JPH0662680B2 - Method for producing carrier for immobilizing antibody - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention immobilizes antibodies to maximize antibody activity,
The present invention relates to a method for producing an antibody, which is suitable for applications such as affinity chromatography, for efficiently separating and purifying a target antigenic substance.

[Prior Art] It has been well known that a dialdehyde such as glutaraldehyde is used by utilizing an amino group of chitosan in order to immobilize an antibody using chitosan. The use of a carbodiimide reagent as a peptide bond is also disclosed in Japanese Patent Publication No. 53-10150. The present inventors
Japanese Patent Application No. 62-92623 has previously proposed a method for obtaining a carrier for immobilizing a physiologically active substance by treating a porous granular chitosan derivative having a carboxyl group with dicyclohexylcarbodiimide and N-hydroxysuccinimide.

[Problems to be Solved by the Invention] The carrier prepared by treating the above-mentioned porous granular chitosan derivative having a carboxyl group previously filed by the present inventors with dicyclohexylcarbodiimide and N-hydroxysuccinimide is A physiologically active substance such as an enzyme can be easily immobilized without denaturation, and non-specific adsorption after immobilization does not occur, so that it can be effectively used. However, when a substance such as an antibody having a molecular weight of more than 150,000 is immobilized, the immobilized antibodies are sterically hindered and the antibody activity cannot be sufficiently exhibited. This is because the density of the immobilized physiologically active substance cannot be controlled. Therefore, the reaction site of the antibody is not immobilized in an exposed form so that it can effectively react with the antigen, so that the recovery amount of the antigenic substance to be separated using the antibody-immobilized carrier is significantly reduced.

An object of the present invention is to solve the above-mentioned drawbacks and to obtain a good carrier for antibody immobilization by controlling the introduction amount of the active group so as not to cause steric hindrance between the antibodies.

[Means for Solving Problems] In the present invention, granular porous chitosan is partially acetylated with acetic anhydride, then treated with succinic anhydride, and the exchange capacity is controlled by the amount of acetic anhydride. The carrier for antibody immobilization is obtained by treating with N-hydroxysuccinimide.

The granular porous chitosan used in the present invention was previously disclosed in
It can be obtained by the method disclosed in 61-40337.
The chitosan used in the present invention is not particularly limited, but it is preferable to use low molecular weight chitosan having an average molecular weight of 10,000 to 230,000. Chitosan is dissolved in an aqueous solution of acetic acid, dichloroacetic acid, formic acid or the like alone or in a mixture to obtain an acidic aqueous chitosan solution. The concentration of the chitosan acidic aqueous solution is 2 to 20%
Is preferred. The chitosan acidic aqueous solution is sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate,
A granular aqueous chitosan is obtained by dropping it into a basic aqueous solution containing an alkaline substance such as ammonia or ethylenediamine under pressure from the discharge hole at a fixed amount to coagulate and regenerate it, and sufficiently wash it until it becomes neutral. At this time, a polar solvent such as alcohol may be used in combination with the basic aqueous solution, and if necessary, the granular porous chitosan may be subjected to a crosslinking treatment with an organic diisocyanate compound or the like in a polar solvent.

The granular porous chitosan obtained as described above is treated with acetic anhydride for acetylation. The amount of acetic anhydride is adjusted to control the density of amino groups, and the desired carboxyl group is introduced with succinic anhydride. Measure the introduction of active groups. After that, wash thoroughly with water until neutral. The amount of the active group introduced at this time is preferably 5 to 140 μmol / ml granular material. When the density of active groups is 5 μmol / ml or less, the amount of immobilized antibody is small,
This is disadvantageous in that only the capacity becomes large. On the other hand, above 140 μmol / ml granules cause steric hindrance to the immobilized antibody, resulting in a drawback that the antibody activity is not sufficiently exhibited.

The granular porous chitosan introduced with a carboxyl group obtained as described above is treated with a polar solvent such as dioxane,
After suspending in dimethylformamide, dimethylacetamide, dimethylsulfoxide, tetrahydrofuran or the like, a binder such as carbodiimide such as dicyclohexylcarbodiimide and N-hydroxysuccinimide are added, and the mixture is stirred and reacted at room temperature for 24 hours. After completion of the reaction, an excellent carrier for antibody immobilization can be obtained by thorough washing with the polar solvent used.

The antibody used for the antibody-immobilizing carrier of the present invention includes
All monoclonal and polyclonal antibodies whose antigens are the target substances to be purified by affinity chromatography can be used.

[Examples] Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to the scope of the Examples.

The active group density and the specific surface area were determined by the following methods.

(A) Active group density Approximately 50 ml of sample is treated in 500 ml of 1N-HCl with gentle stirring for 1 hour and washed thoroughly with deionized water until it becomes neutral so that carbon dioxide gas in the air is not absorbed. Accurately and quickly weigh 30 ml of the dehydrated sample carefully, and use 1/5 N-NaOH
Add to 500 ml and leave for 5 hours with gentle stirring. This supernatant is used as a test solution. Exploit 10 ml of this,
The methyl red solution was used as an indicator and was neutralized and titrated with 1/10 N-HCl to obtain the value by the following formula.

a: Amount of 1/10 N-HCl required to neutralize 10 ml of the test solution b: Amount of 1/10 N-HCl required to neutralize 10 ml of 1/5 N-NaOH before adding the sample f: 1 / 10 N-HCl titer (B) Specific surface area BET method was used to measure the specific surface area.

Example 65 g of chitosan having a deacetylation degree of 77% and an average molecular weight of 42,000
Was dissolved in 935 g of a 3.25% acetic acid aqueous solution, and the solution was dropped into a mixed solution of 7% sodium hydroxide, 30% ethanol and 63% water from a nozzle having a hole diameter of 0.15 mm and coagulated and regenerated. , Washed with water until neutral, average particle size 0.3m
A mφ granular porous chitosan 0.9 was obtained.

100 ml (wet state) of each of the obtained granular porous chitosan
The mixture was replaced with 00 ml of ethanol four times to remove water. Then, add 100 ml of ethanol to each, and add 1.9 g and 2.6 g of acetic anhydride.
4.0 g, 6.4 g, 10.0 g and 12.8 g were added and the reaction was carried out at room temperature for 24 hours. Then, add 100 ml of 1N-sodium hydroxide and add 1
After stirring for a period of time, the mixture was thoroughly washed with water until it became neutral, replaced with 100 ml of ethanol 4 times again, 100 ml of ethanol and 10 g of succinic anhydride were added, and the mixture was reacted at room temperature for 24 hours. After treatment with 100 ml of 1N-sodium hydroxide, 100 ml of 1N-hydrochloric acid was added, and the mixture was stirred for 1 hour and washed thoroughly with water until neutral. The active group densities of the obtained chitosan granules were measured and found to be 140 μmol / ml granules, 66 μmol / ml granules, 55 μmol / ml granules, 37 μmol / ml, respectively.
They were ml granules, 25 μmol / ml granules, and 15 μmol / ml granules.

Then, 100 ml of dioxane was added to each of the above 6 samples.
After replacement with twice with 100 ml of dioxane, 5.0 g of dicyclohexylcarbodiimide and 2.5 g of N-hydroxysuccinimide were added, and the mixture was stirred at room temperature for 24 hours. Then 100 ml of dioxane twice, 100 ml of methanol twice, dioxane 10
The sample was washed twice with 0 ml and twice with isopropyl alcohol to obtain 80 ml of the antibody-immobilizing carrier for each sample. Measurement of the specific surface area of each 69.0m ² /g,73.0m ² /g,85.0
was ^{m 2 /g,63.3m 2 /g,91.5m 2 /g,80.5m 2} / g.

Application Example A carrier for antibody immobilization obtained from those having an active group density of 15 μmol / ml granules obtained in the above Example, 3 ml stored in isopropyl alcohol was collected, and isopropyl alcohol was removed by centrifugation. It was washed with pure water. 0.3M immediately
6 ml of a 7 mg / ml monoclonal anti-lactoferrin antibody solution dissolved in 0.2 M sodium hydrogen carbonate containing sodium chloride was added, and the mixture was gently stirred overnight at 4 ° C. Then, after removing unreacted antibody solution by centrifugation, it was repeatedly washed twice with 0.2 M ethanolamine-hydrochloric acid solution (pH 8), and the same solution 6
After adding ml, the mixture was stirred at 4 ° C. for 2 hours and washed with a phosphate buffer to obtain an antibody-immobilized carrier. When the amount of immobilized antibody was determined from the protein concentration before and after the reaction, the amount immobilized was 7.4 mg antibody / ml carrier. Hold this at 4 ° C, fill 3 ml of the obtained antibody-immobilized carrier into a column with a diameter of 1 cm and a length of 5 cm, and wash thoroughly with 0.02 M phosphate buffer (pH 7.2) containing 0.15 M sodium chloride. 20 ml of a 0.1% lactoferrin solution dissolved in the above-mentioned phosphate buffer was flowed at a flow rate of 2 ml / min.

The carrier was thoroughly washed with the above-mentioned phosphate buffer, and the adsorbed lactoferrin was eluted with 0.2 M acetate buffer (pH 3.5) containing 0.15 M sodium chloride. The volume of the recovered lactoferrin solution was immediately measured, and the protein concentration was measured with a protein measurement kit (manufactured by Bio Rad). As a result, 8.4 mg of lactone was recovered. If the reaction rate is calculated by dividing the amount of recovered lactoferrin by the amount of immobilized antibody (8.4
/7.4 x 3) x 100 = 37.9%. Similarly, the active group density is 140μmol / ml carrier, 66μmol / ml carrier, 55μmol / ml
The same procedure was performed for the antibody-immobilized carriers obtained from the carrier, 37 μmol / ml carrier, and 25 μmol / ml carrier, and the reaction rates were determined to be 23%, 29%, 32%, 35%, and 37%, respectively. there were.

Comparative example Detoxification degree 77%, average molecular weight 42,000 chitosan 65g
Was dissolved in 935 g of a 3.25% acetic acid aqueous solution, and the solution was dropped into a mixed solution of 7% sodium hydroxide, 30% ethanol, and 63% water from a nozzle having a hole diameter of 0.15 mm to coagulate and regenerate the medium. Washed with water until it becomes stable
Granular porous chitosan 0.9 was obtained. 50 ml of the obtained granular porous chitosan (wet state) was added to dimethylformamide 50.
After washing by displacement twice with ml, 50 g of a dimethylformamide solution containing 0.5 g of adipic acid active ester was added and wetted overnight. Then, it was washed 4 times with 50 ml of dimethylformamide, 1.0 g of succinic anhydride was added and allowed to infiltrate overnight, and washed 4 times with methyl alcohol to obtain granular porous chitosan having a carboxyl group. After that, 5.0 g of acetic anhydride was added and allowed to infiltrate overnight, the amino groups were acetylated to remove the alcohol, 50 ml of 1N sodium hydroxide was added, and the mixture was wet for 1 hour and washed until neutral. The active group density of this granular material is 35.
0 μmol / ml granular material, which was suspended in 100 ml of dioxane, added with dicyclohexylcarbodiimide and N-hydroxysuccinimide so that both of them became 0.1 M, and allowed to infiltrate overnight, and washed thoroughly with dioxane and methyl alcohol to obtain a ratio. Surface area
40 ml of an antibody immobilizing carrier of 85 m ² / g was obtained. The monoclonal anti-lactoferrin antibody was immobilized in the same manner as in the application example, and the reaction rate was determined to be 16%.

As shown in this comparative example, if the density of the active groups is large, the reaction rate is lowered and it becomes substantially unsuitable for use.

[Effects of the Invention] The present invention controls the density of amino groups in advance with acetic anhydride in porous granular chitosan as described above, and introduces a carboxyl group with succinic anhydride, thereby making the active group density 5 to 140 μm.
Since it can be controlled to be ol / ml particles, there is an effect that an antibody immobilizing carrier suitable for affinity chromatography, which is excellent in immobilizing an antibody having a large molecular weight, can be obtained.

Claims (1)

[Claims] 1. A method for producing a carrier for immobilizing an antibody, which comprises partially acetylating granular porous chitosan with acetic anhydride, then treating with succinic anhydride, and further treating with dicyclohexylcarbodiimide and N-hydroxysuccinimide. .