the second aspect of the present invention, the target substance-containing liquid is bound to a ligand of a target substance and hybridized with a first nucleotide immobilized on a carrier packed in a column. By flowing together with the two nucleotides, the target substance can be separated and recovered efficiently and without any damage. Further, in the apparatus of the present invention, the target substance captured in the column by hybridization is dehybridized at a predetermined temperature, so that simultaneously with the recovery of the target substance, the nucleotide immobilized on the carrier is easily regenerated. be able to.

In a third aspect of the present invention, a first nucleotide having a complementary strand length of 30 or more to which a ligand of a target substance is bound is hybridized at a first temperature and dehybridized at a second temperature. A soybean is a pair of nucleotides consisting of a second nucleotide having a complementary strand length of 30 or more. The pair of nucleotides of the third aspect of the invention hybridize to each other by raising the first temperature and dehybridize by raising the temperature to the second temperature. Such nucleotides that hybridize and dehybridize in a temperature-dependent manner are exemplified in the method of the first aspect of the present invention. In the pair of nucleotides according to the third embodiment of the present invention, one of the paired nucleotides is bound to a ligand of a target substance.
Due to these features, the pair of nucleotides of the third aspect of the present invention can be suitably used in the method of the first aspect of the present invention and the device of the second aspect of the present invention. In addition,
In the examples described below, an example of a pair of nucleotides according to the third aspect of the present invention was used.

[0024]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the present invention is limited thereto. (Examples 1-3, Comparative Examples 1 and 2) CD4 positive cells as target cells and substances having specific affinity for target cells in order to confirm the effectiveness of the method for separating and recovering target substances of the present invention. An experiment was performed using an anti-CD4 antibody (Leu-3a).

[0025] CNBr-activated Seph
arose 6MB (Amersham Pharmacia Biote
Amount of CN required for immobilization of streptavidin on
Br-activated Sepharose 6M
B was measured and swelled with 1 mM HCl for 15 minutes, and then washed with 1 mM HCl (200 ml / g). 20 ml of coupling buffer (0.1 M NaHCO containing 0.5 M NaCl) per 1 ml of swollen gel
₃ , pH 8-9,4 ° C). After adding streptavidin (manufactured by Wako Pure Chemical Industries, Ltd.), the gel suspension was gently stirred at a predetermined temperature with an end-over-end type mixer. After incubation, unbound streptavidin was added to the wash buffer.
r (0.1 M acetate buffer containing 0.5 M NaCl) and coupling buffer (10 ml / ml gel)
For 3 times. After masking with a coupling buffer containing 0.2 M glycine to mask the remaining active groups, the plate was equilibrated with PBS containing 0.1% sodium azide.

Immobilization of Nucleotides on Beads The above-mentioned streptavidin-immobilized beads had a biotinylated single-stranded DNA (all base sequence is adenine (A)) having a DNA length of 75, 55 and 35, respectively.
Of 50 μg to 100 μg per ml of beads
Immobilized. As a comparative example, DNA having a chain length of 10 was similarly immobilized. In addition, experiments were performed using beads on which DNA was not immobilized.

Preparation of a column A 5.0 ml syringe (manufactured by Terumo Corporation) was added to Ex in advance.
A column having a filter washed with tran MA02 (MERCK) was packed with 1 ml of avidin (2 mg / ml) immobilized beads.

Cell Separation Operation When cells are manipulated with a pipette or the like, they are coated with PBS-A (containing 2% albumin and 0.1% sodium azide) to prevent non-specific adsorption of cells to the pipette inner surface. And then sucked up the cells. 50 ml of human fresh blood was collected, and a mononuclear cell component (mixture of Th cells, B cells, mononuclear cells, etc.) separated by Ficoll (manufactured by Amersham Pharmacy Biotech) was collected using a 15 ml centrifuge tube.
After rinsing twice with BS-A, the volume was increased by 1.5 ml with PBS-A. The number of cells was measured using an automatic hemocytometer (Sysmex Ne-6000, manufactured by Toa Medical Electronics Co., Ltd.). The thiol-terminal single-stranded DNA (all base sequences are thymine (T)) having a sequence complementary to the single-stranded DNA (chain lengths of 75, 55, and 35, respectively) immobilized on the beads is used as the amino group of the antibody. The bound DNA-bound antibody was mixed in an amount of 20 μl per 1 × 10 ⁶ cells, and reacted on ice on a light-shielded plate for 30 minutes. Then, the plate was washed twice with PBS-A to remove excess DNA-bound antibody, and then washed with PBS-A.
And the cell number was measured using Sysmex. The number of cells used in one column was adjusted to 3.5 × 10 ⁴ / μl. 100 μl of the above cell suspension was added to a column which had been incubated at 4 ° C. for 20 minutes.
(3.5 × 10 ⁶ cells) and incubated at 4 ° C. for 10 minutes. Wash unadsorbed cells and remove 15m
Pooled in 1 tube (Flow Through solution). Then, PBS-A 3 kept at 37 ° C.
5 times (2 ml / min) and pooled (E
lution solution).

Analysis Cell suspension before separation / collection, Flow Through
Solution, the Eluton solution, spin down and
Suspended in 0 μl of PBS-A. Automatic blood cell counter (Sysmex NE-6, manufactured by Toa Medical Electronics Co., Ltd.)
000), the number of cells was mixed with 20 μl of PE-labeled apidin and biotinylated CD3 / CD8 antibody per 1 × 10 ⁶ cells, incubated on ice for 30 minutes under light-shielded conditions, and analyzed by a flow cytometer. went. According to the following equation, CD4 positive cell recovery rate and El
The CD positive rate in the union solution was calculated. CD4 positive cell recovery rate (%) = (number of CD4 positive cells in Elution solution / number of CD4 positive cells in cell suspension before separation / recovery) × 100 CD4 positive rate in Elution solution (%) = (Elu
Number of CD4-positive cells in the tion solution / Elution
Table 1 shows the recovery rate of CD4 positive cells and the recovery rate of CD4 in the Elution solution.

[0030] As can be seen from Table 1, the complementary strand length is 35 or more,
It showed excellent recovery and selectivity. When the number of the complementary strand is 10, the recovery rate and the selectivity are lower than those in the examples. Beads without nucleotide binding further reduced target cell recovery and selectivity.

(Examples 4 and 5, Comparative Example 3) Poly (9-vinyladenine) and poly (1-vinyluracil) synthesized according to a previous report (Baba et al., Analytical Chemistry, No. 64, p. 1920, 1992) Were performed according to Examples 1 to 3 using artificial nucleotides having an average complementary chain length of 150 or 80, respectively. In the examples, poly (9-vinyladenine) was bound to beads and poly (1-vinyluracil) was bound to target cells. As a comparative example, an experiment was performed by binding the same poly (9-vinyladenine) to beads and target cells. Table 2 shows the results.

[0032] As is clear from Table 2, the system that forms complementary base pairs has excellent selectivity, but the system that does not form complementary base pairs has no selectivity.

Example 6 Example of Separating and Recovering Target Substance as Aggregate Biotinylated DNA used in Example 2 was applied to 0.1 μm-diameter polystyrene beads (manufactured by Molecular Probes) on which streptavidin was immobilized. (Chain length 55) to bead 1
30 μg was immobilized per ml. 100 μl of a suspension of the DNA-immobilized beads in PBS-A and 100 μl of a suspension of CD4-positive cells bound to the DNA-binding antibody (chain length 55) used in Example 2 (cell number 3.5 × 10 ⁶ ), stirred gently at 4 ° C., and incubated for 30 minutes. Thereafter, as a result of observation with an optical microscope, aggregates of beads and cells were confirmed. In addition, DNa is added to the aggregate.
buffer solution (pH 7.5, 10 mM Tris-H) in which seI (manufactured by Amersham Pharmacia Biotech) has been dissolved.
Cl, 10 mM CaCl ₂ , 10 mM MgCl ₂ ,
After adding 1% glycol), the mixture was incubated at 4 ° C. for 10 minutes. After that, observation with an optical microscope confirmed that the aggregates were dissociated due to DNA degradation.

[0034]

According to the method and apparatus for separating and recovering a target substance of the present invention, a target substance can be separated by hybridization of nucleotides, and can be recovered by dehybridization with a small temperature change. Since there are almost no contact sites, it is easy to collect. As a result, damage to cells and the like is reduced, and the function is favorably maintained. The method and apparatus for separating and recovering a target substance according to the present invention can be used to separate target cells and microcellular microstructures easily and in a short time without deteriorating quality, characteristics, etc. Can be recovered. For this reason, from the mixed solution of multi-components, only the target substance can be quickly and selectively selected without complicated operation.
Separation and collection can be performed efficiently without causing damage. Further, the method and apparatus for separating and collecting a target substance of the present invention can selectively separate and collect necessary cells or cells to be removed from a cell-containing solution containing cells to be collected and cells to be removed, depending on the purpose. it can. Furthermore, the target substance separation / recovery apparatus of the present invention can simultaneously perform the recovery of the target substance by dehybridization and the nucleotide immobilized on the carrier, so that the apparatus can be easily regenerated.
Excellent maintainability. Due to these features, the method and apparatus for separating and recovering a target substance of the present invention can be used in the field of cell medicine such as hematopoietic stem cell transplantation, and can also be effective in the fields of gene therapy and regenerative medicine. Become.

Continued on front page F-term (reference) 4B024 AA20 BA80 CA09 HA12 4B029 AA09 AA21 BB20 CC03 HA10 4B063 QA01 QQ01 QQ42 QR32 QR56 QR58 QR82 QS15 QS34 QS36 QX02 4B065 AA90X BA22 BA30 CA44 CA46

Claims (10)

[Claims] 1. A target substance-containing solution is mixed with a first nucleotide having a complementary strand length of 30 or more and bound to a ligand of a target substance, and the target substance is mixed with the first nucleotide via the ligand. A step of binding to a nucleotide ligand, wherein at a first temperature, a first nucleotide bound to the target substance and a complementary strand length including a base sequence capable of hybridizing to the first nucleotide have a length of 30. The above is a step of contacting the second nucleotide fixed to the carrier, hybridizing the first nucleotide and the second nucleotide bound to the target substance, and separating the target substance. Setting the second temperature to dehybridize the hybridized first nucleotide and second nucleotide to collect the target substance. A method of separating and recovering a target substance, wherein the door. 2. The method according to claim 1, wherein the ligand of the target substance and the first nucleotide are separated and mixed with the liquid containing the target substance to bind the ligand of the target substance to the first nucleotide. The method for separating and recovering a target substance according to claim 1. 3. The method according to claim 1, further comprising, after the step of binding the target substance to the first nucleotide via the ligand, removing the first nucleotide not bound to the target substance. 3. The method for separating and recovering a target substance according to 2. 4. The method for separating and recovering a target substance according to claim 1, further comprising a step of removing the unhybridized first nucleotide in the step of separating the target substance. . 5. A complementary solution comprising a target substance-containing solution, a first nucleotide having a complementary strand length of 30 or more, which binds to a ligand of the target substance, and a base sequence capable of hybridizing to the first nucleotide. A step of mixing a second nucleotide having a chain length of 30 or more and binding to a ligand of a target substance to bind the target substance to the first nucleotide and the second nucleotide via the ligand, respectively; Contacting the first nucleotide bound to the target substance with the second nucleotide bound to the target substance at a first temperature and hybridizing to obtain an aggregate of nucleotides And a second temperature, wherein the aggregate of nucleotides is dehybridized to recover the target substance. Away recovery method. 6. The method according to claim 1, wherein the ligand of the target substance, the first nucleotide and the second nucleotide are separated from each other.
The method for separating and recovering a target substance according to claim 5, wherein the target substance-containing liquid is mixed with the target substance-containing solution to bind the ligand of the target substance to the first nucleotide and the second nucleotide, respectively. 7. The method for separating and recovering a target substance according to claim 1, wherein the second temperature is higher than the first temperature. 8. The target according to claim 1, wherein the first temperature is a temperature in a range of 1 to 20 ° C., and the second temperature is a temperature in a range of 25 to 40 ° C. How to separate and collect substances. 9. A column having an inlet and an outlet, a first nucleotide having a complementary strand length of 30 or more and bound to a ligand of a target substance, and a base sequence capable of hybridizing to the first nucleotide. An apparatus for separating and recovering a target substance, comprising a second nucleotide having a complementary strand length of 30 or more and fixed to a carrier packed in the column, and means for controlling the temperature in the column. 10. A complementary strand having a complementary strand length of 30 or more, which hybridizes with a first nucleotide bound to a ligand of a target substance at a first temperature and dehybridizes at a second temperature. A pair of nucleotides consisting of 30 or more second nucleotides.