JPS61226059A - Adsorbent for anti-deoxyribonucleic acid antibody - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed Description of the Invention] Industrial Application Field The present invention provides an anti-DNA comprising deoxyribonucleic acid (hereinafter abbreviated as DNA) stably immobilized on a polymer carrier.
This invention relates to an adsorbent for antibodies. More specifically, the present invention is easy to sterilize, has excellent selectivity, has a large adsorption capacity, is easy to handle, and has anti-1' ) Nnor antibody or DNA-anti-DNA
The present invention relates to an adsorbent with excellent stability that can be suitably used to remove anti-1 antibody immune complexes from +fn plasma of patients with the disease.

Conventional technology In recent years, with the remarkable development of medical engineering technology, treatment of autoimmune diseases such as systemic lupus erythematosus, chronic rheumatoid arthritis, and scleroderma requires the removal of the causative substances of these diseases from the patient's blood. Blood purification therapy is being established. This blood purification therapy has so far included plasma exchange, filtration,
Adsorption methods have been tried, but all methods have some drawbacks and are not necessarily satisfactory.
.

For example, plasmapheresis is a method of replacing a patient's plasma with fresh frozen plasma or plasma preparations, or it is difficult to maintain large amounts of replacement fluid, and there is a risk of infection such as bloody hepatitis. There are important issues such as: As for filtration methods, attempts have been made to use two types of filters, such as a double vortex filter that fractionates and removes only substances with specific molecular weights, but no filter with sufficient fractionation ability has been found. Moreover, since this method involves fractionation based on molecular size, useful substances are removed at the same time, and there are problems such as the need for plasma replenishment fluid, so it has not yet been put to practical use. - Method For the adsorption method, activated carbon, styrene-novinylbenzene copolymer, anion exchange resin, etc. were used as the adsorbent! (Engineering liver and adsorbent saints) [Kidneys have already been put into practical use.However, these adsorbents selectively adsorb and remove relatively high-molecular components such as anti-DNA antibodies. does not have the ability.

Among these methods, the adsorption method is advantageous in various respects for removing disease-causing substances from the blood. Therefore, it is desirable to develop adsorption +4, which can adsorb and remove only specific disease-causing substances. It is rare.

By the way, in the field of biochemistry, antigen-antibody, enzyme-
Separate specific protein components such as substrates, enzyme-1!11 harmful agents, hormone receptors, etc. by utilizing the special pJ-like Kawahara action that exists only in the amount of each substance! 'Made by i'
The adsorption method used in affinity chromatography is the affinity chromatography method used in affinity chromatography.
This is a method in which a substance having 1 is immobilized on a carrier and dissolved.

Applying the principle of affinity chromatography, we are actively researching the use of substances that specifically react with immune substances to remove immune substances such as anti-1'NA antibodies from the blood. is being carried out. For example, with regard to systemic lupus erythematosus, the adsorbent substances are anti-DNA anti-I, anti-I-) NA-anti-1, and X-antibody immune complex 14. , (1) Methylated albumin immobilized on polysaccharide gel particles (JP-A-55-120875), (2
) When 1) NA adsorbed on cellulose was encapsulated in agar gel [[Clinical and Experimental Immunology (CIin.

hl], l+nmuno1. ) l 2nd.・Volume 1, Volume 2
31-237 Penn (1976 1')], (3) DNA adsorbed on activated carbon and encapsulated with Freon [[Clinical Immunology and Immunopathy (CIin.

11111111111O1,1mmunopalho
1. )l Volume 8, No. 9 ('l -page 96 (19
1977)], (,1) I') One complex of NA and divalent metal ions [Journal Offa Immunological Methods
: l+ o d s Month 1'i () Volume 1 No. 89-9.1
Page (1981)], (5) DNA covalently bonded to a polymeric collagen membrane with acylat [[Biotechnology and Bioengineering (LI)].
iot, ecl+no1. Bioeng, ) I 2nd
Volume 6, pages 665-669 (9984), etc. have been proposed.

In adsorbents with immobilized biopolymers,
It is necessary to have excellent stability during sterilization, storage, and use, and especially during use, if immobilized DNA or protein is eluted into the 1111 solution, serious side effects may occur. There is that. therefore,
When using biopolymers as adsorbents, it is necessary to immobilize them as strongly as possible.

However, the above-mentioned adsorbents are not always satisfactory in terms of the stability of the immobilized material, and the means for sterilization is unclear, which poses a practical problem.

Problems to be Solved by the Invention The object of the present invention is to obtain a microorganism that has excellent selectivity, excellent adsorption capacity, and Anti-1)N is easy to handle and is a factor in autoimmune diseases such as systemic lupus erythematosus.
An object of the present invention is to provide an adsorption agent 4 (4) with excellent stability that can be suitably used to remove A antibody and l)NA-anti-r)NA antibody immune complex from the plasma of patients with the disease.

Means for Solving the Problems As a result of extensive research, the present inventors found that using a polymer compound having a functional group capable of bonding with an epoxy group as a carrier,
An epoxy group is introduced into this, and D
The present invention was completed based on the findings 1 and 2 of the present invention by discovering scales that are compatible with chemical bonding of NA.

That is, the present invention provides an adsorbent for anti-1')NA antibodies, which is formed by chemically bonding l',)NA to a polymer compound into which an epoxy group has been introduced via the epoxy group. It is.

Has the method of immobilizing NA on a carrier via an epoxy group been used in affinity chromatography adsorbents for the purification of proteins? There are no examples of it being applied to adsorbents for plasma purification.

In the present invention, it is preferable to use a polymer compound having a functional group, such as a hydroxyl group or a thiol group, into which an epoxy group can be introduced as a carrier, or a nonionic material that does not have nonspecific adsorption ability. ).

For example, hydroxyl group-containing polymer compounds such as agarose, Dekis 1 run, cellulose, and polyvinyl alcohol are suitable as such carriers. There are no particular limitations, and for example, those having a bead-like, fibrous, or hollow fiber-like shape may be used.

As a method for introducing epoxy groups into the polymer compound, epichlorohydrin, 1,11
-Via, (2+:(-epoxypropoquine)7) Among the known methods of acting with a bisoxirane compound such as (2+:(-epoxypropoquine)7tan), there are two methods using the arbitrary Jj method.

Next, the epoxy 7Jζ was introduced into the polymer compound. - For example, pfl! ]-13, preferably H),
In an alkaline aqueous solution of 5 to 12, the epoxy group-introduced polymer compound and the desired 3().
-80°C1 preferably 1. fl -G fi 'C
Just keep it at 261 degrees and let it react for the required time.'': Yes.

If the temperature is too high, the reaction rate will be slow, or -) Immobilization when heated 1) Stability at 1 minute of N Immobilization of A 1j
), decreases. In addition, the reaction time depends on '(A +' + 1 degree), usually 5 = 4. ('1 hour is good, and it takes about 1 hour for the polymer compound.') N
A (formerly 1, epoxy J introduced into the polymer compound)
1 (of 1) is selected as appropriate, and its concentration is higher or immobilized 1j) or more.

By washing the polymer compound with immobilized DNA on the filter for 1 minute, an adsorption column of the present invention can be obtained.

Effects of the Invention Unlike conventional adsorption methods, DNA or epoxy groups are released and bonded stably to the polymer compound of the carrier. When stored at a temperature of 1°C for 70 Bl...1% or less, 8
It has excellent characteristics, such as the amount of adsorption is only 2% or less when heat treated at 0°C for 1 hour, and the adsorption activity for anti-DNA antibodies is not impaired by storage or heat treatment. There is.

Furthermore, since the epoxy group is introduced into the carrier via a stable ether bond or thioether bond, there is no risk of the epoxy group falling off, and furthermore, crosslinking of the carrier occurs simultaneously when the epoxy group is introduced. Therefore, the carrier becomes strong and has excellent stability against heating and the like. Therefore, a simple heating method can be applied as a sterilization method.

As described above, the adsorbent of the present invention has excellent stability and is easy to manufacture and sterilize.In addition, its selectivity and adsorption performance are comparable to those of conventional ones. As blood purification therapy for autoimmune diseases such as sexual lupus erythematosus, anti-1) NA antibodies and D
NA-Anti-I) NA antibody immune complexes are suitably used to remove from the plasma of the disease. .

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

Example 1 Sepharose, I (manufactured by Pharmaciali) was treated with shrimp chlorohydrin.
%, epoxy group-introduced food 8 mol/g 5 g of dried beads and the DNA of salmon shield liquid mountain rice
0.5M sodium carbonate [manufactured by Wako Pure Chemical Industries, Ltd.]
Lithium hydroxide (molar ratio '7:, E) dissolved in aqueous solution, N Ai concentration 4.7% by weight, pH 10
, 7 solution 1 f-1rIρ and iiR iγ and 1.1
Shake for 2.1 hours at il'C. Next, the pieces were extracted with 0.1 pe 1 acid lJ&t! After repeated washing with IIj solution (pl+4.0) and 0,2 M carbonated paper jΦj solution (++I19.0), 0.184 boric acid mild 1,000 J solution (pH 7)
, 3), 111 is a bead-like adsorbent on which DNA is immobilized by washing with a 1M sodium chloride solution and distilled water.

The DNA content of this product was determined by measuring phosphorus using the molybdenum blue method.Il'i, dry adsorption 441
23Il per g! It was g.

Next, 0.01 M borate buffer-physiological saline (pH 7.5:l 1F) mp was added to 1.5 g (dry weight 87) of the adsorbed J' obtained in this way, and 8 (1 After the treatment, the solution was shaken for 1 h at
By measuring the absorbance in and measuring the phosphorus in the adsorbent,
The DNA dropout rate was determined to be 1.8%. Note that no change in the shape of adsorbed H was observed even after heat treatment.

Example 2 DNA mackerel prepared in the same manner as in Example 1 was stored for a while and then washed with 1M sodium chloride solution and distilled water.

The DNA content of this product is I per gram of dry adsorbent.
,'+ fl thg was there.

Example 3 Toyopearl INV (i5 [manufactured by Toyo Soda 1゜Gyo Co., Ltd.] was treated with 1,4-bis(2,:-epoxypropoxy)phthane to give a 1:1 ratio, and the moisture content was 89.6'. i'
ji%, Eboxino, (Introduction 1 time 0.19 vno l
/ y dry 1. %i, piece 5y of beads and 15% of N concentration 5.1 heavy h196.1 + 1IIl,)! → J Kel II The same treatment as in Example 1 was carried out to obtain bead-like adsorbed particles with immobilized DNA.
1 unit containing A is transferred to Category 1.
It was 9.

Example 3.1 Epoxy group introduction meter treated with epichlorohydrin 0
．． Gauze 1.29 of 06ml + ol/g dry gauze,
20 m0 of a salmon semen-derived DNA solution with rlNA concentration of 5.2% by weight and 1 . The DNA content of this product was 3.9% per 1g of dry 1.v adsorbent.

Example 5 Shrimp treated with chlorohydrin, the amount of epoxy group introduced was 0.
07+0+nol/g dry fiber, finely divided polyvinyl alcohol fiber 1.0g, DNA concentration 5.1% by weight
, 1] Salmon semen DNA solution 207 of 1111.3
1ρ was added and treated in the same manner as in Example 1 to obtain adsorbed 4·A on which DNA was immobilized. The DNA content of this product was SIng per gram of dry adsorbent.

Example 6 Using Sepharose 4.8 as a carrier, bead-like adsorbent 5 (Ji9 (DNA content 0.7hy) with a water content of 92.7% by weight obtained by the same treatment as in Example 1) was used. ,0
．． After washing with 0 gM phosphate buffer-saline (pH 7.3, hereinafter abbreviated as PBS), this was mixed with 250 μp of serum from a whole body + L lupus erythematosus patient and shaken for 1 hour.

Create a series of diluted serum before and after treatment with PBS,
Anti-native DNAh+DNA) antibody, anti-nDN
Measurement was carried out by a fluorescent antibody method using an A antibody test kit (manufactured by Kalestad), and the dilution ratio at which the result was negative was determined. The results are shown in the table below.

Reference Example A bead-like adsorbent with a water content of 94.1% by weight obtained by using Sepharose 4B as a carrier and treated in the same manner as in Example 1. 50 f'l mg (1') NA content 4.5
After washing 6~) with PBS, 1()ηg each of bovine serum albumin and γ-globulin were added to F'B.
S ] (Mix with the solution dissolved in '1ytrl 25
After shaking at °C for 1 hour, adsorption of bovine serum albumin and γ-globulin was examined by Jv absorption, but no adsorption was observed in either case.

Claims (1)

[Scope of Claims] 1. An adsorbent for anti-deoxyribonucleic acid antibodies, which is formed by chemically bonding deoxyribonucleic acid to a polymer compound into which an epoxy group has been introduced via the epoxy group. 2. The adsorbent according to claim 1, wherein the polymer compound contains a hydroxyl group. 3. The adsorbent according to claim 2, wherein the hydroxyl group-containing polymer compound is an agarose-based, dextran-based, cellulose-based, or polyvinyl alcohol-based compound.