JPS6412280B2 - - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for producing an immunoadsorbent.

BACKGROUND ART In specific separation and purification methods for biological substances, separation of biological substances based on immunological principles is being actively researched and developed. When a substance (antigen) that is completely foreign to the organism is given to the organism,
Substances that react with antigens (antigens) are produced within the body. The reaction between an antigen and its corresponding antibody is called an antigen-antibody reaction, and this reaction is very specific.
The immunoadsorption method utilizes the specificity of this antigen-antibody reaction in a separation and purification method. For example, an antibody (or antigen) present in the body fluid of a living body is ) is adsorbed and separated by contact with
In such methods, the use of adsorbents to which antigens or antibodies are bound is
This is advantageous because antigen-antibody conjugates can be more easily separated than free antigen or antibody. Various methods have been proposed for producing immunoadsorbents to which immunoreactive substances such as antigens and antibodies are bound. For example, Campbell et al. created an immunoadsorbent by binding an antigen to a water-insoluble solid. That is, ovalbumin is immobilized on diazotized aminobenzylcellulose, and this adsorbent is used to purify anti-ovalbumin antibodies. Furthermore, recently, a method of binding antigens or antibodies using polysaccharides activated by bromic cyanide as water-insoluble solids has become common practice. In these methods, water-insoluble solids such as cellulose, cross-linked dextran, agarose, nylon, and polyamino acids that have active groups on their surfaces or that can be easily introduced with active groups by polymer reactions are used.

However, these methods have the disadvantage that they cannot be applied to materials such as activated carbon and silicone resins, which do not have active groups that can directly bind liquid-reactive substances, and it is difficult to introduce active groups by polymer reaction. It was hot.

In view of the current situation, the present inventors have conducted intensive research on a treatment method that can bind immunoreactive substances to any solid, including solids that do not have active groups such as activated carbon and silicone resin. , this invention has been achieved.

That is, the present invention provides at least two functional groups capable of reacting with amino groups and/or hydroxyl groups.
A reagent in which at least one of two or more functional groups is an epoxy group is reacted with chitosan on a solid surface to form a chitosan film on the solid surface, and then the chitosan film is formed on the solid surface. This is a method for producing an immunoadsorbent, which is characterized by binding an immunoreactive substance.

Examples of solid surfaces in the present invention include surfaces of inorganic substances such as activated carbon, glass, asbestos, mica, kaolinite bentonite, and polyphosphazene, surfaces of natural polymers such as natural rubber, proteins, starches, cellulose, collagen, agarose, and dextran, and surfaces of natural polymers such as polystyrene. Examples include synthetic polymer surfaces such as polyamide, polyester, polyamino acid, polyethylene, polypropylene, silicone, polyvinyl chloride, polymethacrylate, polyvinyl alcohol, and polyurethane.

A reagent having at least two functional groups capable of reacting with an amino group and/or a hydroxyl group used in the present invention, and at least one of the two or more functional groups being an epoxy group (hereinafter referred to as an epoxy-containing polyfunctional reagent) ) include polyepoxides such as diglycidyl ether of tetramethylene glycol and diglycidyl ether of diethylene glycol, and epoxy halides such as epichlorohydrin and epibromohydrin.

Chitosan used in the present invention is an aminopolysaccharide, specifically β-1,4'-polyglucosamine, which is a deacetylated form of natural chitin (β-1,4'-poly-N-acetyl-glucosamine). refers to something that has become

In the present invention, it is first necessary to react this chitosan with an epoxy group-containing polyfunctional reagent to form a chitosan film on the solid surface.
In this case, in the present invention, it is necessary to leave a terminally reactive amino group, imino group, or hydroxyl group on the chitosan film, and therefore a functional group that can react with the amino group and/or hydroxyl group of the epoxy group-containing polyfunctional reagent. It is necessary to use chitosan in excess.

To form a chitosan film on a solid surface, for example, the solid surface is brought into contact with an epoxy group-containing polyfunctional reagent solution to adsorb the epoxy group-containing polyfunctional reagent onto the solid surface, and then a large excess of chitosan solution is added to the solid surface. The epoxy group-containing polyfunctional reagent adsorbed on the surface may be reacted with chitosan, or a large excess of chitosan solution may be brought into contact with the solid surface to adsorb chitosan on the solid surface, and then
A chitosan film can also be formed on the solid surface by treatment with a dilute epoxy group-containing multifunctional reagent solution.

As a solvent for dissolving the epoxy group-containing polyfunctional reagent or chitosan, any solvent may be used as long as it does not dissolve the solid to be treated and is inert to the epoxy group-containing polyfunctional reagent. It is okay to add acids, alkalis, salts, etc.

Adsorption of the epoxy group-containing polyfunctional reagent or chitosan onto the solid surface is preferably carried out at a temperature of 0 to 50°C, while renewing the surface as necessary.
In addition, the reaction between the epoxy group-containing polyfunctional reagent and chitosan is carried out at a temperature of 0 to 150°C using an acid or
It is desirable to carry out the reaction in the presence of a catalyst such as a base and a deoxidizing agent.

In the present invention, it is then necessary to bond the immunoreactive substance to the chitosan film formed on the solid surface. The reaction between the amino group, imino group, or hydroxyl group on the chitosan film formed on the solid surface and the immunoreactive substance can be carried out using a known method. For example, N,
N'-dicyclohexylcarbodiimide, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide using a dehydrating agent such as hydrochloride. The amino groups on the chitosan film and the carboxyl groups of the immunoreactive substance can be condensed. Furthermore, the above-mentioned epoxy group-containing polyfunctional reagent or polyaldehyde such as glutaraldehyde, terephthalaldehyde, isophthalaldehyde, dialdehyde starch, hexamethylene diisocyanate, toluene diisocyanate, xylene diisocyanate, phenylene diisocyanate , polyisocyanates such as isocyanate derivatives of aniline-formaldehyde, acid chlorides such as adipoyl chloride, isophthaloyl chloride, terephthaloyl chloride, cyanuric chloride, polythioisocyanates such as hexamethylene lentioisocyanate, N,N'-ethylene bis Iodoacetamide, N,N'-polymethylene biiodoacetamide such as N,N'-hexamethylene iodoacetamide, maleic anhydride-methyl vinyl ether copolymer, maleic anhydride-ethylene copolymer, maleic anhydride-styrene copolymer Polycarboxylic acid anhydrides such as polymers, divinyl compounds such as divinyl sulfone and divinyl ketone, bismaleimides such as N,N-ethylene bismaleimide, N,
N'-methylenebisacrylamide, N,N'-hexamethylenebisacrylamide, N,N'-hexamethylenebismethacrylamide, N,N',
A substance immunoreactive with an amino group, an imino group, or a hydroxyl group on the film using a reagent having at least two amino groups and/or hydroxyl groups, such as a poly(meth)acryloyl compound such as N″-triacryloylhexahydrotriazine. To do this, first, a reagent having at least two functional groups capable of reacting with an excess amount of amino groups and/or hydroxyl groups relative to the amino groups on the chitosan film is used. It is sufficient to react, leave some functional groups of the reagent unreacted, and then react the unreacted functional groups with the immunoreactive substance. The reaction is preferably carried out at a temperature of 0 to 80°C.

The method of the invention is applicable to all immunoreactive substances. For example, the immunoreactive substances used in the present invention include antigens extracted from the glomerular basement membrane of the kidney, nuclear antigens, protein antigens, haptens, enzymes, hormones, cells, viruses, bacteria, vaccine antigens, hepatitis antigens, etc. antibody against, e
Examples include antibodies, antibodies to angiotensin, antibodies to cortisol, and the like.

The method of the present invention can be applied to solid surfaces of various shapes, such as powders, beads, films, membranes, permeable membranes, sheets, tubes, hollow fibers, fibers, cloth, knitted fabrics, and the like.

By bringing a sample into contact with an immunoadsorbent having an immunoreactive substance bound to its surface by the column method or batch method according to the method of the present invention, antigens and antibodies can be separated and purified based on antigen and antibody reactions. For example, if an immunoadsorbent using an antigen as an immunoreactive substance is used, the antibodies to be separated and purified are selected based on antibody activity, regardless of the class of immunoglobulin G, immunoglobulin A, immunoglobulin M, etc.

In the present invention, since chitosan having two functional groups, an amino group and a hydroxyl group, is used,
A chitosan film produced from chitosan and an epoxy group-containing polyfunctional reagent has excellent film strength, is stable even in highly ionic solutions, and does not easily peel off from the immobilized surface.

Since the immunoadsorbent produced according to the present invention has a large amount of immunoactive substance bound to it, it is possible to perform immunological separation, purification, and analysis using this adsorbent with high precision and for a long time. Since it can be manipulated and uses bio-derived chitosan, it is advantageous in terms of both toxicity and stability of the immunoactive substance.

The immunoadsorbent produced by the method of the present invention can be used not only to separate and purify biological substances, but also to selectively remove substances (antigens, antigen-antibody conjugates, etc.) that are closely related to disease activity and progression of lesions into body fluids. It can also be used for measurements in clinical tests based on antigen and antibody reactions. The immunoadsorbent thus produced by the method of the present invention is particularly useful in the fields of medical care, medicine, and clinical testing.

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

Example 1 30 g of activated carbon with a particle size of about 0.8 mm was placed in 600 ml of a 0.3% chitosan (acetate) aqueous solution, stirred at 30°C for 2 hours to adsorb chitosan on the activated carbon, filtered, and washed with water. It was immersed in a specified caustic soda solution for several minutes, then thoroughly washed with water, and dried under reduced pressure. Chitosan-adsorbing activated carbon obtained in this way
10g of 1% epichlorohydrin dioxane solution 25
After stirring at 60°C for 1 hour, the mixture was thoroughly washed with dioxane and dried under reduced pressure. 5g of activated carbon with a chitosan film formed on its surface in this way
Contains 2.5ml of 25% glutaraldehyde aqueous solution
The mixture was poured into a 27.5 ml solution of 0.05M phosphate buffer (PH7.0), stirred at 20°C for 2 hours, and then the activated carbon was filtered and thoroughly washed with water. This activated charcoal was mixed with human immunoglobulin [an antibody titer of 1:32,000 by passive hemagglutination method].
HBs (hepatitis B) antibody-containing globulin] solution containing 50 ml of HBs (hepatitis B) antibody] solution and stirred at 20°C for 2 hours to react. After that, it was filtered, washed with 0.05M phosphate buffer (PH7.0) containing 1M NaCl, and washed with water.
% sodium borohydride aqueous solution,
Then, after thorough washing with water, it was dried under reduced pressure to obtain activated carbon with human immunoglobulin bound to its surface. The amount of globulin bound to this activated carbon was quantified by amino acid analysis and was found to be 0.26% by weight based on glycine, which is an amino acid that is relatively abundant in globulin.
of globulin was bound to the activated carbon surface.

This human immunoglobulin-bound activated charcoal is used to generate HBs using the reverse passive hemagglutination method (RPHA method).
It was placed in blood cells showing an antigen titer of 1:8192, and after stirring at room temperature for 1 hour, the activated charcoal was removed.

When the HBs antigen in this blood was measured by the RPHA method, it was negative ( ^1:20 ).

For comparison, the HBs antigen titer was 1 using the RPHA method:
Activated charcoal to which no human immunoglobulin was bound was added to the same blood as above showing 8192, and the same procedure was performed. As a result, the HBs antigen titer of the blood was 1:8192 by the RPHA method.

Example 2 2 g of activated carbon with a chitosan film formed on its surface in the same manner as in Example 1 was placed in 11 ml of 0.05M phosphate buffer (PH7.0) containing 1.0 ml of 25% glutaraldehyde aqueous solution, and heated at 20°C. After stirring for 2 hours, the activated carbon was filtered and thoroughly washed with water.

This activated charcoal is used for hepatitis B vaccine (RPHA method).
Pour into 20ml of HBs antigen titer 1:32000) solution and heat at 20°C.
The mixture was stirred for 2 hours to react. Then filter
0.05M phosphate buffer (PH7.0) containing 1M Nacl
The carbon was then washed with water, treated with 10 ml of a 1% aqueous sodium borohydride solution, washed again with water, and dried under reduced pressure to obtain activated carbon with the hepatitis B vaccine bound to its surface.

The activated carbon obtained was placed in a globulin solution containing an HBs antibody that showed an antibody titer of 1:8000 using the passive hemagglutination method, and after stirring at 7°C for 24 hours, the activated carbon was filtered, and then phosphoric acid containing 3M sodium iodide was added. Washed with buffer. The HBs antibody titer of this washing solution was measured by passive hemagglutination method: 1:
It was 320,000.

Example 3 Ten silicone resin disks with a thickness of 300 μm and a diameter of 15 mm were washed with acetone and then with distilled water, and then treated as follows.

(1) Pour into 100 ml of 0.5% chitosan (acetate) aqueous solution and shake at room temperature for 2 hours to adsorb chitosan onto the silicone disc. Wash the silicone disc with water and soak in 1N caustic soda solution for several minutes. After immersion, it was thoroughly washed with water and dried under reduced pressure.

(2) Then, it was poured into 100 ml of 1% epichlorohydrin dioxane solution and shaken at 60°C for 1 hour.
After cleaning the obtained disc with dioxane,
Dry under reduced pressure.

(3) Next, 4 g of maleic anhydride-methyl vinyl ether copolymer was dissolved in 50 ml of acetone dehydrated with dryarite, and the resulting solution was immersed for 1 hour while shaking at room temperature, and then washed with dehydrated acetone. , and then dried under reduced pressure.

(4) Next, human immunoglobulin G (human IgG)
Phosphate buffer (PH) containing the IgG fraction (anti-human IgG) prepared from rabbit antiserum against
7.0) After soaking in 30ml and leaving overnight at room temperature,
It was washed with the above phosphoric acid buffer.

(Anti-human IgG) IgG obtained as above
The silicone disc bound to was hydrolyzed with dilute aqueous hydrochloric acid at 40°C for 1 hour, and the color of the solubilized (anti-human IgG) IgG was measured using the Lowry method. As a result, 12 μg/cm ² of silicone disc area was obtained.
(anti-human IgG) IgG was found to bind.

For comparison, when silicone disks without chitosan treatment were used, ² μg/cm2 was used.
Only IgG was bound.

Note that (anti-human IgG) a silicone disk to which IgG is bound is used for measuring human IgG.

Claims (1)

[Claims] 1. A reagent having at least two functional groups capable of reacting with an amino group and/or a hydroxyl group, in which at least one of the two or more functional groups is an epoxy group, and chitosan are combined on a solid surface. A method for producing an immunoadsorbent, which comprises reacting the above to form a chitosan film on the solid surface, and then bonding the chitosan film and an immunoreactive substance. 2. The manufacturing method according to claim 1, wherein the immunoreactive substance is an antigen. 3. The manufacturing method according to claim 1, wherein the immunoreactive substance is an antibody.