JPH0741167B2 - Production method of bioactive substance-immobilized adsorbent and production kit - Google Patents

Description

The present invention relates to a method for producing a physiologically active substance-immobilized adsorbent and a kit for producing a physiologically active substance-immobilized adsorbent.

The physiologically active substance-immobilized adsorbent obtained by the present invention is
Utilizing the activity of immobilized physiologically active substances, as a medical adsorbent for treating diseases by purifying body fluids, as an adsorbent for affinity chromatography to separate and acquire target substances It is useful.

[Conventional technology]

JP-A-64-68398 discloses a method for producing an adsorbent capable of adsorbing an autoantibody against a nicotinic acetylcholine receptor, which is considered to be a causative agent of myasthenia gravis, with cyanogen bromide. Activated agarose particles contain a monoclonal anti-idiotype antibody specific for a monoclonal antibody having specificity for the nicotinic acetylcholine receptor at concentrations of 0.5 mol / min and 0.1 mol / mol of sodium chloride and sodium bicarbonate, respectively. Immobilizing in an aqueous solution (pH: 8.3), treating the obtained agarose particles on which the monoclonal anti-idiotype antibody has been immobilized with a phosphate buffer containing glycine, and then washing with a phosphate buffer And a cellulose particle activated with a succinimidooxycarbonyl group. , A monoclonal anti-idiotype antibody similar to the one described above was immobilized in a 10 mM phosphate buffer (pH: 7.4), and the obtained cellulose particles on which the monoclonal anti-idiotype antibody was immobilized were subjected to phosphorus immobilization. A method consisting of washing with a salt buffer is described.

As a method for producing an adsorbent for affinity chromatography, which comprises immobilizing a ligand having an amino group such as a protein on an agarose particle having an active ester group, the particle contains 0.1 mol of sodium hydrogen carbonate and 0.1 mol of sodium chloride, respectively. And immobilized in a carbonate buffer (pH: 8) dissolved in water at a concentration of 0.5 mol / min, and the obtained ligand-immobilized agarose particles are treated with an ethanolamine aqueous solution or Tris-HCl buffer. After blocking, Tris buffer (pH: 8) prepared by dissolving Tris and sodium chloride in water at a concentration of 0.1 mol / and 0.5 mol / acetic acid or acetic acid or formic acid and sodium chloride respectively 0.1 mol / and 0.5 mol / Alternating 4 to 5 times with acetate buffer or formate buffer (pH: 4) dissolved in water at the concentration of Process comprising washing are known.

Further, as a method for producing an adsorbent for affinity chromatography which comprises immobilizing a ligand having choline, a hydroxyl group such as sugar, an amino group or a mercapto group on an agarose particle activated with an epoxy group, the particle is provided with a ligand. After immobilizing in a solvent, the obtained ligand-immobilized agarose particles were blocked with an aqueous solution of 1 mol / ethanolamine, washed with the same solvent as used for immobilization, and further acetic acid and sodium chloride were added. Acetic acid buffer solution (pH: 4.0) dissolved in water at a concentration of 0.1 mol / and 0.5 mol /, respectively, and borate prepared by dissolving boric acid and sodium chloride in water at a concentration of 0.1 mol / and 0.5 mol /, respectively. Acid buffer (pH:
A method is known which consists of alternating cleaning with 8.0).

[Problems to be Solved by the Invention]

In the adsorbent obtained according to the production method described in the above-mentioned JP-A-64-68398, the monoclonal anti-idiotype antibody that should have been immobilized during use may be released from the adsorbent slightly. It has been found. When such an adsorbent is used as a therapeutic adsorbent, it is necessary for safety to suppress the release of the monoclonal anti-idiotype antibody as much as possible, and therefore it is necessary to subject the adsorbent to a further sufficient washing treatment. is there.

On the other hand, the adsorbent obtained by the method for producing an adsorbent for affinity chromatography, which comprises immobilizing a ligand on agarose particles having an active ester group or an epoxy group, should also be immobilized during use. It was found that the above ligand may be liberated from the adsorbent, though slightly. When such an adsorbent is used as an adsorbent for affinity chromatography, it is desirable to suppress the liberation of the ligand as much as possible in order to prevent the mixture of the ligand into the target substance, and the adsorbent is used for therapeutic purposes. Even when it is applied to an adsorbent, it is safer to suppress the liberation of the ligand as much as possible.

Accordingly, one object of the present invention is to provide a method for producing a physiologically active substance-immobilized adsorbent in which the released amount of the physiologically active substance is extremely small, by a simple operation. Another object of the present invention is to provide a kit for easily producing the physiologically active substance-immobilized adsorbent.

[Means for Solving the Problems]

According to the present invention, the above object is to immobilize a physiologically active substance on a carrier having an active group by a covalent bonding method, and after contacting the obtained physiologically active substance-immobilized carrier with a blocking agent, if necessary, It is achieved by providing a method for producing a physiologically active substance-immobilized adsorbent characterized by alternately washing with a buffer solution having a pH of 3.5 or less and a buffer solution having a pH of 5.5 or more, and an active group This is achieved by providing a kit for producing a physiologically active substance-immobilized adsorbent, which comprises a carrier, a buffer solution having a pH of 3.5 or less, and a buffer solution having a pH of 5.5 or more.

The physiologically active substance means a nucleic acid, a protein, a hormone, a lipid, a saccharide and a compound thereof having physiological activity such as an enzyme activity and an antigen-antibody reaction activity; an amino acid; a peptide and the like, and the physiologically active substance is an enzyme, Coenzyme, antibody,
Antigens, antigen-antibody complexes, complements, haptens, tissues, cells etc. are used. Specific examples of the physiologically active substance include calf thymus DNA, anti-β ₂ -microglobulin antibody, insulin, blood group antigen, tryptophan, phenylalanine,
Superoxide dismutase, coenzyme Q, DNA-anti-DNA
Antibodies, Clq, penicillin and the like. The physiologically active substance has a portion directly related to the expression of the activity such as an active center and a substrate binding site, but has a functional group such as an amino group, a hydroxyl group and a mercapto group in the other portion. . The active group possessed by the carrier is a group capable of reacting with a functional group possessed by these physiologically active substances to form a covalent bond, and examples of the active group include a succinimidooxycarbonyl group and p-nitrophenyloxycarbonyl group. Group, dinitrophenyloxycarbonyl group, pentafluorophenyloxycarbonyl group, trichlorophenyloxycarbonyl group, pentachlorophenyloxycarbonyl group and other active ester groups; epoxy group; formyl group and the like. Among these active groups, an active ester group capable of reacting with an amino group of a physiologically active substance to form a strong amide bond is preferable, and among the active ester groups, a succinimidooxycarbonyl group is a reaction between this and a physiologically active substance. N-hydroxysuccinimide that is eliminated by the method is particularly preferable because it is easily removed from the carrier. The concentration of the active group of the carrier, in order to increase the immobilization rate of the bioactive substance, is preferably a carrier 3 × 10 ^-8 mol or more per 1g of ^{wet, 1 × 10 -7 ~5 × 10} - It is more preferably within the range of ⁵ mol. It is desirable that the carrier having an active group is a porous water-insoluble substance having a hydrophilic surface because the immobilization rate of the physiologically active substance is high and the adsorption performance of the obtained adsorbent is excellent. The shape of the carrier is
It is not particularly limited, and examples thereof include particles, films, solid fibers, hollow fibers, and the like, but for the purpose of producing an adsorbent to be packed in a column, a liquid to be flowed through the column is used. Spherical particles are preferable because of low resistance. Examples of the carrier include cellulose-based carriers such as CM-celluloin CH (sold by Seikagaku Corporation); polyvinyl alcohol-based carriers such as CM-Toyopearl 650C (manufactured by Toso Corporation); CM-Trisacryl M (CM
-Trisacryl M) [Farmassier LKB, Sweden]
(Pharmacia-LKB), etc.]; polyacrylamide type carriers; Sepharose CL-4B (Sepharose CL-4B), Activated CH-Sepharose 4B (Activated CH-Seph).
arose 4B), Epoxy activated cepharose
An organic carrier such as an agarose-based carrier such as 6B (Epoxy-activated Sepharose 6B) (all manufactured by Pharmacia-LKB of Sweden); and
Examples of the inorganic carrier include porous glass such as CPG-10-1000 [average pore size: 100 nm; manufactured by Electro-nucleonics of the United States]. Among these carriers, the cellulosic carrier is preferably high in mechanical strength, excellent in acid resistance and alkali resistance, less susceptible to decomposition by microorganisms, and low in toxicity.

Immobilization of a physiologically active substance on a carrier having an active group can be carried out according to a usual method, and it is preferably carried out in a buffer solution such as a phosphate buffer solution or a carbonate buffer solution. The buffer solution has a pH in the range of 5.5 to 8.5 and a salt concentration of 10
It is preferable to use 0 mmol / mol or less from the viewpoint of suppressing deactivation of active groups and physiologically active substances during the immobilization operation. The salts contained in the buffer solution include phosphates such as sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate; carbonates such as sodium carbonate, sodium hydrogen carbonate, potassium carbonate; sodium chloride, etc. However, it is desirable because it does not deactivate the active group.

The physiologically active substance-immobilized carrier thus obtained,
Since the unreacted active group usually remains, the carrier is brought into contact with a blocking agent capable of reacting with the unreacted active group to deactivate the active group, if necessary. The blocking agent is arbitrarily selected and used according to the type of active group. For example, when the active group is capable of reacting with an amino group, a compound having an amino group such as glycine, ethanolamine or tris is used as a blocking agent. The contact between the physiologically active substance-immobilized carrier and the blocking agent can be carried out according to a usual method, and is generally carried out in a buffer solution. The buffer solution is not particularly limited, and examples thereof include glycine hydrochloric acid buffer solution and Tris hydrochloric acid buffer solution. The concentration of the blocking agent in the buffer solution is preferably 50 mmol / mol or more because the reaction between the blocking agent and the active group proceeds rapidly.
If the concentration of the blocking agent is too high, a large amount of unreacted blocking agent remains, so the concentration is
More preferably, it is in the range of 70 to 1000 mmol /. As the buffer solution, in the state containing the blocking agent, the pH is in the range of 5.5 to 8.5, and the salt concentration is
It is preferable to use one having a concentration of 200 mmol / mol or more because the inactivation of the immobilized physiologically active substance is suppressed and the reaction between the blocking agent and the active group proceeds rapidly. Here, the concentration of the salt in the buffer containing the above-mentioned blocking agent is such that when a blocking agent that forms a salt in the system such as glycine is used, the salt derived from the blocking agent is It includes the concentration. It is desirable that the physiologically active substance-immobilized carrier and the blocking agent are contacted with each other for a sufficient period of time since the blocking of the unreacted active group is completed. From this viewpoint, it is preferable that the physiologically active substance-immobilized carrier is brought into contact with the blocking agent in a buffer solution for about 15 minutes to about 20 hours.

The blocking of unreacted active groups in the physiologically active substance-immobilized carrier can be performed simultaneously with the washing operation by using a washing solution containing a blocking agent in the washing step described below. In order to completely carry out the blocking, it is preferable that the physiologically active substance-immobilized carrier is contacted with the blocking agent for a sufficient time prior to washing.

To wash the physiologically active substance-immobilized carrier thus obtained, a buffer solution having a pH of 3.5 or less and a buffer solution having a pH of 5.5 or more are used as a washing solution. Instead of a buffer solution with a pH of 3.5 or less, p
When H uses a buffer solution higher than 3.5 and when pH uses a buffer solution having a pH of less than 5.5 instead of the buffer solution having a pH of 5.5 or more, the physiologically active substance physically adsorbed on the carrier,
For example, physiologically active substances other than physiologically active substances that are bound to the carrier by covalent bonds, such as physiologically active substances that are bound to the carrier by ionic bonds, are difficult to release from the carrier, and the washing operation is repeated many times. It is not preferable because it is necessary. Since the washing capacity is high and the deactivation of the physiologically active substance immobilized on the carrier through a covalent bond can be suppressed, the washing solution has a pH of 1.5 to 3.0 and a buffer solution having a pH within the range of 3.0.
It is preferable to use a buffer solution having a ratio of 6.0 to 8.5.
The buffer solution having a pH of 3.5 or less is not particularly limited, and examples thereof include glycine hydrochloric acid buffer solution and formate buffer solution. The buffer solution having a pH of 5.5 or more is not particularly limited, but a phosphate buffer solution, a carbonate buffer solution and the like are exemplified. The salt concentration in the buffer solution having a pH of 3.5 or less and the buffer solution having a pH of 5.5 or more is preferably 400 mmol / or less.

The physiologically active substance-immobilized carrier is washed by alternately using a buffer solution having a pH of 3.5 or less and a buffer solution having a pH of 5.5 or more. The buffer used in the first washing operation may be either a buffer having a pH of 3.5 or less and a buffer having a pH of 5.5 or more, but the buffer used in the last washing operation is immobilized on the carrier. A buffer solution having a pH in the range of 5.5 to 8.5 is desirable from the viewpoint of suppressing the inactivation of the bioactive substance that has been made into a form. One washing operation is carried out in accordance with a usual procedure by bringing the buffer solution into contact with the physiologically active substance-immobilized carrier and then allowing the buffer solution to flow out of the system. For example, when a carrier in which particulate physiologically active substances are immobilized is used, it is carried out by dispersing the carrier in a buffer solution and then separating the buffer solution from the carrier by filtration or decanting. As the buffer solution having a pH of 3.5 or less and the buffer solution having a pH of 5.5 or more, one kind or two or more kinds of buffer solutions are used, respectively. The washing operation using a buffer solution having a pH of 3.5 or less, the washing operation using a buffer solution having a pH of 5.5 or more, and the washing operation using a buffer solution having a pH of 5.5 or more are each performed once or more, preferably 2 to 4 times each. . A washing operation using a washing solution different from these buffers may be additionally performed during or before or after repeating the washing operation using these buffers.

The kit for producing a physiologically active substance-immobilized adsorbent of the present invention comprises a carrier having the above-mentioned active group, a buffer solution having a pH of 3.5 or less, and a p-solution.
Since H consists of a buffer solution of 5.5 or more, the physiologically active substance is immobilized on a carrier having an active group by a covalent bond method, and the obtained physiologically active substance-immobilized carrier is contacted with a blocking agent if necessary. The method for producing a physiologically active substance-immobilized adsorbent of the present invention, which comprises alternately washing with a buffer solution having a pH of 3.5 or less and a buffer solution having a pH of 5.5 or more, can be easily applied. The kit has the above-mentioned physiologically active substance for convenience of immobilizing the physiologically active substance on a carrier having an active group and contacting the physiologically active substance-immobilized carrier with a blocking agent. An immobilization buffer such as the buffer used in the immobilization procedure described above and a blocking buffer such as the buffer containing the blocking agent used in the above-described blocking procedure may be added. In addition, even if a column capable of being packed with an adsorbent is added to the kit so that it is convenient when the produced adsorbent is used as a therapeutic adsorbent or a therapeutic test adsorbent. Good. As an example of the column, as shown in FIG. 1, filling the liquid inlet part 1 and the liquid outlet part 2 and the physiologically active substance-immobilized adsorbent which are easily connected to a tube for flowing a liquid such as blood, plasma or serum. The liquid inlet part is located at one end of the body part, and the liquid outlet part is located at the other end of the body part. The adsorbent on which the physiologically active substance-immobilized material has supporting portions 4 and 5 that cannot pass through are provided between the main body portion and the liquid inlet portion and between the main body portion and the liquid outlet portion, respectively. Examples of the material of the main body portion, the liquid inlet portion and the liquid outlet portion include polyethylene, polypropylene, polycarbonate, polyester, polymethylmethacrylate and the like.
Of these, polypropylene and polycarbonate are particularly preferable in that the column can be subjected to sterilization such as autoclave sterilization and γ-ray sterilization. A filter made of polyester fiber or the like is used as the supporting portion.

〔Example〕

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

Example 1 Preparation of Support Having Active Group 10 g of cellulose particles (Seikagaku Kogyo Co., Ltd., CM-cellulophane CH) were suspended in 50 ml of dioxane obtained by distillation in the presence of metallic sodium to obtain a carrier. 0.5 g of N-hydroxysuccinimide and 1.0 g of dicyclohexylcarbodiimide were added to the obtained suspension, and the mixture was shaken and stirred overnight at room temperature. A gel containing dioxane was obtained by repeatedly washing the obtained mixture with methanol and dioxane. 1g of this gel
Was dispersed in 1 ml of 20 mmol / phosphate buffer (pH: 7.4) containing 150 mmol / sodium chloride for 3 days, and the resulting mixture was sucked through. N in the liquid was measured based on the measured absorbance at a wavelength of 230 nm.
As a result of obtaining the concentration of -hydroxysuccinimide, it was found that about 30 μmol of succinimidooxycarbonyl group was present per 1 g of the gel containing dioxane.

Immobilization of antibody After washing 10 g of the gel containing dioxane described above with 20 mmol / phosphate buffer (pH: 7.4), mouse IgG ₁
Mixed with 20 ml of 20 mM phosphate buffer (pH: 7.4) containing 20 mg of the HA7 type monoclonal antibody HA-7 (Ministry of Industrial Science and Technology No. 2471; see JP-A-62-44199) did. The mixture was stirred overnight at 4 ° C. and then suctioned to give a gel. Immediately after mixing and after stirring overnight, the concentration of the antibody in the supernatant of the mixture was measured by the protein assay [Protein-Assay Kit; US-Bio-Rad].
Manufactured by Rad). The antibody immobilization rate on the carrier is 90% based on this quantitative value.
It was.

Blotting 10 g of the carrier gel on which the antibody obtained by the above-mentioned immobilization operation is immobilized is 20 mmol / phosphate buffer solution (pH:
After washing with 7.4), 20 mM phosphate buffer (pH: 7.4) containing glycine and sodium chloride at a concentration of 100 mM / and 150 mM / respectively.
Mixed with 50 ml. The mixture was stirred at room temperature for 4 hours and then suctioned to obtain a gel.

Wash 1 g of 10 g of the gel obtained by the above blocking procedure.
100mM of 00mmol / glycine hydrochloric acid buffer (pH: 2.5)
It was turbid in 1 and then the suspension was washed by aspiration (first wash). The gel obtained by the first washing was washed with 20 mM phosphate buffer (pH: 7.4) containing sodium chloride at a concentration of 150 mM.
It was suspended in 100 ml and then the suspension was washed by aspiration (second wash). The gel obtained by the second washing was washed with the same glycine-hydrochloric acid buffer as in the first washing and the same phosphate buffer as in the second washing. Up to the sixth washing was carried out by alternately applying the washing operation twice each in this order.

Example 2 Preparation of a carrier having an active group, immobilization of an antibody, in the same manner as in Example 1 except that 10 g of polyvinyl alcohol particles (CM-Toyopearl 650C manufactured by Tosoh Corporation) were used as the carrier instead of 10 g of the cellulose particles. Blotting and washing were performed. The amount of succinimidooxycarbonyl groups present per 1 g of the gel obtained by the procedure for preparing the carrier having active groups was about 20 μmol. In the antibody immobilization procedure, the immobilization rate of the antibody on the carrier was 85%.

Example 3 Immobilization of Antibody Agarose particles activated with a succinimidooxycarbonyl group [Farmasia-LKB (Pharm, Sweden)
acia-LKB) Activated CH-Self-Allose 4B
(Activated CH-Sepharose 4B)] was swollen in 100 ml of ice-cold hydrochloric acid (1 mmol / ml) for 15 minutes and washed with ice-cold hydrochloric acid of the same type to obtain a gel. After washing 10 g of this gel with a carbonate buffer solution (pH: 80) prepared by dissolving sodium hydrogen carbonate and sodium chloride in water at concentrations of 0.1 mol / and 0.5 mol / respectively, the same carbonate buffer solution And mouse IgG type ₁ monoclonal antibody HA-7
Suspended in a mixture with 20 mg. The suspension was stirred at 4 ° C. for 4 hours and then suctioned to obtain a gel. Immediately after suspension and after stirring for 4 hours, the concentration of the antibody in the supernatant of the suspension was quantified in the same manner as in Example 1, and as a result, the immobilization rate of the antibody on the carrier was 80%. There was found.

Blocking A carbonate buffer prepared by dissolving 10 g of the gel of the carrier on which the antibody was immobilized by the immobilization procedure described above in water at concentrations of 0.1 mol / mol and 0.5 mol / mol, respectively. After washing with liquid (pH: 8.0),
It was mixed with 50 ml of 0.1 mol / tris hydrochloric acid buffer solution (pH: 8). The mixture was stirred at room temperature for 1 hour and then suctioned to obtain a gel.

Washing 10 g of the gel obtained by the above-mentioned blotting operation was subjected to a washing operation up to the sixth time by the same washing method as in Example 1.

Reference Example 1 The same procedure as in Example 3 was repeated except that antibody immobilization and blocking were carried out.
g containing sodium chloride at a concentration of 0.5 mol /.
1 mol / acetic acid buffer (pH: 4) and 0.5% sodium chloride
This was washed three times in this order with 0.1 mol / tris buffer solution (pH: 8) contained at a mol / concentration.

Reference Example 2 1 g of the gelled physiologically active substance-immobilized adsorbent obtained in Examples 1, 2 and 3 and Reference Example 1 contains sodium chloride at a concentration of 150 mmol / 20 mmol /
Was mixed with 1 ml of the phosphate buffer (pH: 7.4) (hereinafter, the phosphate buffer containing sodium chloride is referred to as PBS), and the mixture was shaken at 37 ° C. for 3 hours. The resulting mixture was centrifuged. The concentration of mouse IgG type ₁ monoclonal antibody HA-7 in the obtained supernatant was determined by enzyme immunoassay.

That is, the IgG fraction of goat antiserum against mouse IgA, IgG and IgM (heavy chain and light chain) [US Organon
Organon Teknika Corpor
ation), goat anti-mouse immunoglobulins (IgA + IgG + IgM) (heavy and light
Chains Species, IgG Fraction (Goat
Anti-Mouse Immunoglobulins (IgA + IgG + IgM) (Hea
vy and Light Chains Specific), IgG Fraction)) 2
PBS containing 5 μg / ml was added to polyvinyl chloride microwell plates [Becton-Dickinson and Cnmpan
y), Falcon 3912, 96-well plate)
The antibody was adsorbed to the plate by dispensing 50 µ / well into each well and allowing it to stand overnight at a temperature of 4 ° C. After removing the solution from each well and washing the well with PBS, 300 μl of PBS solution containing 5% by volume of fetal bovine serum was prepared.
Each well was dispensed, and the solid phase surface on which the antibody was not adsorbed was blocked by standing at 37 ° C for 2 hours. After washing each well with a PBS solution containing 5% by volume of fetal bovine serum, 50 μl of the above supernatant (sample) was washed.
Each well was dispensed into each well and allowed to stand at a temperature of 37 ° C. for 1 hour, and then each well was washed with a solution of PBS containing 5% by volume of fetal bovine serum. Antibodies against mouse immunoglobulin labeled with horseradish peroxidase [Anti-mouse Ig, Peroxidase-l, manufactured by Amersham, UK
inked, Species-specific Whole Antibody)] is dissolved in a solution of PBS containing 5% by volume of fetal bovine serum at a concentration of about 2 μg / ml, and this solution is dispensed into each well at 50 μ / well, Let stand for 1 hour at temperature. After washing each well with PBS, a Tris buffer solution containing 1 mmol / mol of 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) and 0.0045% by weight of hydrogen peroxide ( pH: 7.4) was dispensed at 100 µ / well to each well, and the color was developed by shaking at room temperature for 30 minutes. The absorbance at wavelengths of 409 nm and 501 nm was measured for the solution in each well, and the difference in absorbance at both wavelengths was obtained.

On the other hand, in the above operation, the mouse IgG ₁ type monoclonal antibody was replaced by 10 ^-3 ng / ml to 10 μg / ml instead of the supernatant (sample) obtained after shaking the physiologically active substance-immobilized adsorbent in PBS. m
The same operation was performed except that PBS containing a known concentration within the range of 1 was used, and a calibration curve was prepared based on the obtained difference in absorbance. From this calibration curve, mouse Ig in the supernatant (sample)
The concentration of G ₁ type monoclonal antibody HA-7 was determined, and the amount of released antibody per 1 g of gel of the physiologically active substance-immobilized adsorbent was calculated based on the concentration. The results obtained are shown in Table 1.

[Effects of the Invention] According to the present invention, as is clear from the above examples, a physiologically active substance-immobilized adsorbent in which the amount of released physiologically active substance such as an antibody is extremely small can be produced by a simple operation. The production method of the present invention is easily applied to the kit for producing a physiologically active substance-immobilized adsorbent of the present invention.

[Brief description of drawings]

FIG. 1 is a cross-sectional view showing an example of a column that may constitute one element of the kit for producing a physiologically active substance-immobilized adsorbent of the present invention. Reference numeral 1 is a liquid inlet portion, 2 is a liquid outlet portion, 3 is a main body portion, and 4 and 5 are all supporting portions.

Claims (2)

[Claims] 1. A physiologically active substance is immobilized on a carrier having an active group by a covalent bond method, and the obtained physiologically active substance-immobilized carrier is optionally contacted with a blocking agent,
A method for producing a physiologically active substance-immobilized adsorbent, which comprises alternately washing with a buffer solution having a pH of 3.5 or less and a buffer solution having a pH of 5.5 or more. 2. A kit for producing a physiologically active substance-immobilized adsorbent, comprising a carrier having an active group, a buffer solution having a pH of 3.5 or less and a buffer solution having a pH of 5.5 or more.