JPH05261281A - Carrier for immobilizing bioactive substance and its production - Google Patents

Abstract

PURPOSE:To provide the method for immobilizing bioactive substance to a water-insoluble carrier without deactivating or modifying the activity and the carrier for immobilizing the bioactive substance by reacting sugar chains of glycoproteins or glycopeptides having biological activity. CONSTITUTION:A bioactive substance having sugar-chain bonds is immobilized to a water-insoluble carrier by forming a Schiff base by the reaction of aldehyde group formed by the chain-cleavage of the glycol part of the sugar chain in the bioactive substance with the water-insoluble carrier bonded with poly (oxyethylene) of 10-50 polymn. degree having amino groups on both ends as a hydrophilic spacer, and then reducing the Schiff base. Thus, the carrier for immobilizing bioactive substance is obtd.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for immobilizing a glycoprotein or glycopeptide having a physiological activity on a water-insoluble carrier and a physiologically active substance-immobilized carrier, which comprises a glycol moiety of a sugar chain in the physiologically active substance. It is characterized in that the aldehyde group formed by cleavage by oxidation is allowed to form a Schiff base with a water-insoluble carrier having a hydrophilic spacer having an amino group at the terminal and is further reduced.

This method is an effective immobilization method without deactivating or denaturing its physiological activity and without blocking the active site. A wide range of areas such as adsorbents for the purpose of adsorbing and removing specific substances, quantification of substances by biosensors and enzyme immunoassays that utilize specific chemical reactions, or bioreactors that continuously perform specific chemical reactions. Can be applied in.

[0003]

2. Description of the Related Art Attempts to immobilize various glycoproteins or glycopeptides having physiological activity such as enzymes, antibodies, hormones, and lectins on a carrier, possibly via a spacer, have been reported for many years, especially with respect to enzymes. There is a report example. Most of them are by way of reaction at the functional groups of the amino acid residues that make up the protein. For example, ε-amino group of lysine, amino group of N-terminal, sulfhydryl group of cysteine, β-carboxyl group of aspartic acid, γ-carboxyl group of glutamic acid, C-terminal carboxyl group, phenolic hydroxyl group of tyrosine, hydroxyl group of serine or threonine, arginine. Guanidino group of, histidine imidazolyl group, tryptophan indolyl group, and methionine methylmercapto group. Especially, the content of hydroxyl group, carboxyl group and amino group is high. In this case, the amino acid residue forming the active center site may be modified or denatured, and the activity is often reduced or inactivated accordingly.

Generally, such protein-based physiologically active substances are susceptible to denaturation by the action of heat, acids, alkalis, organic solvents, etc., which are frequently used in ordinary chemical synthesis. Therefore, in order to allow the physiologically active substance that is a ligand to retain the physical or chemical properties that are as close as possible to those before immobilization, there are many restrictions in various immobilization reaction conditions.

Further, the immobilized ligand is firmly immobilized even in various salt concentrations and a wide pH range and leakage of the ligand does not occur, and the immobilized ligand can firmly bind to a target substance. Is a condition. In principle, the methods for immobilizing various physiologically active substances are roughly classified into a carrier binding method, a cross-linking method, and an entrapping method. Each of these methods has advantages and disadvantages, and is properly used according to the type and purpose of the ligand.

Proteins contain highly reactive amino acid residues, ionic amino acid residues, and hydrophobic moieties. Of these, the hydroxyl group, carboxyl group, and amino group, which do not adversely affect the expression of physiological activity as much as possible, are selected, and the covalent bond, ionic bond, hydrophobic bond, biochemical specific bond, etc. are attached to the carrier. What is immobilized by the method is the carrier binding method. For immobilization by covalent bond, activation with cyanogen bromide, derivatization of carboxylic acid with azide, condensation reaction with carbodiimide reagent or Woodward reagent K, diazo coupling reaction, two or more reactivity such as glutaraldehyde are rich. There is a method of crosslinking with a compound having a functional group. Although these methods have advantages such as strong bond and may increase stability,
There are disadvantages such as the possibility of protein denaturation and the difficulty of interaction with the target substance.

In JP-A-58-53757, an antibody having an aldehyde group and a carrier having an amino group or a hydrazide group in its side chain, which are further oxidatively cleaved at the carbohydrate site, is -C.
Methods for immobilization via the H2-NH- or -CH2-NH-NH-CO- structure are described. However, even in this method, since the carrier and the antibody are directly bound to each other, it is difficult to sufficiently retain the function of the antibody and immobilize it. Moreover, non-specific adsorption of other proteins cannot be prevented.

Immobilization by ionic bonding is advantageous in that it is easy to operate and reproducible, but it is easily affected by the type of buffer solution used in the reaction solution, pH, ionic strength, temperature and the like. Bonding is generally often weak in immobilization by physical adsorption.

The cross-linking method is a method in which a reagent having two or more functional groups such as glutaraldehyde, toluene diisocyanate, hexamethylene diisocyanate, and cyanuric chloride is reacted with a ligand to cross-link between molecules to form a macromolecule. Is. Although this method is often used for immobilizing microbial cells, it is not very effective for immobilizing protein-based physiologically active substances.

The entrapment method is a method of entrapping a ligand in a polymer gel. These include a lattice type in which the ligand is confined in the gel of natural polymers such as proteins and polysaccharides or various synthetic polymers, a microcapsule type in which the ligand is wrapped by a semipermeable polymer coating, and a phospholipid type. There is a liposome type in which a ligand is enclosed in a different liquid film. There is also a method of confining the ligand in the hollow child membrane or in the space partitioned by the ultrafiltration membrane. These methods have the advantage that ligand modification is less likely to occur due to immobilization, and immobilization is possible while maintaining the natural state, but it is less susceptible to the action of high molecular weight substances, and deactivation occurs depending on conditions. There are drawbacks.

[0011]

DISCLOSURE OF THE INVENTION The present invention solves the above-mentioned various drawbacks in the prior art, and enables a glycoprotein or glycopeptide having physiological activity to be efficiently and moreover reduced or denatured. The present invention is intended to provide a method for immobilizing a physiologically active substance whose property is as small as possible. Further, the immobilization technique in the present invention can be applied in an extremely wide range of fields.

[0012]

Means for Solving the Problems The immobilization method according to the present invention, which has achieved the above-mentioned object, is a physiologically active glycoprotein or glycopeptide in which an aldehyde group is introduced by modifying the sugar chain portion of the glycoprotein or glycopeptide. The gist is to perform an immobilization reaction by forming a Schiff base with a water-insoluble carrier to which a hydrophilic spacer having a terminal is bound and further reducing the Schiff base. INDUSTRIAL APPLICABILITY The present invention can be applied to any type of substance regardless of the type of enzyme, antibody, hormone, etc., as long as the bound sugar chain portion is a glycoprotein or glycopeptide that does not strongly participate in the expression of its activity.

The type of the glycoprotein or glycopeptide having physiological activity according to the present invention is not particularly limited, but it can be used in vivo including enzymes, antibodies, hormones, lectins and receptors having a sugar chain binding site. It contains various protein-based physiologically active substances that are involved in existing immune phenomena and cell adhesion. The biochemical significance and role of the sugar chain moiety in these glycoproteins and glycopeptides has not been elucidated in detail, but it is considered to be unique and various, and generally, heat and various denaturants are considered. , It is said that they are often involved in the action of enhancing or protecting the stability against degrading enzymes (proteases), or in maintaining the higher-order structure. However, it is rarely considered that the expression of its physiological activity has a large effect, and the effect on the change in physiological activity by modifying the sugar chain part is that the amino acid residue that constitutes the protein part is modified. It is small compared to the case of doing.

The water-insoluble carrier has a functional group reactive with an amino group, such as an aldehyde group or an epoxy group, or these functional groups have been introduced by modification, and It is not particularly limited as long as it has characteristics such as gel strength, particle diameter and pore diameter according to its purpose.
Examples include crosslinked polyacrylic acid, polyvinyl alcohol, polystyrene and derivatives thereof. The form of the carrier is not particularly limited, and may be in the form of beads, fibers, membranes (including hollow fiber membranes), etc., depending on the use and purpose. A poly (oxyethylene) having amino groups at both ends (hereinafter referred to as PEO amine) as a hydrophilic spacer bonded to these carriers is further added to the above-mentioned physiologically active substance. Is introduced as a ligand.

A higher content of amino groups in the carrier leads to an improvement in the rate of ligand introduction, but if it is too high, the gel strength decreases, which is not suitable. Therefore, it is desirable to introduce it in the range of 0.01 to 0.80 meq / g, and it is necessary to adjust the value according to the size of the ligand and the target substance. The chain length of the hydrophilic spacer (corresponding to n in Chemical formula 1) also needs to be appropriately adjusted according to the size and properties of the target substance to be bound to the ligand, but the degree of polymerization is in the range of 10 to 500,
Preferably 30 to 400, more preferably 100
It is suitable to use a poly (oxyethylene) having amino groups at both ends of the amino acid groups of 1 to 300. By introducing a hydrophilic spacer, steric hindrance is reduced, and the bondability with a target substance can be greatly improved. Furthermore, non-specific adsorption can be suppressed by the excluded volume effect of the hydrophilic spacer and the improvement of hydrophilicity.

[0016]

[Chemical 1]

On the other hand, introduction of an aldehyde group into a sugar chain portion of a glycoprotein or a glycopeptide, which is a ligand, is carried out by oxidizing with periodic acid or a salt thereof to cleave the glycol moiety to form an aldehyde group. preferable. The oxidation reaction with periodate occurs between the hydroxyl groups adjacent to the glycol moiety, and 1 mol of IO4− is reduced to IO3− to form 2 mol of an aldehyde group. The structure of the resulting reaction product varies depending on the type of glucosidic bond, but the structure shown in Chemical formula 2 is considered.

[0018]

[Chemical 2]

It is desirable that the amount of periodic acid is used in an excess amount equal to or more than the molar amount of the substance to be oxidized. The optimum pH value during the reaction varies depending on the stability of the substance introduced as a ligand and the ease with which side reactions occur.
It is preferable to carry out at pH 6 to 6, more preferably at pH 4 to 5. Further, it is suitable to use acetic acid or citrate buffer solution of about 0.1 M as the reaction solution. The reaction temperature is preferably room temperature or lower, and ideally 5 ° C. for substances that are unstable at room temperature. The reaction time of 1 hour or less is sufficient at room temperature, but it is 1 at 5 ° C.
It is necessary to do it for about 2 to 24 hours.

After the completion of the oxidation reaction, it is necessary to add substances such as ethylene glycol, glycerin and sodium sulfite to remove the excess oxidizing agent. Also, using the buffer solution used in the oxidation reaction as the external solution, perform dialysis treatment at 5 ° C for 12 hours.
It is more preferable to remove these substances by carrying out the treatment for a longer time.

The thus-obtained aldehyde group-introduced ligand is bound to the above-mentioned carrier to which PEO amine is bound by forming a Schiff base. This reaction is carried out at room temperature or 5 ° C. for 10 to 30 hours and pH is 6 to 10
It is more preferable that the pH is in the range of 8 to 9.5. As the reaction solution, it is preferable to use a carbonate buffer solution of about 0.1M.

The Schiff base thus obtained has poor stability and is susceptible to decomposition, especially under acidic or alkaline conditions. Therefore, as shown in the following equation (R1 is a carrier, R2 is
Is used as a ligand), and it is suitable to stabilize by carrying out a reduction reaction. R1N = CHR2 → R1NH-CH2R2

The reduction reaction of the Schiff base is preferably carried out by adding sodium borohydride (NaBH4) or sodium cyanoborohydride (NaBH3CN). The reaction is carried out at room temperature or 5 ° C at pH 8-9.
It is desirable to carry out the treatment in the above carbonate buffer for 3 to 30 hours.

In order to introduce a glycoprotein or a glycopeptide having an aldehyde group introduced into a sugar chain as a ligand into a water-insoluble carrier to which a hydrophilic spacer having an amino group at the terminal in the present invention is bound, the above method is used. The basic method is not particularly limited, but the following method is preferable when the antibody is introduced using cellulose as a carrier.

(1) Modification of Cellulose A solution of sodium periodate dissolved in sulfuric acid of 0.1 to 5.0 N, preferably 0.5 to 1.5 N, has an average particle size of 20 to 2000 μm and an average particle size. Pore size is 2
Add 00 to 30,000 * granular porous cellulose, and add 5 to 10 ℃ to 50 ℃, preferably 20 ℃ to 30 ℃.
The reaction is performed for 30 hours to 30 hours, preferably 10 hours to 24 hours. The sodium periodate concentration of the sodium periodate-sulfuric acid solution is 2% by weight to 15% by weight, preferably 4% by weight to 10% by weight. The bath ratio of the granular porous cellulose to the sodium periodate solution is 10
% To 30% by volume, preferably 15 to 25% by volume.

The reaction mixture is filtered to recover the product, washed thoroughly and swollen to give an aldehyde content of 0.1.
0 to 4.00 meq / g, preferably 0.20 to 1.00 meq / g [aldehyde]-[cellulose]
To get This [aldehyde]-[cellulose] has a structure in which some glucose units are ring-opened as shown in Chemical formula 2 above.

(2) Introduction of hydrophilic spacer A PEO amine having a degree of polymerization of 10 to 500, preferably 30 to 400, more preferably 100 to 300 as shown in Chemical formula 1 is dissolved in a carbonate buffer solution having a pH of 9.5. I'll do it. The [aldehyde]-[cellulose] obtained in (1) above was added to this and stirred at 10 ° C to 50 ° C.
The reaction is carried out at a temperature of preferably 20 ° C. to 30 ° C. for 5 hours to 30 hours, preferably 10 hours to 24 hours. Above PE
The concentration of O amine is 0.2% to 5.0% by weight, preferably 0.4% to 3.0% by weight, and the bath ratio of [aldehyde]-[cellulose] to this solution is 3% by volume. 20% by volume, preferably 5% to 15% by volume.
The reaction mixture is filtered to collect the product, which is washed with water [P
EO amine]-[cellulose] is obtained.

(3) Modification of sugar chain in antibody Anti-h obtained by purifying antiserum obtained by immunizing rabbit with human albumin (hereinafter referred to as hAlb) by ammonium sulfate salting out
Alb antibody 10 to 20 mg, 20 to 50 ml
The solution is dissolved in an acetic acid buffer solution having a pH of 4.0, 1 to 5 mg of sodium periodate is added, and the mixture is stirred at room temperature and reacted for 30 minutes to 1 hour. Further, ethylene glycol was added to have a concentration of 0.1 M and reacted at 5 ° C. for 5 to 10 hours, and the reaction solution was dialyzed at 5 ° C. for 12 to 24 hours using the above-mentioned acetate buffer as an external solution. ..
Thus, an [aldehyde]-[antibody] solution is obtained.

(4) Introduction of antibody The pH of the [aldehyde]-[antibody] solution obtained in (3) above is adjusted.
The pH is adjusted to 8.0 to 9.0 by adding 9.5 carbonate buffer.
Then, 3 g (swelled state) of [PEO amine]-[cellulose] obtained in (2) above was added, and the mixture was added at 20 ° C. at 20 ° C.
Allow to react for hours to 30 hours to form a Schiff base. The reaction mixture is filtered to recover the product, washed thoroughly, and carbonate buffer pH 9.0 in swollen state is added,
Further, 0.2 to 1 g of sodium borohydride was added and the reduction reaction was carried out by stirring at 5 ° C. for 5 hours to 15 hours. The reaction mixture is filtered to recover the product,
By thorough washing, an anti-hAlb antibody immobilized on cellulose via PEO amine as a spacer is obtained.

[0030]

EXAMPLES The present invention will be described below with reference to examples. <Example 1> Immobilization of invertase Yeast (Saccharomyces cerevisiae)
e), and the immobilase (β-D-fructofuranosidase) obtained by lyophilizing the culture broth purified by gel filtration, anion and cation exchange columns, and immobilizing it on cellulose was as follows. Was carried out.

400 mg of sodium periodate was dissolved in 50 ml of 1N sulfuric acid, 5 g of porous cellulose (Cellulofine GCL-1000m, manufactured by Chisso) was added, and the mixture was reacted for 20 hours by stirring, and then the reaction mixture was filtered to produce. The product was recovered and thoroughly washed to obtain [aldehyde]-[cellulose]. The aldehyde content was determined by the oxime method and was 0.97 meq / g.

PH of 20 g of PEO amine having a molecular weight of 600
It is dissolved in 50 ml of 9.5 carbonate buffer solution, the whole amount of [aldehyde]-[cellulose] is added and reacted by stirring, and the reaction mixture is filtered to collect the product, which is thoroughly washed [ PEO amine]-[cellulose] was obtained. Amino group content is determined by potentiometric titration with hydrochloric acid (COM by Hiranuma Sangyo
Quantification by TITE101), 0.31
It was meq / g.

On the other hand, 30 mg of the above invertase was added to pH.
Dissolved in 50 ml of citrate buffer of 4.5, added 50 mg of sodium periodate, and reacted for 45 minutes at room temperature with stirring. Further, glycerin was added to have a concentration of 0.1 M, and the mixture was stirred at 5 ° C. for 7 hours to react,
The reaction solution was dialyzed at 5 ° C. overnight using the above buffer solution as an external solution to obtain an [aldehyde]-[enzyme] solution.

Carbonate buffer was added to the above [aldehyde]-[enzyme] solution to adjust the pH to 8.0, 1 g of the above [PEO amine]-[cellulose] was added, and the mixture was stirred at 5 ° C. for 30 hours. To form a Schiff base. The reaction mixture was filtered to collect the product, which was thoroughly washed, 50 ml of a pH 8.0 carbonate buffer solution and 200 mg of sodium borohydride were added, and the reduction reaction was carried out at 5 ° C. for 7 hours. The reaction mixture was filtered to recover the product, which was thoroughly washed to obtain the immobilized invertase. The invertase immobilization amount was calculated by precipitating the residual solution of the Schiff base reaction with a trichloroacetic acid solution and dissolving the solution in a sodium hydroxide aqueous solution to quantify the remaining amount with a BCA protein quantification reagent (Pierce). Hereinafter referred to as TCA-BCA method),
18.5 mg had been immobilized.

The enzyme activity was quantified by suspending the above-mentioned immobilized invertase in a sucrose-citrate buffer solution (pH 5.5) having a concentration of 0.1% so that the immobilized amount would be 1 mg. It was carried out by reacting for 30 minutes. The concentrations of glucose and fructose, which are decomposition products in the reaction solution, were quantified by high performance liquid chromatography (LC-6A system manufactured by Shimadzu Corporation) using a normal phase column having an amino group. The results are shown in Table 1. In addition, the amount of glucose and fructose in the same experimental result in 1 mg of invertase in a free state not immobilized is shown as 100.

[0036]

[Table 1]

The effect on the thermostability of the present enzyme was examined by quantifying the residual activity of the immobilized enzyme suspension treated at 55 ° C. in the same manner as above.
The results are shown in FIG. Residual activity is p in free state
The amount of glucose produced by the reaction at 30 ° C. in H5.5 was expressed as 100%.

<Example 2> Immobilization of anti-mIgG (mouse immunoglobulin) antibody Immobilization of anti-mIgG antibody obtained by purifying rabbit antiserum immunized with mIgG (Kappel) by ammonium sulfate salting-out. Was carried out as follows.

200 mg of sodium periodate was dissolved in 50 ml of 1N sulfuric acid, 5 g of porous cellulose (Cellulofine GCL-1000m manufactured by Chisso) was added, and the mixture was reacted for 20 hours with stirring, and then the reaction mixture was filtered to produce. The product was recovered and thoroughly washed to obtain [aldehyde]-[cellulose]. The aldehyde content was 0.43 meq / g as determined by the oxime method.

PH of 4 g of PEO amine having a molecular weight of 1000 was adjusted to pH.
It is dissolved in 50 ml of 9.5 carbonate buffer solution, the whole amount of [aldehyde]-[cellulose] is added and reacted by stirring, and the reaction mixture is filtered to collect the product, which is thoroughly washed [ PEO amine]-[cellulose] was obtained. The amino group content was 0.22 meq / g as determined by potentiometric titration with hydrochloric acid.

On the other hand, 20 mg of the above antibody was dissolved in 50 ml of a citrate buffer solution having a pH of 3.8, 50 mg of sodium periodate was added, and the mixture was reacted for 45 minutes at room temperature with stirring. Further, ethylene glycol was added to have a concentration of 0.1 M, and the mixture was reacted by stirring at 5 ° C for 7 hours, and the reaction solution was dialyzed overnight at 5 ° C using the above buffer solution as an external solution [aldehyde]-[ Antibody] solution was obtained.

A carbonate buffer was added to the above [aldehyde]-[antibody] solution to adjust the pH to 9.0, 1 g of the above [PEO amine]-[cellulose] was added, and the mixture was stirred at 5 ° C. for 30 hours. To form a Schiff base. The reaction mixture was filtered to collect the product, which was thoroughly washed, 50 ml of a pH 8.0 carbonate buffer solution and 200 mg of sodium borohydride were added, and the reduction reaction was carried out at 5 ° C. for 7 hours. The reaction mixture was filtered to collect the product, which was thoroughly washed to obtain the immobilized antibody. The immobilized amount of the antibody was calculated by quantifying the remaining amount with a BCA protein quantification reagent in the same manner as in Example 1, and 17.1 mg was immobilized.

Cellulose gel 1 m on which the above antibody is immobilized
1 was packed in an open column tube and the antigen mIgG
Was dissolved in 50 mM phosphate buffer of pH 8.0 at a concentration of 0.2% and 10 ml was applied to the column. The antibody concentration of the column passage solution was quantified by the TCA-BCA method, and the adsorption rate to the column was calculated from the amount of non-adsorbed mIgG. The results are shown in Table 2.

[0044]

[Table 2]

<Example 3> Immobilization of anti-hLDL (human low-density lipoprotein) antibody [0045] Rabbit antiserum immunized with hLDL (Chemicon) was purified by ammonium sulphate salting out to give an anti-hLDL antibody on cellulose. Immobilization was performed as follows. Sodium periodate (200 mg) is dissolved in 1N sulfuric acid (50 ml) to give porous cellulose (Cissro cellulofine CPCm) 5 g
Was added and reacted for 20 hours by stirring, then the reaction mixture was filtered to collect the product, which was thoroughly washed to obtain [aldehyde]-[cellulose]. The aldehyde content was determined by the oxime method and was 0.55 meq / g.

PH of 2 g of PEO amine having a molecular weight of 5000 was adjusted to pH.
It is dissolved in 50 ml of 9.5 carbonate buffer solution, the whole amount of [aldehyde]-[cellulose] is added and reacted by stirring, and the reaction mixture is filtered to collect the product, which is thoroughly washed [ PEO amine]-[cellulose] was obtained. The amino group content was 0.20 meq / g as determined by potentiometric titration with hydrochloric acid.

On the other hand, 40 mg of the above antibody was dissolved in 50 ml of a citrate buffer solution having a pH of 3.8, 50 mg of sodium periodate was added, and the mixture was reacted at room temperature for 45 minutes by stirring. Further, ethylene glycol was added to have a concentration of 0.1 M, and the mixture was reacted by stirring at 5 ° C for 7 hours, and the reaction solution was dialyzed overnight at 5 ° C using the above buffer solution as an external solution [aldehyde]- An [antibody] solution was obtained.

A carbonate buffer was added to the above [aldehyde]-[antibody] solution to adjust the pH to 9.0, 1 g of the above [PEO amine]-[cellulose] was added, and the mixture was stirred at 5 ° C. for 30 hours. To form a Schiff base. The reaction mixture was filtered to collect the product, which was thoroughly washed, 50 ml of a pH 8.0 carbonate buffer solution and 200 mg of sodium borohydride were added, and the reduction reaction was carried out at 5 ° C. for 7 hours. The reaction mixture was filtered to collect the product, which was thoroughly washed to obtain the immobilized antibody. The immobilized amount of antibody was calculated by quantifying the remaining amount with a BCA protein quantifying reagent in the same manner as in Example 1, and 27.9 mg was immobilized.

Cellulose gel 1 m on which the above antibody is immobilized
3 ml of pig serum (LDL value 150.5 mg / ml)
With 20 ml of vial and incubate by shaking at 30 ° C for 30 minutes, and add L of the centrifuged supernatant.
The DL value was quantified by β-lipoprotein C Test Wako (manufactured by Wako Pure Chemical Industries). LDL from the obtained residual LDL value
The results of calculating the adsorption rate are shown in Table 3.

[0050]

[Table 3]

<Example 4> Wheat germ lectin (WG)
Immobilization of A) Immobilization of WGA (manufactured by Vector Laboratory) on cellulose was carried out as follows. Sodium periodate (100 mg) was dissolved in 1N sulfuric acid (50 ml) to prepare porous cellulose (Chisso Cellulofine GCL-1000).
m) 5 g was added and reacted for 20 hours by stirring,
The reaction mixture was filtered to collect the product, which was thoroughly washed to obtain [aldehyde]-[cellulose]. The aldehyde content was determined by the oxime method to be 0.22 meq / g
Met.

PH of 4 g of PEO amine having a molecular weight of 1000 was adjusted to pH.
It is dissolved in 50 ml of 9.5 carbonate buffer solution, the whole amount of [aldehyde]-[cellulose] is added and reacted by stirring, and the reaction mixture is filtered to collect the product, which is thoroughly washed [ PEO amine]-[cellulose] was obtained. The amino group content was 0.15 meq / g as determined by potentiometric titration with hydrochloric acid.

On the other hand, 20 mg of the above WGA was dissolved in 50 ml of a citrate buffer solution having a pH of 4.5, 50 mg of sodium periodate was added, and the mixture was reacted for 45 minutes at room temperature with stirring. Further, ethylene glycol was added to have a concentration of 0.1 M, and the mixture was reacted by stirring at 5 ° C for 7 hours, and the reaction solution was dialyzed overnight at 5 ° C using the above buffer solution as an external solution [aldehyde]- I got [lectin]. The above-mentioned [aldehyde]-[lectin] solution is added with carbonate buffer to adjust the pH.
Was adjusted to 8.5, 1 g of the above [PEO amine]-[cellulose] was added, and the mixture was stirred at 5 ° C. for 30 hours to form a Schiff base. The reaction mixture is filtered to recover the product,
After thorough washing, 50 ml of a pH 8.0 carbonate buffer and 200 mg of sodium borohydride were added, and a reduction reaction was carried out at 5 ° C. for 7 hours. The reaction mixture was filtered to collect the product, which was thoroughly washed to obtain the immobilized lectin. The immobilized amount of lectin was calculated by quantifying the remaining amount with a reagent for quantifying BCA protein, and 13.7 mg was immobilized.

Cellulose gel 1 on which the above WGA is immobilized
Fill the open column tube with ml and
The column was dissolved in 8.0 of 50 mM phosphate buffer at a concentration of 0.2% and 10 ml was applied to the column. The antibody concentration of the column passage solution was quantified by the TCA-BCA method, and the adsorption rate to the column was calculated from the amount of non-adsorbed mIgG. The results are shown in Table 4.

[0055]

[Table 4]

<Comparative Example 1> Immobilization of invertase 30 mg of invertase in Example 1 was adjusted to pH 8.0.
1 g of [PEO amine]-[cellulose] prepared in Example 1 was added, and the mixture was stirred at 5 ° C. for 30 hours to form a Schiff base. The reaction mixture is filtered to collect the product, washed thoroughly to ensure pH
50 ml of a carbonate buffer solution of 8.0 and 200 mg of sodium borohydride were added to carry out a reduction reaction at 5 ° C. for 7 hours.
The reaction mixture was filtered to recover the product, which was thoroughly washed to obtain the immobilized invertase. The amount of immobilized invertase was determined in the same manner as in Example 1, and 21.3 mg was immobilized. The enzyme activity and its thermal stability were determined in the same manner as in Example 1. The results are shown in Table 1 and FIG.

<Comparative Example 2> Immobilization of anti-mIgG antibody 20 mg of the anti-mIgG antibody in Example 2 was adjusted to pH 9.0.
1 g of [PEO amine]-[cellulose] prepared in Example 2 was added and stirred at 5 ° C. for 30 hours to form a Schiff base. The reaction mixture is filtered to collect the product, washed thoroughly to ensure pH
50 ml of a carbonate buffer solution of 8.0 and 200 mg of sodium borohydride were added to carry out a reduction reaction at 5 ° C. for 7 hours.
The reaction mixture was filtered to collect the product, which was thoroughly washed to obtain the immobilized antibody. The amount of antibody immobilized was determined in the same manner as in Example 2, and 18.3 mg was immobilized. Example 2
A column was prepared in the same manner as above, and the mIgG adsorption rate was determined.
The results are shown in Table 2.

<Comparative Example 3> Immobilization of anti-hLDL antibody 40 mg of the anti-hLDL antibody in Example 3 was adjusted to pH 9.0.
1 g of [PEO amine]-[cellulose] prepared in Example 3 was added, and the mixture was stirred at 5 ° C. for 30 hours to form a Schiff base. The reaction mixture is filtered to collect the product, washed thoroughly to ensure pH
50 ml of a carbonate buffer solution of 8.0 and 200 mg of sodium borohydride were added to carry out a reduction reaction at 5 ° C. for 7 hours.
The reaction mixture was filtered to collect the product, which was thoroughly washed to obtain the immobilized antibody. The amount of antibody immobilized was determined in the same manner as in Example 2, and 35.1 mg was immobilized. Example 3
The LDL adsorption rate in pig serum was determined in the same manner as in. The results are shown in Table 3.

<Comparative Example 4> Immobilization of anti-hLDL antibody In the same manner as in Example 3, 5 g of CPCm was oxidized to prepare [aldehyde]-[cellulose] having an aldehyde content of 0.50 meq / g. Further, 50 ml of 28% ammonia water was added thereto and reacted at room temperature for 8 hours, and the product was collected by filtration and sufficiently washed to obtain [amino]-[cellulose]. The amino group content was determined by potentiometric titration with hydrochloric acid and found to be 0.28 meq / g.

On the other hand, the anti-hLDL antibody 40 in Example 3
mg was dissolved in 50 ml of a carbonate buffer having a pH of 9.0, 1 g of the above [amino]-[cellulose] was added, and the mixture was added at 30 ° C. at 30 ° C.
Stir for hours to form the Schiff base. The reaction mixture was filtered to collect the product, washed thoroughly, and washed with 50 ml of a pH 8.0 carbonate buffer and 200 mg of sodium borohydride.
Was added and the reduction reaction was carried out at 5 ° C. for 7 hours. The reaction mixture was filtered to collect the product, which was thoroughly washed to obtain the immobilized antibody. The immobilized amount of antibody was determined in the same manner as in Example 2,
35.1 mg was immobilized. The LDL adsorption rate in pig serum was determined in the same manner as in Example 3. The results are shown in Table 3.

Comparative Example 5 Immobilization of WGA 20 mg of WGA in Example 4 was dissolved in 50 ml of a carbonate buffer having a pH of 8.5, and 1 g of [PEO amine]-[cellulose] prepared in Example 1 was added to give 5 The Schiff base was formed by stirring at 30 ° C. for 30 hours. The reaction mixture was filtered to collect the product, washed thoroughly to pH 8.0.
50 ml of carbonate buffer, sodium borohydride 200
mg was added and the reduction reaction was performed at 5 ° C. for 7 hours. The reaction mixture was filtered to collect the product, which was thoroughly washed to obtain the immobilized lectin. The immobilized amount of lectin was determined in the same manner as in Example 2, and 15.9 mg was immobilized. A column similar to that in Example 4 was prepared and the mIgG adsorption rate was determined. The results are shown in Table 4.

[0062]

EFFECTS OF THE INVENTION According to the present invention, it becomes possible to immobilize various sugar chain-bonded physiologically active substances on a water-insoluble carrier without deactivating or denaturing the activity thereof, and before immobilization. It has become possible to provide a physiologically active substance-immobilized carrier that maintains a similar activity. The present invention is useful in a wide range of fields including adsorbents, carriers for affinity chromatography, biosensors, enzyme immunoassays and bioreactors.

[Brief description of drawings]

FIG. 1 is a diagram showing thermal stability of Example 1 and Comparative Example 1.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Masakazu Tanaka 2-1-1 Katata, Otsu City, Shiga Prefecture Toyobo Co., Ltd.

Claims (3)

[Claims] 1. A physiologically active substance-immobilized carrier, wherein a physiologically active glycoprotein or glycopeptide is immobilized at a sugar chain site on a water-insoluble carrier via a hydrophilic spacer. 2. A Schiff base is formed with a water-insoluble carrier to which an aldehyde group formed by cleavage of a glycol moiety in a sugar chain of a glycoprotein or glycopeptide having a physiological activity is bound, and a hydrophilic spacer having an amino group at the end is bound. A method for producing a physiologically active substance-immobilized carrier, which comprises reducing and then reducing the concentration. 3. The hydrophilic spacer has a polymerization degree of 10 to 5.
The method for producing a physiologically active substance-immobilized carrier according to claim 2, which is poly (oxyethylene) having amino groups at both ends, which is 00.