JPS5853757A - Adsorbent for immuno-affinity chromatography and manufacture of same - Google Patents

Abstract

PURPOSE:To obtain an adsorbent for an affinity chromatography with a strength enough to withstand repeated use thereof, by immobilizing an antibody used as ligand on a carrier with a high yield without lowering the activity thereof. CONSTITUTION:An antibody as ligand is oxidized by periodic acid or a salt thereof to cleave a glycol unit in the sugar chain moiety to form an aldehyde group. Then, the antibody is bonded to an immobilizing carrier having an amino group or a hydrazide group via a Schiff's base formation reaction and the Schiff's base is reduced by a reducing agent such as sodium borohydride to stabilize it. The oxidation by periodic acid occurs between adjacent hydroxyl groups of a glycol, a reaction under which 1mol of IO4<-> is reduced to IO<-> while 2mol of aldehyde groups are generated. Structurally, the product is obtained by cleavage of a glycol unit followed by oxidation of the resulting adjacent hydroxyl groups. A relationship between the adsorption level of this adsorbent and the repeated use thereof is as illustrated, indicating no lowering of the adsorption capacity.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an adsorbent for immunoaffinity chromatography that has durability that can be used repeatedly, and a method for producing the same.
More specifically, the present invention relates to an adsorbent for immunoaffinity chromatography that uses an antibody as a ligand and is immobilized on a carrier in high yield without reducing its activity, and a method for producing the same.

Affinity chromatography, which utilizes biological specificity such as enzyme-substrate reactions and antigen-antibody reactions, is an excellent method for separating and purifying specific proteins. , by Yamazaki et al., published by Kodansha, “Affinity Chromatography” and rMethods in En
ZymologyJ 34.1974゜Academi
It is described in detail in c Press etc.

In addition, immunoaffinity chromatography uses antigen-antibody reactions as a specific reaction, and is used to isolate, purify, and quantify trace amounts of active ingredients contained in living organisms with high purity, as well as perform various types of rapid and quantitative analysis and clinical diagnosis. It can be used for systems, etc., and is useful, for example, as described in Sasaki's "Chemistry and Biology" magazine [IO], page 658.

In affinity chromatography, ligand immobilization methods are broadly classified into carrier binding method, crosslinking method, and entrapment method.
For practical use, especially in mm on an industrial scale, it can be said that the covalent bonding method, which belongs to the carrier bonding method, is the most excellent method.

A specific example of the covalent bonding method is a method in which agarose, a multipotent substance contained in agar, is used as a carrier, activated with cyanogen bromide, and then bonded with the amino group of the ligand (as described by Chibata et al. Known methods include "Affinity Chromatography" (Kodansha) and a method in which the amino group of ω-aminoalkylamine-agarose, which is a derivative of agarose, and the carboxyl group of a ligand are bonded to a peptide using a carbodiimide reagent. (Chibata et al. "Affinity Chromatography" Kodansha) However, even in these methods, active functional groups such as amino groups and carboxyl groups in the polypeptide are used to bind biologically active proteins such as antibodies, enzymes, and hormones. However, it is not practical because it has negative effects such as a decrease in the expression of biological activity of the immobilized protein.

In addition, 71hler and Milstein reported that the lymphocyte cell fusion method introduced in the field of immunology has made it possible to collect large amounts of highly pure monoclonal antibodies with specificity. .

(Nature, 256.495.1975) Conventional antibodies are obtained from the serum or ascites of immunized animals and are used as they are, or after being purified using a cold ethanol method, etc., and are a mixture of antibodies with different specificities. It has been difficult to produce antibodies with constant quality such as antigen specificity and antibody titer. However, by using the above cell fusion method, monoclonal antibodies can be
Currently, they are produced for various antigenic determinants such as polysaccharides, lipids, enzymes, cell surface antigens, viruses, haptens, etc., and are commercially available and easy to obtain or produce.

Monoclonal antibodies can be obtained with higher specificity than antibodies obtained from conventional antisera, and can be used for antigen purification, immunoassays, etc.; If an adsorbent was prepared, it would be expensive, and highly specific antibodies would be lost during the treatment process.

Biologically active proteins are often glycoproteins, but it is generally believed that sugar chain moieties are not directly related to biological activity.In particular, antibodies are glycoproteins, and the bonding positions of sugar chains have not been elucidated. ing.

For example, IgG, the main human antibody, has sugar chains in the constant region (
It is located in the CH2 domain of the constant region) and is known not to be directly involved in antigen recognition or complement fixation. Therefore, JP-A-52-104591 discloses a method for chemically modifying and bonding sugar chains in antibodies.

The present inventors attempted to apply an adsorbent formed by bonding an antibody modified with a sugar chain site to a carrier for affinity chromatography, but the method disclosed in JP-A-52-104591 did not When chemically modified, the modified site and the active group of the polypeptide site react simultaneously, resulting in self-association, etc., which inhibits antibody activity or significantly reduces the antibody immobilization yield. there were.

In immunoaffinity chromatography, when immobilizing expensive monoclonal antibodies, improving the immobilization efficiency, maintaining antibody activity, and ultimately improving the antigen recovery rate are important conditions that determine the success or failure of industrial application. Furthermore, in affinity chromatography, it is necessary to fill a column and use it repeatedly, so it is essential to have strength that can withstand repeated adsorption and desorption and a suitable particle size.

The present inventors have conducted extensive research to satisfy the above conditions, and researched conditions for chemically modifying sugar chains and carriers that can withstand repeated use in order to improve immobilization efficiency and prevent decreases in antibody activity. As a result, we have completed a practically usable adsorbent for immunoaffinity chromatography and a method for producing the same. That is, the present invention involves oxidizing the antibody as a ligand using periodic acid or a salt thereof under conditions of pH 1 to 5 to form an aldehyde group on the sugar chain moiety, and then immobilizing the antibody with an amino group or a hydrazide group. The Schiff base is brought into contact with a carrier for use in the reaction, and a Schiff base is formed and bonded, and the Schiff base is reduced and stabilized using a reducing agent such as sodium borohydride.

According to the present invention, an antibody adsorbent having a high immobilization yield and antibody activity as well as strength enough to withstand repeated use can be obtained, making affinity chromatography possible on a practical and industrial scale.

The present invention will be explained in detail below.

In the present invention, an antibody as a ligand is oxidized with periodic acid or a salt thereof to cleave the glycol moiety of the sugar chain moiety to form an aldehyde group, and then brought into contact with an immobilization carrier having an amino group or a hydrazide group. They are bound by a Schiff base reaction and stabilized by reducing the Schiff base with a reducing agent such as sodium hydride.

Oxidation by a periodate trap occurs between adjacent hydroxyl groups of glycol, and is a reaction in which 1 mole of 104- is reduced to IO3- and 2 moles of aldehyde groups are generated at the same time.

The product of this reaction varies depending on the type of glycosidic bond, but the glycol moiety is cleaved and the adjacent hydroxyl group is oxidized, yielding a product with the structure shown in the following formula.

C120HCH20H The formation of Schiff's base largely depends on the reaction PR, and the pH at which Schiff's base formation reaches its maximum varies depending on the basicity of the amino group-containing compound, but in the case of antibodies, formation is large near neutrality; (Immunochemiatry15.52
3 ("781) This can also be determined from the following experimental example.

Experimental Example 0: Human IgG (Seikagaku Kogyo Co., Ltd., 13 mgf) was dissolved in 10 ml of 1 M buffer, 1 mg of NaIO was added, and the mixture was reacted for 30 minutes at room temperature.
Ark and Lubs buffers, PH4-6 are acetate buffers and PH6-8 are phosphate buffers.

Next, ethylene glycol was added to a final concentration of 0.1 M, and 4B? The reaction solution was reacted for a period of time, and the optical density (OD 660 nm) of the reaction solution was measured and is shown in FIG.

As can be seen from Figure 1, 4 degrees is large near neutrality, and IgG
This is thought to be due to a self-association reaction involving the amino groups of the molecule. This is because a Schiff base is formed between the aldehyde group in the sugar chain of the IgG molecule and the amino group in the polypeptide during the oxidation reaction, resulting in the self-association of h molecules. Therefore, the oxidation reaction of the saccharide of the antibody with periodic acid or its salt is preferably carried out at a pH at which the formation of Schiff bases between antibody molecules, which is a side reaction, is suppressed. Japanese Patent Publication No. 52-104
In the method of No. 591, the oxidation reaction of sugar chains is carried out at around PH6, but in the case of antibodies, a self-association reaction of antibody molecules occurs, which has the disadvantage that antibody activity and immobilization yield are significantly reduced. The present invention enables the oxidation reaction of antibody sugar chains with periodic acid or its salts to be carried out at a pH that suppresses the self-association reaction between antibody molecules, which is a side reaction during the oxidation reaction. It is possible to obtain a carrier that significantly improves antibody binding efficiency and has a high antigen recovery rate.

Periodic acid or a salt thereof used for oxidizing sugar chains is used in an amount of about 10,000 to 100,000 to 100,000 times the molar amount of the antibody. When using 100 times the molar amount or more of the antibody, a reaction time of one hour or less is sufficient. Since periodic acid solution decomposes by 9,
It is better to carry out the reaction while blocking light, but if an excess amount of periodic acid solution is used and the reaction takes place within a few hours, there is no problem even if the reaction is not blocked from light.

In order to maintain a specified pH, a buffer is generally used, but any buffer can be used except those containing functional groups that react with aldehyde groups formed in saccharide chains during oxidation reactions. It is. However, it may be better to avoid using phosphate buffer as it reacts with periodic acid. The concentration of the buffer solution is 0.001 to LM, preferably 0.001 to LM.
It is preferable to use it at 01 to 0.5M. P of buffer solution
H is PH 1 to 5 in order to suppress side reactions when the sugar chain of the antibody is oxidized with periodic acid or its salt, but it is preferably as low as PH 1 to 5 so as not to cause acid denaturation of the antibody molecule.
．． It is 5 or less, more preferably 2 to 4.

If the pH is below 1, the acid denaturation of the antibody molecules will be significant, and if the pH is above 5, a self-association reaction of the antibody molecules will occur as a side reaction, which is not preferable.

The oxidation and expansion temperature of the sugar chains of the antibody is 0 to 60°C, preferably 5 to 20°C. The antibody is inactivated at temperatures above 60'(:') and does not react at temperatures below 0°C.

In addition to periodic acid or its salt, the oxidizing agent includes acid potassium, chlorine, nitric acid, etc., but periodic acid or its salt provides the best results.

Excess oxidizing agent remaining after the oxidation reaction must be removed to avoid excessive oxidation of the sugar chain moiety and the effect of the oxidizing agent on the carrier. Ethylene glycol, glycerin,
Chemicals that can be oxidized by an oxidizing agent, such as glucose or sodium sulfite, can be added or removed by dialysis against the same buffer used in the oxidation reaction, or by other conventional methods.

When an antibody having an aldehyde group in its sugar chain is bound to a carrier having at least one reactive amino group or hydrazide group, the PR of the reaction is 5 to 8, preferably PH 6 to 7. Outside this pH range, the main reaction, the formation of a Schiff base between the aldehyde group in the sugar chain of the antibody and the amino group or hydrazide group of the carrier, is suppressed. The antibody and carrier are allowed to coexist in a buffer solution with a pH of 5 to 8, preferably 6 to 7, and a pH of 0 to 8.
By reacting at 25° C. for several hours to several days, the antibody and the carrier are bound by a thick base reaction. However, the thick bases formed are unstable and easily decompose under acidic or alkaline conditions. In immunoaffinity chromatography, antibody-bound carriers are used repeatedly, and the elution of adsorbed proteins is often performed under acidic conditions.Therefore, it is necessary to stabilize the thick base between the antibody and the carrier. This is done by reduction as shown in the following formula.

Reduction R1CH=NR2-R1CH2-NHR2 (7 Tuff bases) (R,; antibody, R2; carrier) The reduction of the thick base between the antibody and the carrier is performed using sodium borohydride (Na
BH4) or sodium cyanoborohydride (NaBH
The reaction is carried out using 3CNI at θ° to 25°C and pH 5 to 10 for several tens of minutes to several tens of hours.

As a reducing agent, monohydric acid, cyanohydrogenated fluorine, etc. ! In addition to -, examples include hydrogen iodide, hydrogen sulfide, aluminum hydride, lithium, sulfite, sodium sulfide, ammonium sulfide, etc., with sodium borohydride or sodium cyanoborohydride being the most preferred.

In addition to derivatives of porous cellulose beads having at least one reactive amino group or hydrazide group, examples of immobilization carriers include aminated or hydrazide polystyrene, crosslinkable aminated or hydrazide poval, aminated or hydrazide Examples include aminated or hydrazidized dextran, aminated or hydrazidized glass beads, etc.

Generally speaking, synthetic polymers with sufficient physical strength tend to cause non-specific adsorption due to hydrophobicity as supports for affinity chromatography, glass beads have poor ligand adsorption efficiency, but porous cellulose beads It has sufficient physical strength, has no non-specific adsorption, has a large surface area, has high adsorption efficiency, and is inexpensive and dust-free.
It is one of the most suitable carriers for the present invention.

Derivatives of porous cellulose beads having at least one reactive amino group or hydrazide group are commercially available products such as aminehexylcellulose (AH-cellulose, manufactured by Merck & Co., Ltd.), carbomethoxycellulose hydrazide (CM-
Cellulose hydrazide manufactured by Merck & Co., Ltd.] may be used,
Further, derivatives may be prepared and used from porous cellulose beads. Examples of methods for preparing derivatives from porous cellulose beads include, for example, a method of epoxidizing porous cellulose beads and further aminating them with ammonia (synthetic organic chemistry, Volume 38, No. 2 128 (= 80 )) or activated with cyanogen bromide and general 2NH2 (CH2) nN
A method for obtaining ω-aminoalkylamine-cellulose by combining diamines represented by H2 (Kosasa et al., "Affinity Chromatography J Kodansha)" or a method in which cyanogen bromide-activated porous cellulose beads are combined with the general formula NH2NHc.
There is a method of obtaining a hydrazide derivative by combining lercihydrazides such as o(CH2)ncoNHNH2. The beads according to the present invention preferably have a particle size of 5° to 1000 μm and a pore size of 5° to 100°, but are not limited to these ranges.

These carriers may be those that satisfy the above conditions,
Commercially available products may be used, or those specially prepared to be more suitable for the purpose of the present invention may be used. (Biot
echnol, Bioeng, 18.1507 (Theory 76)) Antibodies used in the present invention can be prepared by known methods. Antiserum obtained from immunized animals and monoclonal antibodies obtained by cell fusion method. In either case, it is used after being purified by ammonium sulfate salting out or cold ethanol precipitation, but it is preferably used after being further purified by column chromatography such as DEAE-cellulose or gel filtration.

As detailed above, the present invention oxidizes sugar chain moieties that are not directly involved in the specific reaction of antibodies and binds them to a carrier via a -cH2-NH- or -CH2-NH-NU-CO- structure. However, it is necessary to control the reaction conditions for binding to prevent antibody inactivation and increase binding efficiency, while maintaining physical strength that can withstand repeated use and high antigen recovery rate. It is something.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

In addition, in the following examples, the antigen recovery rate was measured by the following method.

1) Antigen radioactive iodine (126I) labeled chloramine T
The pellets were labeled with 125 µ according to the method (Nature, 194.495 ('62)) and purified by gel filtration.

2) Measurement of antigen-bound amount of soluble antibody Add a constant amount of antigen Jt (125I-labeled antigen solution in a diluted series) in an equal volume, stir well, let stand for a certain period of time, centrifuge, and irradiate the supernatant or precipitate. The activity was measured and the maximum amount of antigen that could be bound was determined.

(Antigen concentration, antibody concentration, and standing time were determined by preliminary experiments.) 3] Measurement of antigen binding amount of adsorbent (immobilized antibody) Fill a column with adsorbent, supply antigen labeled with 12J, and perform affinity chromatography. The radioactivity of the adsorbed antigen was measured to determine the amount of antigen bound to the adsorbent.

4) Calculation of antigen recovery rate It was calculated as the ratio between the amount of antigen adsorbed in the actual column reaction (X) and the maximum amount of antigen (Yl) that can be bound by the soluble antibody used for tabulation of the adsorbent.

That is, antigen recovery rate = X x , i D, Q-4%
).

Example 1-1 Preparation of aminated porous cellulose beads Porous cellulose beads (Seikagaku Corporation, Cellulofine 700m
l1g (solid content J to distilled water 8m42NNaOH21ml
, 6mt of shrimp chlorhibirif was added one after another, and 3
The reaction was carried out for a period of time to obtain epoxidized porous cellulose beads having epoxy groups of sso μmote/g (solid content). Furthermore, the epoxidized porous cellulose beads were reacted with concentrated ammonia water at 40°C for 2 hours to obtain sOOp.
Total mote/g (solid content) of aminated porous cellulose beads having amine groups were obtained.

The epoxy group can be quantitatively determined using a titration method using hydrochloric acid (J, C
chromatogr, 90.87 ('740), the method of Inman and Pintzia (Bioclemistry 几4074 + '69)
) according to ).

Example 1-2 Introduction of an aldehyde group into anti-bovine albumin antibody Purified anti-bovine albumin antibody was converted into rabbit antiserum (Fuji Organ)
It was prepared by salting out ammonium sulfate.

This purified anti-bovine albumin antibody 15mg f, 10Tn
Dissolved in 0.1 M acetate buffer (PH3,7) at t and N
0.1 mg of al was added, and the mixture was allowed to react at room temperature for 30 minutes. Next, ethylene glycol was added to a final concentration of 0.1 M, and the mixture was reacted at 4°C for 4 hours. Furthermore, this reaction solution was incubated at -4°C for 18 hours with 0.1 M acetate buffer (PH
3,7) Dialyzed against 1t.

Example 1-3 Binding of aldehyde group-introduced anti-bovine albumin antibody to aminated porous cellulose beads Add 0.4g (solid content) of cellulose beads and make 0.1M
The pH was adjusted to 1 to 6 using sodium carbonate buffer (PH93. After reacting overnight at 4°C, 45 mg of NaBH was added to the formed Schiff base and incubated at 4'C for 5 hours in lP.
It was reduced and stabilized by treatment under Has conditions. The amount of bovine albumin adsorbed by the anti-bovine albumin antibody-bound porous cellulose beads thus obtained was 9
At 15 mg, the antigen recovery rate was 91%.

For comparison, a purified anti-bovine albumin antibody not treated with NaIO47 was brought into contact with aminated porous cellulose beads in exactly the same manner as above, but no binding of the antibody was observed.

Comparative Example I Binding of anti-bovine albumin antibody to CNBr-activated agarose Commercially available Sepharose 4B (manufactured by Pharmacia) 8rrL
Transfer the sample onto a glass filter and add 200rrLt of distilled water.
After washing and suctioning, the solution was suspended in 20 mt of distilled water. While stirring, adjust the pH to 11-12 with tONNaOH, and add CN to this.
An aqueous Br solution (L 5 g CNBr150rrLt distilled water) was slowly added. During this time the pH was maintained at 11-12 by adding 1ON NaOH. The temperature of the reaction solution was controlled so as not to exceed 30'C. After the reaction is complete,
Filtered through a glass filter. Then 200m of cold distilled water
l s further cooled 0,25 M NaHCOalo
%CNBr-activated agarose was obtained by washing with orrLt.

0.4 g of CNBr-activated agarose obtained by boiling (
Solid content) was suspended in 20 mt of 0.25 M sodium carbonate buffer (containing α5 MN & C1 NaC1, 5) containing 15 mg of the purified anti-bovine albumin antibody prepared in Example 1, and reacted overnight at 4°C. The active groups remaining after the reaction were removed by treating with 0.2M glycine (PH&5)'t' at room temperature for 2 hours.

The amount of bovine albumin adsorbed by the thus obtained anti-bovine albumin antibody-bound agarose was 3 m
g, and the antigen recovery rate was 17 g.

Example 2-1 Purification of albumin in bovine serum using the affinity adsorbent for immunoaffinity chromatography prepared in Example 1 and Comparative Example 1 Experimental example Anti-bovine albumin antibody-bound porous cellulose prepared in Example 1 0.4 g (solid content) of beads and 0.6 g (solid content) of the anti-bovine albumin antibody-conjugated agarose prepared in Comparative Example 1 were placed in a column (1 mφ x 2.
．． 5crrI)k, 0.15mt of bovine serum was supplied from the top of the column, and 0.02M borate buffer (PH8,
0.15M NaCl) was allowed to flow down. After passing the same buffer as above until the OD280nm of the eluate from the column became 0.03 or less, a 2 M KSCN solution was passed through the column to elute the adsorbed bovine albumin. The results are shown in the table and FIG.

In addition, the protein amount was measured according to the method of Lowry.

(Lowry, O,)L, Rowebrough
, N., J., Fall.

A, L, and Randall, R, J, J
our own of Biological Chemistry
The same applies to Fig. 4 (Stry 193265 (1951)).

In addition, in Comparative Example 1, purification was impossible when the flow rate exceeded 10 mt/d・h, whereas in Example 1, the flow rate exceeded 2 mt/d・h.
Good results were obtained even at 5 mt/d·h.

In addition, it was confirmed that the elution fraction with 0.2M glycine-hydrochloric acid buffer was bovine albumin by the Ophthalony method (New Experimental Chemistry Course 20-CI) - Biochemistry, Maruzen), and this elution fraction was analyzed with SDS. When analyzed by polyacrylamide electrophoresis, only an albumin band was shown.

Example 2-2 When the same column operation as in Example 2-1 was repeated, the anti-bovine albumin antibody-conjugated agarose prepared by the method of Comparative Example 1 showed a significant decrease in bovine albumin adsorption capacity after 5 uses. However, the anti-bovine albumin antibody-conjugated porous cellulose beads prepared by the method of Example 1
Even after 5 uses, no decrease in bovine albumin adsorption capacity was observed. The results are shown in FIG.

Comparative Example 2 Antibody oxidation reaction t-When carried out at PH5 or higher Example 1-
,．． In 2, purified anti-bovine albumin antibody Nal0
．． Oxidation with t0.1 M IJ acid buffer.

(PH6). That is, 10 ml, (D
15 mg of antibody in O1IMIJ acid buffer (PH6)
It was dissolved, 41 mg of NaIO was added, and the mixture was reacted at room temperature for 30 minutes.

At this point, the reaction solution became cloudy and self-association of antibodies was clearly observed. Next, add ethylene glycol to a final concentration of 0.1
M and reacted at 4°C for 4 hours. 0
．． 1 M IJ acid buffer (PH6) 1/4"
4 g (solid content) of aminated porous cellulose beads prepared by the method of Example i-1 after 18 hours dialysis at
Add NaBH45m and react overnight at 4°C.
The reduction treatment with g was carried out at 4°C for 5 hours under PH&50 conditions. The amount of bovine albumin adsorbed by the anti-bovine albumin antibody-bound porous cellulose beads thus obtained was 1 g.
(Solid content) was 1 mg, and the antigen recovery rate was 6.

Example 3-1 Preparation of hydrazide porous cellulose beads Porous cellulose beads (manufactured by Seikagaku Corporation, Cellulofine?0)
0 m) 1 ge Epoxidized by the method of Example 1-1, 5
Epoxidized porous cellulose beads with 50 μmate/g (solid content) of epoxy groups were obtained. Add this to a saturated solution of adipic acid dihydrazide (approximately 90 g/lt・
The suspension was suspended in 10 mt of 0.1M sodium carbonate buffer (PH9) and reacted overnight with 4'Cf. 0.2 M after reaction
Washed with NaCl until the wash was negative for the 2°4.6-)IJ nitrobenzene sulfonic acid (TNBS) reaction.

The hydrazide porous cellulose beads thus obtained had hydrazide groups of 150 tt mole/g (solid content). Quantification of hydrazide groups was performed by the TNBS method (Mo1. Ce11. Biochem,
, 4, 181 ("74)).

Example 3-2 Introduction of aldehyde group into anti-human urokinase antibody A purified anti-human urokinase antibody was prepared from rabbit antiserum (manufactured by Midori Juji) by salting out ammonium sulfate. 0.1 of this purified anti-human urokinase antibody 10mgt, 10mt
Dissolved in M acetate buffer (PH3,73, N!LIO41
mg was added and allowed to react at room temperature for 30 minutes. Next, ethylene glycol was added to a final concentration of 0.05M, and the mixture was reacted at 4°C for 3 hours.

Furthermore, this reaction solution was dialyzed against 1 t of 0.1 M acetate buffer (PH17) at 4'C for 18 hours.

Example 3-3 Binding of aldehyde group-introduced anti-human urokinase antibody to hydrazide porous cellulose beads The aldehyde group-introduced anti-human urokinase antibody prepared in Example 3-2 was bonded to the hydrazide porous antibody prepared in Example 3-1. 0.5 g (solid content) of cellulose beads were added, and the pH was adjusted to 6 with Q.1M sodium carbonate buffer (PH9). 4
After overnight reaction at °C, 4 °C using 5 mg of NaBH,
The Schiff base was stabilized by treatment under conditions of S time and PH&5 to obtain an adsorbent for immunoaffinity chromatography.

Example 4 Purification of urokinase in human urine using the adsorbent for immunoaffinity chromatography prepared in Example 3 0.4 g (solid content) of porous cellulose beads bound to the anti-human urokinase antibody prepared in Example 3 was applied to a column. (lcr
nφxz! 0.2 g of urokinase bulk powder (700 international units/mg) obtained from human urine was supplied from the top of the column, and 0.1 MIJ of acid-buffered saline (PH7,
5) was allowed to flow down. OD280nm of eluate is 0.03
After passing the same buffer as above until it reaches 1.5MK
Urokinase adsorbed by SCN was eluted. The results are shown in FIG.

When the KSCN eluate was dialyzed to remove KSCN, concentrated at a low temperature, and further freeze-dried, urokinase with a specific activity of 130,000 international units/mg protein was obtained, and the activity recovery rate was 92%. . Test results for pyrogenic substances were also negative.

Although this column operation was repeated 10 times, no decrease in the adsorption capacity of human urokinase was observed.Urokinase activity was measured using the fibrin plate method (P,
L, Walton, Cl1n, Chim, A
cta, 13.680 ("66)), and the test for pyrogenic substances was conducted in accordance with the Japanese Pharmacopoeia General Test Methods Section 27 Pyrogenicity Test Method, and the dose was 60,000 international units/Ky.

[Brief explanation of drawings]

Figure 1 is a graph showing the relationship between PH and optical density of the reaction solution when antibodies are oxidized with NaIO4, Figures 2 and 4 are graphs showing the purification results of bovine albumin and urokinase using an affinity column, respectively. FIG. 3 is a graph showing the relationship between the amount of adsorption and the number of repetitions in column operation. Procedural amendment 1Q/3/1982! Mr. Haruki Shimada, Director General of the % Correction Agency 1. Indication of the case, Patent Application No. 151462, filed in 1982. 2. Name of the invention and its manufacturing method 1. Relationship with the amendment person case. Address of the patent applicant: 1-chome Yurakucho, Chiyoda-ku, Tokyo, 100. 4 No. 1 Self-issue 5, Detailed explanation of the invention column 66 in the subject specification of supplement r Contents of amendment (1) Specification, page 3, 2 lines from the bottom ``...also in the method, 'J' er... In the method, it is corrected to ``. (2nd. Same, p. 6, lines 4-5, "For immobilizing expensive monoclonal antibodies," should be corrected to "For immobilizing antibodies." (3) Same as above. , page 8, line 6. (4) ``Antigen solution'' on page 17, line 8 of the same ``antibody solution'' should be corrected. " should be corrected to "epichlorohydrin." (6) Same, page 23, line 4 should be corrected to "r 0.2 M glycine-hydrochloric acid buffer J f r2M' KSCN solution."

Claims (2)

[Claims] (1) An antibody having a CHO group formed by oxidative cleavage of the glycol moiety of a carbohydrate moiety and a carrier having an amino group or hydrazide group in the side chain have a -CH, -NH- structure or -CH
, -NH-NH-CO- structure, and even if the column is packed and adsorption/desorption is repeated more than 10 times, 8
An adsorbent for immunoaffinity chromatography that has an antigen recovery rate of 0 or more. (2) allowing an oxidizing agent to act on the antibody in the pH range of 1 to 5;
After oxidizing the glycol moiety of the sugar chain site of the antibody to an aldehyde group, it is bonded to a carrier having an amino group or a hydrazide group, and the bonded portion is reduced. A method for producing an adsorbent for immunoaffinity chromatography having an antigen recovery rate of 1 or higher.