JPS6048524B2 - Biologically active substance reagent and its manufacturing method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a biologically active substance reagent in which a biologically active substance is bound to a water-insoluble polymer compound, and a method for producing the same.

In recent years, new techniques have increasingly been used in the field of biochemical manipulation, whose main feature is the use of the affinity of biologically active substances bound to carriers for reactions to be selectively carried out. . The biologically active substance reagent of the present invention can be widely used in such fields of biochemical manipulation, and can be particularly suitably used in affinity chromatography.

Through specific complex formation of the substance bound to the carrier and a second substance present in the mixture, the substance can be removed from the mixture and then optionally isolated by desorption.

The use of carrier-bound enzymes has the advantage that the material conversion can be carried out as a continuous production process and that the reaction products can be obtained enzyme-free. In biochemical enzymatic analysis, a water-insoluble enzyme bound to a carrier is
It can be used as a reagent that can be used repeatedly.

Due to the nature of enzymes, which exhibit an affinity not only for substrates but also for specific inhibitors, it is particularly suitable for the collection of enzyme inhibitors to be carried out by affinity chromatography with carrier-bound enzymes. be.

On the other hand, if the inhibitor is bound to a water-insoluble matrix,
The corresponding enzyme can be prepared and collected. - So-called immunoadsorbents bind antigens or antibodies to water-insoluble matrices, which make it possible to isolate the corresponding antibodies or antigens.

As the biologically active substance, in vivo (I
Mention may be made of natural and synthetic substances active in vivo and in vitro, which can be referred to in the broadest sense as enzymes, activators, inhibitors, antigens or antibodies, vitamins and hormones.

Since these biologically active substances depict the principle of action of water-insoluble systems, the term ``effector'' (or expression substance) has been introduced for them. Most of the effectors produced are inherently more stable than the corresponding bioactive substances in solution.

As carrier material, so-called matrix, it is advantageous to use substances which, in addition to being insoluble in aqueous systems, have as low a non-specific adsorption as possible.

This requires sufficient prevention of hydrophobic, hydrophilic and ionic interactions between the matrix and the reaction partner of the effector. Binding should not occur to substances that are undesirable to bind to the effector, eg, substances that are not specific reaction partners of the effector. The matrices traditionally used as carriers for biologically active substances are those that bind the effectors by physical adsorption (this includes, for example, polystyrene, activated carbon and glass spheres), and those that bind the effectors to each other via covalent bonds. It is broadly divided into things.

The latter matrices include, for example, vinyl polymers (for example homo and copolymers) such as polyacrylic acid, polyacrylic acid amide and amino-, carboxy- or sulfonyl-substituted polystyrene, and cellulose and its derivatives, and also natural and synthetic polystyrenes. Examples include peptides and proteins. Carbohydrates, especially cellulose, dextran, starch, agar or their derivatives, appear to be hampered by the non-specific affinity of the carboxyl groups often present in these natural products. Most commonly used because the interaction between matrix and effector is average.
Furthermore, these materials have relatively low thermal and chemical stability. Many of these drawbacks could be sufficiently overcome by using a synthetic polymer called polyvinylene glycol as a matrix.

Polyvinylene glycol, also called polyhydroxymethylene, is produced by acidic or basic hydrolysis of polyvinylene carbonate. Each carbon atom has one hydroxyl group and one hydrogen atom.
The penetrating -C-C- bonds endow the carrier with particularly high stability. According to the invention, it has been found that vinylene glycol copolymers can be advantageously used as carrier matrix instead of polyvinylene glycol.

By incorporating suitable comonomers into polyvinylene carbonate by polymerization, the physical and chemical properties of the polyvinylene glycols produced therefrom can be varied widely. Thus, for example, the "hydrophilicity" or "swellability" of polyvinylene glycol can be altered by polymerizing incorporation of hydrophobic monomers such as ethylene, vinyl chloride or styrene into polyvinylene carbonate. By copolymerization of vinyl acetate, the vinylene glycol groups are replaced by vinyl alcohol groups in the saponified target product, resulting in increased swelling in aqueous media. It is also possible to crosslink by using comonomers such as diethylene glycol divinyl ether and benzene, and thus improve the mechanical properties of the support.

Electrophilic; functional groups such as oxirane groups (epoxy groups) from vinyl glycidyl ether comonomers can be incorporated by polymerization into the matrix directly with nucleophilic functional groups (-NH
2, -0H, etc.). A carboxyl group can be introduced through an acrylate comonomer trimer. After the copolymerization is complete, the ester groups are saponified to carboxyl groups. -NH. group or -COO
Introduction of the H group enables activation with carbodiimide, isoxazolium salt, glutaraldehyde, etc. 3. Vinylene glycol/vinyl alcohol copolymers have a relatively large specific surface area, so for example,
*The binding ability to the white polymer powder surface is greater than that of vinylene glycol homopolymer.

The subject of the present invention is therefore a bioactive compound consisting of a preferably water-insoluble copolymer of polyvinylene glycol and a bioactive substance bound to the copolymer while maintaining biological activity.

In these compounds, the carrier matrix is 45%
Polyvinylene glycol copolymers containing up to 5% to 20% comonomer, preferably from 5% to 20%. Biologically active substances can be, for example, enzymes, activators, inhibitors, antigens or antibodies, other plasma proteins, blood group substances, phytohemagglutinins, antibiotics, vitamins or hormones, peptides or amino acids, or synthetically produced substances. It is a substance such as an effector. The polymeric carrier and the biologically active substance are linked to each other by covalent bonds, either directly or via side chains (spacers). Linking biologically active compounds via side chains (spacers) is useful when the molecular weights of the partners with which they have affinities are very different, or when using proteins or molecules with extremely high molecular weights in processes that exhibit biospecificity. This is advantageous in cases where proteins composed of these subunits are involved.

A spacer is a side chain of a certain length that is anchored to a polymer matrix and is added or incorporated (
(binding of di- or polyfunctional compounds such as tripeptides to the matrix) or by incorporation of functional groups and sequential chain extensions on said groups. The subject of the invention is furthermore a method for covalently linking said biologically active substance to a carrier.

A series of commonly known reactions can be used for this.

The method of the present invention first relates to a method for producing the polyvinylene glycol copolymer.

The method involves converting vinylene carbonate into a compound of the general formula (wherein R means hydrogen, methyl, or ethyl; R.

means methyl, ethyl, propyl, and n is 1 to 4
It is characterized in that it is polymerized with a comonomer represented by (e) (meaning an integer of ).

The polymerization of polyvinylene carbonate is carried out using up to 45% comonomer, preferably from 5 to 20% comonomer. The bioactive compound can be prepared, for example, by introducing an electrophilic group into the bioactive substance or polyvinylene glycol copolymer and reacting said bioactive substance with the polyvinylene glycol copolymer via said group. can be manufactured.

Introduction of electrophilic groups into the carrier matrix can be achieved by copolymerizing vinylene carbonate with an electrophilic group-containing comonomer or by copolymerizing vinylene carbonate with an electrophilic group-containing comonomer. Alternatively, it is carried out by reacting a carrier matrix with a low molecular weight compound having an activated electrophilic group. In general, chemically bonding reaction partners on a carrier matrix is straightforward when nucleophilic and electrophilic functionalities are present on the one hand and electrophilic on the other hand.

Reactive nucleophilic functionalities include amino groups, sulfhydryl groups and hydroxyl groups, among others, which are usually already present in the matrix or reaction partner.
Electrophilic functionalities must be introduced. Furthermore, carboxyl groups can be introduced into acid halides, acid azides, acid anhydrides, imigzolides, or can be activated directly with carbodiimides. As the reactive electrophilic group C, an isocyanate group, an isothiocyanate group, a diazonium group, or a cyclic imidocarbonate ester can also be used. It is particularly suitable to introduce the reactive groups by copolymerization. By copolymerizing monomers having epoxy groups, such as epoxy alkyl vinyl ethers, into polyvinylene carbonate, the difference in saponification rate between the cyclic carbonate groups and the epoxy groups is also extremely large. A polyvinylene glycol matrix having reactive epoxy groups capable of reacting with nucleophilic functionalities can be easily obtained. That is, it is possible to attach biologically active substances directly and/or to introduce (extend) side chains (spacers). Therefore, another advantage of using copolymers is that electrophilic functionalities can be introduced directly (incorporated by polymerization), so that the reaction can be carried out by known methods. . By copolymerizing vinylene carbonate with ethyl acrylate and then saponifying the polymer, a matrix with -0H and -COOH functional groups is obtained.

The carboxyl group can be activated with a carbodiimide, in which case an amide bond is generated to the amino group of the effector. Furthermore, the carboxyl group is
The isoxazolium salt proposed by WOOdward enables the formation of an amide bond. Another method for attaching bioactive compounds to carboxyl group-containing polymers is N-ethoxycarboxyl-2-ethoxy-1,2-dihydroquinolite (EEDQ).
This is done using

The carboxyl group is esterified by a known method,
The ester can then be converted into a hydrazide and finally the resulting azide can be coupled to a polyvinylene glycol matrix via the amino group of an effector protein.

If the comonomer introduced into the matrix by polymerization has an amino group that can be liberated by a subsequent reaction, the aryl can be prepared using a vinyl sulfone derivative containing an arylamino group or a sulfate ester of β-hydroxy5-ethyl sulfone. Amino groups can be introduced.

The arylamino group can then be diazotized by the following known methods and then bonded to the corresponding reactive group of the effector. An example is shown below. This method also makes it possible to "extend" the spacer.

Another method of attaching proteins through their amino groups is done using glutardialdehyde.

Attachment of the effector to the matrix is achieved by activating the hydroxyl or amino groups of the polyvinylene glycol copolymer with a cyanide halide, preferably bromocyan, and then reacting the amino group-containing bioactive effector through this activated group. It's easy to do.

Attachment of effectors to polyvinylene glycol or polyvinylene glycol copolymer matrices also
This can also be accomplished by acylating the hydroxyl group with promacetylbutamide and then alkylating the amino group of the effector.

For example, a similar reaction can be carried out by reacting the hydroxyl group of the carrier with a reactive triazine.

In that case, part of the reactive groups of the triazine reacts with the polyvinylene glycol compound, and the other part reacts with the amino group of the effector. On the one hand, diazotizable aromatic amines which can be coupled to the hydroxyl groups of the carrier via other reactive groups can be combined with activated amino acids capable of coupling, such as tyrosine residues or Allows coupling with histidine residues.

Vinyl sulfone derivatives containing arylamino groups and sulfuric acid half esters of β-hydroxyethyl sulfone can be combined with the hydroxy groups of the carrier.

They allow the binding of effectors by the aforementioned diazotization reaction. The hydroxyl groups of the polyvinylene glycol copolymer are substituted with a nonion-forming epoxide containing at least two reactive groups, such as epihalohydrin or polyepoxide, e.g. Particularly stable ether bonds result when reacted with, for example, epichlorohydrin or bisepoxides.

In another variation, a comonomer (e.g. vinylpyrrolidone) is polymerized into the polyvinylene carbonate chain and the cyclocarbonate ring of the polyvinylene carbonate is then partially reacted with an amine such as hexamethylene diamine. A polyvinylene carbonate copolymer substituted with a spacer or effector via a urethane bond.

The remaining cyclocarbonate is then saponified to hydroxyl groups. That is, methods for creating a covalent bond between a phoribnylene carbonate carrier matrix and a protein effector or other effector include:
In addition to the methods already exemplified, there is also another method in which the hydroxyl groups or specific groups of the two copolymers are reacted with an effector to form a covalent bond between the two.

For example, known reactions using complex-forming metal compounds, such as titanium compounds, are one example. All of these methods are within the knowledge of those skilled in the art. When binding small bioactive compounds, it may be advantageous to activate the effector rather than the carrier.

In principle, in polymer chemistry, synthesis or... Any method known for modifying natural polymeric compounds can be used.

Polyvinylene glycol copolymers are distinguished not only by their chemical and thermal stability, but also by the fact that they have advantageous properties in terms of operational technology, and are therefore the most suitable natural Much superior to carbohydrate-based carrier matrices.

They can be produced, for example, in the form of fibres, threads, foils or spherical particles, so that the appropriate shape can be selected depending on the intended use of the effector to be coupled. Preferably, these copolymers are used in the form of finely divided powders with a large specific surface area. Polyvinylene glycol copolymers are also superior to known carrier materials in that the surface area available for attachment of effectors or spacers can be adjusted on an industrial scale.

The bioactive compounds according to the invention can be used in most methods known to date for other effectors coupled to hydrophilic, water-insoluble carriers.

The enzyme can therefore be rendered water-insoluble. This insoluble enzyme is increasingly being used for substrate determination by automated analysis and as so-called enzyme electrodes. Many carrier-bound enzymes are highly stable and therefore suitable for carrying out enzymatic reactions on an industrial scale. Biologically active substances bound to carriers are widely used in affinity chromatography due to their properties as specific adsorbents. A high degree of enzyme purification is possible using carrier-bound natural or synthetic enzyme inhibitors, while enzymes as effectors have been found to be particularly suitable for recovering natural enzyme inhibitors from crude extracts. Served. A water-insoluble antigen bound to a carrier is used to isolate antibodies against it;
The antibodies thus obtained are free of other serum components and other antigens. In affinity chromatography, antibodies that cannot be precipitated or antibodies that cannot be precipitated because their concentration in serum is low can be isolated and quantitatively measured. Example Preparation of vinylene carbonate copolymer The vinylene carbonate used in the production of the copolymer (CP) was diluted with 33 w on sodium borohydride (NaBH. 10 parts of vinylene carbonate was used per heel part) before use.
Reflux under the pressure of rIn for 1 hour, then heat to 75°C.
/ 50 cm long Laschig (R) at a pressure of 337-Irm.
aschig)-distilled through a silver barrel column filled with glass rings and used for copolymerization as quickly as possible.

Similarly, the comonomers used were previously purified by distillation. (
Example 1 Polyvinylene glycol/vinyl alcohol copolymer from saponified vinylene carbonate/vinyl acetate copolymer (a) 0.05 part by weight of azo to 9 parts by weight of vinylene carbonate and 1 part by weight of vinyl acetate Dissolve the bisisobutyronitrile under nitrogen.

The monomer mixture was flattened under nitrogen in an aluminum tube with a screw stopper (thickness 4W0n, width 60Twt) length 150.
WrIrL) (total filling volume is approximately 35ml),
Seal under nitrogen (N2) and suspend in a 50°C hot water bath for 48 hours. The resulting hard and brittle synthetic resin plate is pre-pulverized using a grinding mill. The granules were then subjected to a pressure of 1
Heating to 120° C. for 5 hours at ~2 rm to separate unpolymerized residual monomers. The monomer-removed granules were further ground in a mill until the particle size was <0.1TnIft, added to 5 parts by weight of 0.5N caustic soda, and stirred at 20°C using a high-speed stirrer. After mixing for 1 hour, the carbonate and acetyl groups are saponified. The water-insoluble vinylene glycol/vinyl alcohol-CP is taken out, washed with water to be free of inorganic salts, and freeze-dried. Vinylene glycol/ having a specific surface area of 34 i/y (measured by BET method)
4.7 parts by weight of vinyl alcohol-CP are obtained.

3 (b) As in Example 1(a), but using 3 parts by weight of vinyl acetate to 7 parts by weight of vinylene carbonate.

Vinylene glycol/vinyl alcohol 4tcol CP4 whose freeze-dried polymer powder has a specific surface area of 62.5y
．． 1 part by weight was obtained.

(c) For comparative experiment purposes, as in Example 1(a), but using m parts by weight of vinylene carbonate and no comonomer.

4.7 parts by weight of a vinylene glycol homopolymer having a specific surface area of 20.5 Ida of freeze-dried polymer powder was obtained.

Example 2 Vinylene glycol/vinyl glycidyl ether copolymer A mixture consisting of 7 parts by weight of vinylene carbonate, 3 parts by weight of vinyl glycidyl ether and 0.1 part by weight of azobisisobutyl nitrile was added to Example 1 (a).
), nitrogen (N2
) and then warm to 50°C.

The resulting synthetic resin plate was pulverized in the same manner as in Example 1(a) to remove the monomer, pulverized until the particle size became <0.1 Tm, and immersed in 5 μg of ice-cold 0.5 N NaOH at high speed. Using a stirrer, the 3 powders were suspended, and then the saponified polymer was immediately collected by centrifugation, suspended in ice water, neutralized to pH 7 using 2NH2SO under ice cooling, and suction filtered. Ice water wash and lyophilize. Contains 1.3 milliequivalents of oxirane groups per gram) [D. Braun et al., “PraktikumdermakrOmOlek
ularen Organischen Chemie, page 221 (published by 196)] vinylene glycol/vinyl glycidyl ether CP3. The tumor mass was obtained. Example 3 Amino-substituted copolymer carrier material 3. The vinylene glycol/vinyl glycidyl ether (from Example 2) in the weighing part was treated with Ultraturax (Ul
tra-Turrax) using a stirrer in 100 parts by weight of H2O and mixed with 10 ml of 30% ammonia.

It is then stirred for a further hour at 50° C. using a small magnetic stirrer and finally filtered with suction, thoroughly washed with water and lyophilized. 3.5 parts by weight of a copolymerized carrier material containing 3-amino-2-hydroxy-propyl groups (which result from the reaction of ammonia and glycidyl groups) with 0.9 meq amino groups per 1 f1 were obtained.

Example 4 Vinylene glycol/acrylic acid-CP 9 parts by weight of vinylene carbonate, 1 part by weight of acrylic acid ethyl ester and 0.05 part by weight of azobisisobutyronitrile under nitrogen as in Example 1(a) polymerized, pulverized, removed monomers,
Grind to 7$, saponify, vacuum filter, wash and freeze dry.

5. Hydrophilic polymer powder containing 1.1 milliequivalent carboxyl groups per carrier 1y and having a specific surface area of 27.2y1y by BET method. The tumor mass was obtained.

Example 55 5 parts by weight of ω-aminohexyl group-substituted polyvinylene glycol/acrylic acid
CP (from Example 4) was suspended in 100 nl H2O using an Ultraturax stirrer and cooled to 5°C for 1 hour.
(2.5y(7)N-cyclohexyl N'- (
N-methylmorpholino) monoethylcarbodiimide p
- Mix with toluene-sulfonate, stir at pH 5 and 5°C, filter, and quickly wash with ice water.

The activated carrier was then immediately suspended in a solution of hexamethylene diamine (5y) in water (100 mι) adjusted to pH 7.5 with 12N HCI and cooled to 5°C.
The mixture is then stirred under these conditions for an additional 24 hours using a magnetic stirrer, filtered with suction, thoroughly washed with water, and freeze-dried. 2 ω- having 0.7 milliequivalents of amino groups per y of carrier
4.5 parts by weight of aminohexyl group-substituted polyvinylene glycol was obtained.

Example 6 Reaction of vinylene glycol/vinyl alcohol-CP with an area of 34Iy by BET method obtained by Example 1(a) (a) Reaction with epichlorohydrin Produced by Example 1(a) Vinylene glycol/
Vinyl alcohol - CP5O da 11 (7) 2N diNa
Suspend in OH, mix with 250 mι of epichlorohydrin and stir at 55-60'C for 2 hours.

The pH value of the suspension drops to 10-11 after some time. This pH value is maintained for a further hour by addition of NaOH.
After a reaction time of 2 hours, the solid material was aspirated. Filter, wash with water, acetone and finally water again.

(b) Reaction with Hexamethylene Diamine Dissolve 50y of hexamethylenediamine in 1.51% water and add HCI until PHIO.

To the solution was added vinylene glycol/vinyl alcohol-CP activated according to Example E and 50
Stir at ~55°C for 6 hours. The product is then filtered with suction and washed free of hexamethylene diamine with water. c) Reaction with 1-aminobenzene-4-β-hydroxyethylsulfone sulfate Example 6 (b
) is stirred with 50y of 1-aminobenzene-4-β-hydroxyethylsulfonic acid ester at 55° C. and PHIO for 1 hour.

The solid material is then separated and washed with water, acetone and again with water. d) Diazotization The product 10y obtained according to Example 6(c) was heated to 0.2007711 on a nutschier. Washed with INHCI and then washed with 300 Tnι
．． Suspend in 5N HCI.

This suspension was heated to 0 to 4°C with stirring. INNaN0
2 solutions are added to the diazotization until a slight excess of nitrite is confirmed by KI-starch paper.
After 1 rinsing, it was filtered on a Nutssie filter and the residue was washed with ice water and then 0.15M sodium phosphate buffer at 0.4°C (
PH7.5).

(e) Covalently bonding 0.8 g of albumin with protein to 350
Dissolved in wL1 phosphate buffer (PH7.5),
Cool to 4° C. and add the product prepared according to Example 6(d).

The suspension was stirred for 2 hours at 4°C, then separated and the solid material was dissolved in IMNaCl and phosphate buffered saline (abbreviated as PBS) (1115 MN of pH 7.2).
a. HP0, -KH. P.O. 0.9 containing one buffer
% NaCl aqueous solution. The albumin test is performed on the laminar fluid and washing fluid using the radial immunodiffusion method. 60 m9 of albumin is bound to the carrier 1y thus produced. Example 7 Reaction of vinylene glycol/vinyl alcohol-CP with a specific surface area of 62.5 ld by the BET method obtained in Example 1(b) Same as the reaction described in Example 6(a) to (e) 80 mg of albumin can be quantitatively bound to the activated carrier of 1f1.

Example 8 1 g as in the reaction described in Example 6(a)-(e)
45m9 of albumin can be quantitatively bound to the activated carrier.

Example 9 ω-aminohexyl group-substituted vinylene glycol/
Vinylpyrrolidone copolymer 0.05 parts by weight of azobisisobutyronitrile was dissolved in 9 parts by weight of vinylene carbonate and 1 part by weight of vinylpyrrolidone under nitrogen, and polymerized in the same manner as in Example 1(a). and post-process.

After removal of the monomer, the granules were dissolved into a 1% by weight dimethylformamide solution, and the solution was dissolved at 15 atmg.
injected into a methanol precipitation bath at a pressure of . The resulting finely fibrous precipitated vinylene carbonate/vinyl pyrrolidone copolymer is filtered off with suction, further washed with methanol and resuspended in 200 parts by weight of methanol. (4) Add 6 parts by weight of hexamethylene diamine dissolved in parts by weight of methanol, stir the suspension for 2 days at room temperature, filter with suction and wash with methanol. The washed filter residue is then suspended for 4 days at room temperature in a solution of w parts by weight of sodium methylate in 30 parts by weight of 96% methanol, washed with methanol followed by a very thorough water wash and lyophilized. . Vinylene glycol/vinyl pyrrolidone copolymer substituted by ω-aminohexyl groups through urethane bonds, containing 1.6 meq of NH2 groups per y of support 5. The volume part was obtained. Example 102 ω using succinic anhydride and water-soluble carbodiimide
- Binding of IgG to aminohexyl group-substituted copolymer (produced according to Example 9) 5 parts by weight of the ω-aminohexyl substituted carrier produced according to Example 9 were added to 2003 parts by weight of water at 10°C and pH 6. Succinoylation is carried out for 4 hours with 2.5 parts by weight of suspended succinic anhydride.

The pH value is adjusted with 2N NaOH. After washing the solid material with water, the product was triturated with 1.25 parts by weight of N-cyclohexyl-N''-(N-methylmorpholino)-ethyl-carbodiyl-3-mido-p-toluene-sulfonate at pH 5 and 5°C. Stir, separate and quickly wash with ice water. 0.5 part by weight of IgG is dissolved in 1 part by weight of phosphate buffer (pH 7.5) and combined with the activated carrier as described above. Stir for 24 hours at 4°C.

After filtration, the product is washed with 1M common salt and PBS. 75 m9 of IgG is covalently bound to 1 g of carrier.

Example 11 Vinylene Glycol/Diethylene Glycol Divinyl Ether Copolymer 0.1 part by weight of azobisisobutyronitrile is dissolved in 9 parts by weight of vinylene carbonate and 1 part by weight of diethylene glycol divinyl ether under nitrogen and carried out. Polymerization is carried out in a flat aluminum tube under nitrogen at 50° C. for 3 days as in Example 1(a).

After post-treatment and saponification as in Example 1(a), 3.5 parts by weight of vinylene glycol/diethylene glycol divinyl ether are obtained. Example 12 Vinylene glycol/N-acryloylaminoacetaldehyde dimethyl acetal (a) 0.05 parts by weight of azobisisobutyronitrile is added to 9 parts by weight of vinylene carbonate and 1 part by weight of N-acryloylaminoacetaldehyde dimethyl acetal with nitrogen. Dissolve below.

The monomer mixture was polymerized in the same manner as in Example 1(a),
After treatment and saponification, 4.1 parts by weight of N-acryloylaminoacetaldehyde dimethyl acetal/vinylene glycol copolymer are obtained. One part by volume of N-acryloylaminoacetaldehyde dimethyl acetal/vinylene glycol copolymer was stirred in 10 parts by volume of 1N HCl for 4-5 hours. The activated support was washed with water and phosphate buffer (PH 7.5). (b) 0.5g of albumin to 200TTLt (7)
Dissolved in PBS and stirred with the product prepared in (a) above for 14 hours at 4°C.

Claims (1)

[Claims] 1 Vinylene carbonate and the general formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formula, R_1 means hydrogen, methyl, ethyl, R_3
means methyl, ethyl, propyl, and n is 1 to
Enzymes, activators, and inhibitors chemically bonded to a vinylene glycol copolymer produced by hydrolyzing a vinylene carbonate copolymer with at least one monomer represented by (meaning an integer of 4) biological activity consisting of biologically active substances selected from substances, antigens or antibodies, plasma proteins, blood group substances, phytohemagglutinin, antibiotics, vitamins or hormones, peptides or amino acids, natural or synthetically produced effectors; substance reagent. 2 (A) Vinylene carbonate and general formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formula, R_1 means hydrogen, methyl, ethyl, R_3
means methyl, ethyl, propyl, and n is 1 to
vinylene carbonate copolymer with at least one monomer represented by (meaning an integer of 4) enzymes, activators, inhibitors, antigens or antibodies, plasma proteins, blood group substances, phytohemagglutinin. , antibiotics, vitamins or hormones, peptides or amino acids, natural or synthetically produced effectors, and then converting the cyclocarbonate groups still present into hydroxy groups or (B ) converting the cyclocarbonate groups of a copolymer of vinylene carbonate into hydroxy groups; and (a) reacting the electrophilic groups present in or introduced into said copolymer with a biologically active substance; or (b) the hydroxy group is reacted (1) first with an electrophilic group-containing compound and then with a biologically active substance, or (2) directly with an electrophilic group-containing biologically active substance. A method for producing a biologically active substance reagent chemically bonded to a copolymer of vinylene glycol.