JPS6128375B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a material in which nucleic acids are immobilized and insolubilized.

The present invention is characterized by achieving immobilization by irradiating glow discharge plasma. Specifically, the component to be immobilized is mixed with a general-purpose polymer material, and the polymer part is exposed to the plasma. It is characterized by being encapsulated in a matrix formed by cross-linking and gelling through irradiation.

As one of the affinity chromatography methods, adsorption materials in the form of biomolecules such as nucleic acids,
Alternatively, methods using adsorbent materials that trap these component molecules have developed dramatically over the past few years, coupled with advances in insolubilization or immobilization technology, and are now useful for isolating specific genes, nucleic acids, etc. It has become widely used. Deoxyribonucleic acid (DNA)-affinity chromatography is one of the most effective in these applications.

In DNA affinity chromatography, several methods have already been reported for immobilizing or binding DNA in or on the surface of a carrier. Among these, the commonly used ones are: (1) Cyanogen bromide (BrCN) activates a carrier with a hydroxyl group such as cellulose or cross-linked polyvinyl alcohol using epichlorohydrin, etc., and activates the active site created in this way. (2) A method in which a phosphoric acid group at the end of a nucleic acid is covalently bound to cellulose or Cephadex using a carbodiimide accompanied by a dehydration reaction; (3) A method in which a nucleic acid is attached to a network of an acrylamide gel. (4) A method in which cellulose impregnated with nucleic acid is dispersed in an ethanol solvent and bonded by light irradiation while stirring; (5) A method in which cellulose infiltrated with nucleic acid is bonded by irradiation with light; (6) A method of solidifying nucleic acids by mixing them with agarose; (7) A method of binding nucleic acids to cellulose derivatives such as carboxymethylcellulose.

Each of these methods differs in the degree of ease of preparation, strength of immobilization, degree of affinity with the adsorbed component, etc., and as a result, each method has advantages and disadvantages. This is what we are doing. For example, among the above methods, in which nucleic acids are immobilized on carriers through covalent bonds, the binding force is so strong that the nucleic acids may be released or detached even during actual use. Although this does not happen easily, since the binding points where nucleic acids are bound are limited to the surface of the carrier, the amount of binding is small, so when used as an Affinity chromatography column or adsorbent, efficiency It is possible that the company was considered inferior to the Furthermore, when a sample is prepared by such a chemical reaction, the reaction conditions are quite severe, so structural destruction of the nucleic acid itself may occur.

On the other hand, methods for confining and immobilizing nucleic acids in carriers such as (3), (5), and (6), of course, differ slightly from each other, but in general, for example, manufacturing Although this method is legally relatively easy and has advantages from a practical point of view, it also has the disadvantage that depending on the manufacturing conditions, the size of the mesh in the carrier matrix makes it difficult for the nucleic acids to be tightly confined. There are issues that must be considered accordingly. That is, if the size is too small, adsorption may not substantially occur, whereas if it is too large, the nucleic acid will become liberated. Therefore, it is necessary to appropriately control the mesh that confines nucleic acids.

Furthermore, in any of these methods, depending on the immobilization conditions, it is possible that the molecular structure of the nucleic acid and its morphology such as single-stranded, double-stranded, etc. are affected.
For example, changes may occur in the form of the nucleic acid during production, or depending on the conditions of use of the immobilized product, specificity in adsorption may be lost, or in some cases, the adsorption ability may be completely lost. It is also possible that there may be a crisis caused by something like this. Therefore, in order to avoid such inconvenience as much as possible,
It is necessary to take into account the conditions under which it will be used. Furthermore, for this purpose, it is necessary to carefully consider how to immobilize and insolubilize nucleic acids and under what conditions to use them.

The present invention was achieved as a result of diligent efforts to obtain nucleic acid immobilization materials having various good performances, taking into account the above points.

The general procedure in the present invention is as follows.

1. Prepare an aqueous solution of a mixture of a nucleic acid and a water-soluble polymer that will become a matrix.

2. A reinforcing base material such as a woven fabric, non-woven fabric, or fibrous material is immersed in the prepared viscous and uniform mixed solution, and then applied thinly thereon, and dried to form a coating made of the mixture.

3. The dried film-treated sample is irradiated with glow discharge plasma to crosslink and insolubilize the polymer film, thereby immobilizing the nucleic acid.

The formulation at each step can be modified as appropriate depending on the chemical structure, molecular weight, etc. of the polymer serving as the matrix for insolubilizing the nucleic acid, and the purpose of using the produced immobilized sample. The raw material polymer serving as the matrix for holding the nucleic acid component needs to be a homogeneous mixed solution of both components in an aqueous system, and any polymer that satisfies these conditions at the minimum is sufficient. Water-soluble resins for this purpose include, for example, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, etc., and monomers of these components and other monomers, such as acrylonitrile, methacrylonitrile, acrylic esters, methacrylic esters, etc. These include copolymers with and natural polymers such as gelatin and agar.

The crosslinked layer produced by irradiating the surface of a polymer with glow discharge gas plasma is extremely thin. Therefore, they are often fragile and lack mechanical strength to be used for direct immobilization. In actual use, it is necessary to reinforce it in some way rather than using it as is. In this case, reinforcing materials include woven fabrics such as gauze and dish towels, non-woven fabrics such as filter paper, fibrous materials such as absorbent cotton and carbon fiber, and granular or powdered materials such as activated carbon, silica gel, and alumina. . From the viewpoint of materials, it is desirable that the laminate is formed in such a way that the mixture of nucleic acid and matrix polymer penetrates into the material of the reinforcing material and becomes as entangled as possible. In this sense, gauze is preferably made of cotton thread, and these materials have the advantage of being versatile and inexpensive, as well as being easy to process during the actual manufacturing process and use, and can be used in various fields. It has the advantage that it can be applied to however,
It goes without saying that the materials that can be used as the immobilized material in the present invention are not limited to these, and include, for example, synthetic fibers such as nylon, polyester, and acrylic; natural fibers such as silk, linen, and wool; carbon fibers; Glass fibers can also be mentioned.

It is necessary to prevent the nucleic acid mixed with the polymer that forms the matrix from dissolving and desorbing it to make it insolubilizable, but in the present invention, this process is carried out by irradiating glow discharge plasma to cause a crosslinking reaction between the polymer segments. What is unique about this project is that it was achieved by carrying out the following steps. As is well known, glow discharge plasma can be generated by oscillating high frequency waves such as 13.56 MHz radio waves under low pressure of several torr or less. The plasma shown in this example is highly separated, electrically neutral, and in equilibrium. In other words, atoms are ionized in a gaseous state, and the atomic nucleus, which has had its electrons stripped away, moves around naked. The plasma utilized in the present invention is a so-called low-temperature plasma in which atoms exist in a mixed state as radicals, ions, and electrons under low pressure and low density. Under this plasma state, the temperature rise is not rapid, and therefore, this low-temperature plasma is used to treat polymer-related substances, which may cause morphological changes due to the processing temperature exceeding the melting point or glass transition point.
It also has the advantage of being able to suppress changes in properties due to this. Alternatively, the penetrating power of the plasma is extremely small, and its action is limited to a very thin surface area of the material being treated. This has the advantage that only the surface layer can be modified while maintaining the bulk properties of the material.

There are broadly two methods for surface treatment of materials using low-temperature plasma. That is, (1) production of a thin film by plasma polymerization via activation of an organic monomer and coating with this thin film, (2) surface etching and plasma surface treatment by irradiation with non-polymerizable gas plasma from an inorganic gas, etc. It is. The purpose of using plasma in the present invention is to confine nucleic acid molecules within a base material, and this can also be achieved by the coating treatment that involves the formation of a plasma-polymerized thin film in (1). In this case, the coated portion used in an aqueous solvent may peel off, which is often inconvenient. On the other hand, surface treatment by non-polymerizable plasma irradiation (2) does not cause such troubles and can be said to be more preferable. Molecules on the surface of polymeric materials are caused by collisions with active species such as radicals, ions, and electrons generated in non-polymerizable gas plasma, or by the action of low-wavelength ultraviolet rays generated in the plasma. Crosslinking occurs on the chains with hydrogen elimination, resulting in gelation. Therefore, they tend to become insolubilized even in solvents in which the original untreated polymer is soluble. The present invention aims to utilize this phenomenon to insolubilize a water-soluble polymer in which nucleic acids are mixed, thereby achieving immobilization. Here, the effect of crosslinking and gelation caused by irradiation with non-polymerizable gas plasma is limited to the surface area, so the reinforcing base material is
When creating a layered sample by applying a mixed solution of water-soluble polymers, the thickness of the deposited layer is not too thick, and is less than a few microns, which allows crosslinking to be formed by plasma irradiation. It is desirable that there be. However, the effectiveness of the treatment depends on the type of polymer component mixed and its combination with the reinforcing base material, and performance such as adsorption is also affected by the amount of nucleic acid in the test. So,
These factors also need to be taken into careful consideration. Furthermore, depending on the size of the mesh of the polymer matrix that confines nucleic acids, nucleic acids may or may not be effectively contained, so it is necessary to sufficiently control the plasma irradiation conditions. be.

The sample prepared according to the present invention can be used as a column packing in affinity chromatography or as an adsorbent using an antigen-antibody reaction, for example.

When a foreign substance enters the body, the substance that stimulates body cells to produce antibodies is called an antigen. These two substances have a unique correspondence, like a key and a keyhole, and this is thought to be the fundamental effect of self-identification in living things.
Antigens are often proteins or polysaccharides;
The reason why bacteria produce antibodies in the body is mainly because bacterial cell proteins serve as antigenic viruses. When bacteria, viruses, or their extracts containing antigens are mixed with serum containing antibodies in a test tube, agglutination and lytic reactions occur. At this time, the type of antibody against the antigen is always the same, and this is called an antigen-antibody reaction.

A test prepared according to the method of the present invention utilizes this antigen-antibody reaction and is effective as a material for adsorbing and removing, for example, anti-DNA antibodies. Collagen diseases are a group of diseases that cause extensive inflammatory changes and fibrinoid degeneration in connective tissues in living organisms, and there is an example of the development of antibodies that are resistant to DNA. This antibody causes an antigen-antibody reaction with DNA, and the resulting complex is a cause of, for example, kidney damage, which is an incurable disease that affects women especially in their 20s to 30s. Measures have been taken to treat this disease, including the administration of steroid-based drugs, but these do not provide a fundamental cure, and currently no effective method has been found. . However, if the anti-DNA antibodies that occur in this abnormal state can be selectively removed by some method, it is expected that a complete cure will be achieved.

The ability of DNA-immobilized materials prepared according to the method of the present invention to adsorb anti-DNA antibodies in serum collected from actual patients was investigated. As a result, it was found that this material has extremely selective antibody adsorption performance.

The adsorption performance was evaluated by immersing the immobilized material in the serum of a patient with a high anti-DNA antibody titer for a certain period of time, and then examining the decrease in the antibody titer in the serum. Specifically, a fixed weight of DNA-immobilized material is brought into contact with serum whose antibody titer has been tested in advance, and after incubation at 37°C for a fixed period of time, the antibody titer in the remaining serum is measured by indirect hemagglutination. The degree of decrease in antibodies observed thereby was used as an index of antibody adsorption performance. The effects of this formulation and adsorption are as specifically shown in Examples and Reference Examples. It is clear from the comparison with the adsorption results for the untreated material that the immobilized material effectively adsorbs the anti-DNA antibody. In addition, the material prepared according to the present invention has a higher DNA binding strength than DNA immobilized cellulose powder that is already commercially available for affinity chromatography.
In other words, it has superior fixation power. For example, after washing with water and immersing it in serum, we measured the amount of DNA that was released as a result, and found that the amount of DNA released from commercially available DNA-immobilized cellulose powder was quite large, whereas the amount of DNA released from the immobilization material of the present invention was quite large. It has the advantage of being able to suppress this.

When the immobilized DNA prepared according to the present invention is actually used in a biological system, it is necessary that adsorption to other components and factors in serum does not cause destruction or other effects. For this purpose, we investigated the concentrations of components such as albumin and globulin, and changes in PH. As a result, these had almost no effect and were found to have selective adsorption to anti-DNA antibodies.

Example 1 0.25 g of commercially available salmon semen deoxyribonucleic acid (DNA) and polyvinyl alcohol (PVA,
Polymerization degree 500) 5.0g is mixed in 50mg of distilled water. In order to achieve uniform dissolution, the mixture was gently stirred at room temperature (22°C) using a magnetic stirrer. In this way, a viscous mixture solution could be obtained.

A washed and dried medical case cloth cut into 15 cm x 15 cm is immersed in 10 ml of an aqueous solution of this mixture and left at room temperature for a while. Take it out, squeeze it slightly to remove excess aqueous solution, and then air dry it. 0.50g to 0.60g per piece of this gauze cloth
Adhesion of the mixture was observed.

A cylindrical reaction tube (inner diameter 4.4
cm, length 40cm) and connect it directly to the vacuum line to perform vacuum evacuation. In this case, it takes a certain amount of time to complete sufficient evacuation, and the remaining air and moisture that has diffused into the sample is desorbed, resulting in a pressure of 10 ^-2 torr.
It took 15-20 minutes to reach the following high vacuum. After this sufficient evacuation, _N2 gas was introduced into the fine adjustment needle valve to a gas pressure of 60×10 ^-3 torr, and a high frequency glow discharge plasma of 13.56MHz was generated at a discharge voltage of 40W. At least I did some plasma treatment. The front side of the sample gauze cloth was irradiated for 5 minutes, and the back side was irradiated for 5 minutes under the same conditions. When processing the back side, the time required for vacuuming and degassing in advance is considerably shortened.
A few minutes was enough.

Example 2 The DNA-PVA mixture aqueous solution prepared in Example 1 was applied thinly and evenly onto filter paper using a brush. This was air-dried at room temperature and then subjected to N ₂ gas plasma treatment in substantially the same manner as in Example 1. Gel insolubilization in the applied area was observed. In an aqueous solvent, the filter paper itself may be destroyed by long-term immersion or mechanical agitation, so care must be taken. Suitable as a material.

Example 3 0.50 g of salmon semen DNA and 5.0 g of PVA were mixed and dissolved in 50 ml of distilled water. Medical gauze was immersed in 10 ml of this mixed aqueous solution, and then lightly rubbed to remove excess aqueous solution and air-dried. As a result, it was possible to deposit 0.14 to 0.16 g of the mixture per 1.0 g of gauze. The sample thus prepared was treated with N ₂ gas plasma in the same manner as in Example 1 to create a DNA immobilization material.

Example 4 1:10 DNA prepared according to Example 3
Gauze cloth coated with PVA mixed solution
A DNA immobilized sample was prepared by plasma irradiation treatment for a total of 10 minutes, 5 minutes each on the front and back sides, under a helium gas of 50×10 ^-3 torr and a discharge voltage of 30 watts.

Reference Example 1 10 mg of the sample prepared in Example 1 was taken, immersed in 0.6 ml of serum collected from a patient, and incubated at 37°C. The patient's original serum is
While hemagglutination was observed up to 320-fold dilution, when adsorption treatment was performed with DNA-fixed samples,
At both treatment times of 30 minutes and 5 minutes, no reaction was observed after a 40-fold dilution. In other words, this means that the antibodies present in the serum are absorbed by
This shows a decrease of 1/8.

On the other hand, when an untreated medical gauze cloth is soaked, there is no change in the antibody titer after 5 minutes of incubation, and even when this period is extended to 30 minutes, there is no substantial change in the antibody titer. No adsorption ability was observed.

Comparative Example 1 A commercially available DNA-immobilized cellulose powder for use in Affinitei chromatographs was treated with anti-inflammatory agents using the same method as in the reference example.
We investigated the adsorption performance of DNA antibodies. This sample is
Each is a combination of undenatured double-stranded and denatured single-stranded DNA, each containing 0.7 to 1.1 mg/
ml, containing 0.3-0.6 mg/ml of DNA. There is a considerable difference in antibody adsorption performance between these two types, with the former showing approximately the same level of antibody adsorption as the sample treated on the gauze in Example 1, whereas the latter sample with denatured DNA immobilized has a high performance. quite inferior in terms of
The antibody titer was only reduced by about 1/2.

Claims (1)

[Claims] 1. A water-soluble polymer material is characterized in that by irradiating glow discharge plasma, the surface layer becomes insolubilized into a cross-linked gel to confine nucleic acids within this matrix. A method for producing an immobilized product. 2. The method according to claim 1, wherein the matrix that confines nucleic acids is reinforced on a reinforcing base material such as a woven fabric, a nonwoven fabric, a fibrous material, a bead-like material, or a powder.