JPS60130526A - Method for purifying antigenic substance - Google Patents

Abstract

PURPOSE:To obtain an antigenic substance corresponding to an immunological antibody in high purity and recovery, by treating the immunological antibody haing a specificity for the antigenic substance to be purified to give a modified purified antibody having a high specificity, and immobilizing the resultant antibody in an insoluble carrier. CONSTITUTION:An immunological antibody, e.g. blood plasma or serum of an immunized animal or monoclonal antibody, having specificity for an antigenic substance to be purified, e.g. urokinase, HBsAg, kallikrein or interferon, is treated with an enzyme. Pepsin, trypsin, amylase, arginase, plasmin, etc. are preferably used as the enzyme. The resultant antibody is then immonilized in an insoluble carrier, e.g. purified agar gel, cellulosic derivative, dextran polymer or glass particles, etc., and the aimed antigenic substance is obtained in high purity and recovery from crude antigenic substance with the immobilized carrier having a high affinity therefor.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for purifying an antigenic substance corresponding to an enzyme-treated antibody using affinity chromatography using the antibody as a ligand. For more details,
The present invention uses the mechanisms of antigens and antibodies to enzyme-treat immune antibodies specific to the antigen substance to be purified, thereby producing modified purified antibodies with extremely high specificity.
The present invention relates to a method for producing an antigenic substance with high purity and high recovery rate from a crude antigenic substance by immobilizing it on an insoluble carrier and using this immobilized carrier with high affinity.

For example, urokinase is a fibrinolytic enzyme that activates plasminogen present in the blood into plasmin. Therefore, by injecting this into the human body, it dissolves blood clots that have formed in blood vessels, so it is not only effective in treating cerebral thrombosis and other thrombosis, but also increases the therapeutic effect when used in combination with anticancer drugs, making its usefulness is expanding.

In particular, urokinase collected from human urine and purified is currently widely used as a medicine because it has almost no side effects.

Conventionally, urokinase purification has been carried out using silica gel, HytuloSupercell, zeolite acrylonitrile, barium sulfate, or various ion exchangers and gel filtration, as well as immunoadsorbents immobilized with anti-urokinase antibodies using the corresponding antibodies. ing.

In addition, hepatitis B surface antigen (HBsAg) has been highly purified,
The inactivated vaccine can be used as a hepatitis B vaccine.

As a method for purifying HBsAg, ammonium sulfate, aerosil, ion exchange resin, gel filtration, ultracentrifugation, and a method using an immunoadsorbent on which an anti-HBsAg antibody is immobilized using the corresponding antibody are used.

Therefore, the present inventors have investigated various purification methods that are better than these known purification methods, and have found that they have a purification efficiency that is superior to any of the known purification methods, are easy to operate, and are We have discovered a method for purifying crude antigenic substances that can be used repeatedly.

In other words, antibodies generally have other contaminant proteins, sugars, etc. bound to them, and even if antibodies are purified using normal purification methods, such as an anion exchanger, these impurities such as contaminant proteins and sugars cannot be cleaved. , cannot be disassembled.

However, if antibodies are treated with enzymes under appropriate conditions,
If these impurities can be removed and the treated antibody is used as a ligand for an adsorbent in affinity chromatography and used to purify the antigenic substance corresponding to the antibody, the antigenic substance can be recovered in high yield and purity. I discovered that.

The present invention was completed based on this new knowledge, and relates to a method for purifying an antigenic substance corresponding to an antibody by using a carrier on which an enzyme-treated antibody is immobilized as an adsorbent.

According to the present invention, antigenic substances can be obtained with high purity and high yield,
Moreover, in addition to being easy to operate, it is possible to improve the functionality of the insoluble carrier on which the immune antibody is immobilized and the biological stability (prevention of spoilage, generation of pyrogenic substances, etc.) during large-scale production.

There are no particular limitations on the antigenic substance to be purified in the present invention, as long as it can be used to immunize animals to produce antibodies, such as urokinase, HBSAg, kallikrein, and interferon.

The antibody used in the present invention is an antibody corresponding to the above-mentioned antigen, and when subjected to enzyme treatment, it does not necessarily have to be a highly purified product, and may be, for example, plasma or serum from an immunized animal. Furthermore, monoclonal antibodies are also preferably used.

The enzyme used for the enzyme treatment is a proteolytic enzyme or a glycolytic enzyme, such as pepsin, trypsin, amylase, arginase, and plasmin. 2-It may be treated with one or more of these enzymes.

Enzyme treatment is carried out under individual water passage conditions, for example as follows.

For example, in the case of pepsin, trypsin, and plasmin, 0.1 μg to 1 mg of antibody depending on the antibody purity.
．． 9112-3.5 using 1-100 μg/ml
p115 for 10 to 30 hours at 25 to 37°C, using 0.1 μg to 100 μg of β-amylase.
．． 5 to 8.5, and treated at 25 to 37°C for 10 to 30 hours. In the case of arginase, add 0.1 μg to 100 μg, and treat at pH 7.5 to 10 for 10 to 30 hours at 25 to 37°C.
Process time.

After this enzyme treatment, the E 280 n is generally concentrated by dialysis, concentration and/or ultrafiltration and gel filtration.
The areas with high m are measured by the HAI method of anti-urokinase using the hemagglutination reaction method and pooled.

As a carrier for immobilizing the antibody thus obtained, any conventionally known immobilization carrier can be suitably used.

Specific examples include purified agar gel, cellulose derivatives, dextran polymers, gels of polymers such as polyacrylamide, and glass particles.

The antibody can be immobilized on a carrier by a conventionally known method [Cu-a
Method CP of trecasas et al., Cuatrec
asas, J., Biol.

Chem, 245.3059 (1970)]
, or a similar method.

According to this method, a pure product with extremely high specific activity can be obtained with a recovery rate of 85%.
% - its specificity is very good,
When using a polymeric urokinase antibody, 100%
A similar high molecular weight urokinase was recovered.

Similarly, when HBsAg was also used after enzyme treatment, high recovery and high purity recovery were obtained.

Examples are shown below, but the present invention is not limited thereto.

Example 1 Specific activity of urokinase purified by a known method, 80.0
00II/E290nm or higher, 100,000 to 200,000 units/ml and Freunds complete ad
Mix the juvant evenly.

This 1 ml was injected subcutaneously or subcutaneously into a 3-4 kg domestic rabbit, followed by booster immunization with 0.5 ml every 4-6 weeks, and then the anti-urokinase titer was determined by hemagglutination method (H
When the value increased, 50 ml of blood was collected from the ear vein of the rabbit at a time.

First, 12 g of ammonium sulfate was added to the anti-blood solution vr4100 II, and then 13 g was added to the centrifuged tube at pH 7.2, and the precipitate was obtained by centrifugation.

This precipitate was dissolved in a 0.04 M phosphoric acid solution containing 0.03 M common salt, pH 8.0, to an absorbance of around 2.0 at an absorbance of 82 Bon m.

Then, this solution was equilibrated with the same buffer and DEAE-
Cellulose was passed through a 70 ml column (3 cm in diameter, 1° in height) and the anti-urokinase moiety was extracted using the HAl method and E29Q.
Approximately 1 g of a highly purified anti-urokinase fraction was obtained as determined by nm.

Per 40 mg of this anti-urokinase fraction, pH 2.5
The reaction was carried out at 37°C for 18 hours at 1 μg/final concentration of pepsin, and then the pH was adjusted to 6.5 with 1N-NaOH, and then β-amylase was added at 5 μg/final concentration at 25°C.
Finally, 1 μg/final concentration of arginase was added at 9°2, treated for 18 hours at 25°C, and then left at room temperature for 1 hour in pHlo. 0. (dialyzed and concentrated with 11M phosphate buffer pH 8.5, gel-filtered with heptaphryl S-500 in the same buffer, and obtained approximately 0.4 g as an anti-urokinase fraction using the PHA method) I got it.

The B 290 nm of this enzyme-treated solution was set to around 5 and 0.
Approximately equal amounts of CNBr-activated 5epharose were diluted with 1°M sodium carbonate buffer pl+9.
4B (manufactured by Falcia) was added, and the mixture was slowly stirred at 4°C for 24 hours.

During this period, the initial HAI value changed from 1:2,000 to 1
The mixed liquid decreased to around 2, indicating that the coupling progressed smoothly.

After the reaction was completed, it was thoroughly washed with a 0.15M salt-added 0.02M boric acid laxative solution pH 8 to obtain enzyme-treated modified anti-urokinase-immobilized 5epharose 4B.

Specific activity 3,000 units/E 2B per 1ml of this product
0 nm crude urokinase can adsorb approximately 300,000 units, and after washing with the same buffer, 0.5
Elution was performed with a 0.17M glycine m tMj solution containing M sodium chloride, and a specific activity of 85,000 units/E 2 go nm or more and a recovery rate of 95% or more were obtained.

Similarly, specific activity i, ooo units/E 280 n
About 200,000 units of tissue culture urokinase of m were also adsorbed, and the specific activity was 100,000 units/E2B6nm.
A recovery rate of 95% or more was obtained.

In addition, purification using anti-urokinase-immobilized 5epharose 4B using an antibody without enzyme treatment resulted in poor adsorption rate, and in particular, the increase in specific activity was 50,000 units/E2QO.
The recovery rate did not exceed 80%.

Example 2 Using the enzyme-treated modified anti-urokinase immobilized 5epharose 4B prepared in Example 1, this product 11
Approximately 10 per 111 of culture medium of tissue culture urokinase
．． 000 units and has a specific activity of 9.
Recovery rate of 0,000 units/E2gQnm or more is 95
%; Note that purification using anti-urokinase-immobilized 5epharose 4B using antibodies without enzyme treatment resulted in poor adsorption;
In particular, the increase in specific activity was 75,000 units/E2o
.

The recovery rate was 75% at nm.

Example 3 HBsAg positive plasma was purified by a known method using reverse passive hemagglutination (RP HA) with a purity of 1:4,000 and a specific activity of E 286 nml: 250,000, and an equal amount of Freunds complete 'a
Mix with djuvant and add 0.1ml of this to 250ml each.
~300 g of guinea pigs were injected subcutaneously or subcutaneously, and then boosted in the same manner with 0.1 ml for 5 weeks, followed by anti-H
Bs titer was measured by hemagglutination method (PHA method), and when the value rose, the entire amount of blood was collected.

Thereafter, it was purified in the same manner as in Example 1 to obtain enzyme-treated modified anti-HBs-immobilized 5epharose.

1 ml of this product can adsorb HBSAg with a specific activity of 1:3,000 in RP HA per m of E2Bo, and a titer of 1:240,000, and after washing and elution, the specific activity is E2Bo. 1 per nm
: 320,000 or more, indicating a recovery rate of 96% or more.

In addition, anti-HBs immobilization 5e with antibody without enzyme treatment
In purification with pl+arose 4B, the adsorption rate was poor, especially the increase in specific activity did not exceed 1:8,000, and the recovery rate was about 75%.

Example 4 Anti-HBs monoclonal antibody 17 purified by a known method
1 HAI method Te 1: 1,600,000, specific activity T
:I per Ez8゜nm: 55,000 cultivation! #:
The solution was purified by the method of Example 1, and anti-HBs immobilized at 5eph.
arose 4B was prepared.

1ml of this product can adsorb HBsA g with a titer of 1:300,000 per Ezaon'm as a specific activity, and after washing and elution, the specific activity is per E2aOnm. 1:35
,000 or more, indicating a recovery rate of 95% or more.

In addition, anti-HBs fixation 5ep with antibody without enzyme treatment
In the purification using l+arose 4B, the adsorption rate was poor, especially the increase in specific activity did not exceed 1:12.000, and the recovery rate was about 73%.

Patent Applicant: Midori Juji Co., Ltd. Letter of amendment, stamped on March B, 1980, 1, Indication of the case: Patent Application No. 237636, filed in 1982, 2, Name of the invention: Method for purifying antigenic substances 3, Review case for amendment Relationship with Patent Applicant Name Mitri Juji Co., Ltd. 4, Agent ■541 Address 406 New Life Hirano-cho, 4-53-3 Hirano-cho, Higashi-ku, Osaka Phone: (06) 227-1156 6.1ili Masano Column 7 of “Detailed explanation of the invention” of the subject specification, contents of amendment (1) r 1 on page 10, line 14 of the specification: 250
,000 J to r]: Correct it to 25,0OOJ.

Method (i1°゛)゛・, 1゛i・discount:

Claims (1)

[Scope of Claims] A method for purifying an antigenic substance corresponding to an antibody, characterized in that a carrier on which an antibody treated with a fil enzyme is immobilized is used as an adsorbent. (2) The purification method according to claim (1), wherein the antigenic substance is urokinase or hepatitis B surface antigen (HBSAg). (3) The purification method according to claim 1 or 2, wherein the enzyme is at least one selected from pepsin, trypsin, amylase, arginase, and plasmin.