JPH0753760B2 - Chicken egg antibody-binding insoluble carrier and method for purifying substance using the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a chicken egg antibody-binding insoluble carrier in which an egg egg antibody as a ligand is bound to an insoluble carrier used in affinity chromatography, and a method for purifying a substance using the same. .

[Conventional technology]

Conventionally, various methods such as ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and gel filtration have been known as protein purification methods. Each of these methods may be used alone or in accordance with the characteristics of the target substance. Purification is made by the above combination.

Among these purification methods, affinity chromatography is used as an effective means for purifying to high purity.

Affinity chromatography uses one of two types of substances that have a high specific biological affinity for each other as a stationary phase, and uses the difference in affinity for the stationary phase to separate the target substance and impurities. Chromatography. For example, one in which one of the interacting substances is covalently bound to an insoluble carrier by utilizing the specific interaction that works between enzymes and substrates, antigens and antibodies, hormones and receptors, lectins and polysaccharides, etc. In this method, a stationary phase for chromatography is used, the other substance is captured and adsorbed during passage through the column, and then the other substance of interest is purified by elution treatment.

As a ligand for affinity chromatography,
Although various ones are known, for example, those in which Protein A is bound to an insoluble carrier, and in immunoaffinity chromatography using an antibody, rabbit Ig is used as a ligand.
G, goat IgG, mouse monoclonal antibody and the like are generally used.

[Problems to be Solved by the Invention]

Since affinity chromatography utilizes the difference in affinity as described above, a certain degree of binding force is required between the ligand bound to the stationary phase and the target substance to be adsorbed.

However, if this binding force is too strong, there is a problem that the conditions for the elution treatment to be performed next become severe. That is, the elution treatment is usually performed by weakening the binding force between the antibody and the antigen by utilizing the change in pH. Therefore, pH conditions under which the target substance is not denatured are desirable as elution conditions, and proteins and viruses are usually susceptible to denaturation under alkaline conditions. Therefore, elution of the target substance by changing the pH to acidic is preferred. However, in general, the antigen-antibody reaction is a very strong bond, and thus it is often impossible to elute the target substance unless elution conditions of pH 2.0 or lower are used. For example rabbit Ig
In immunoaffinity chromatography using G and goat IgG as ligands, elution conditions of pH 2.0 or less are usually used. However, elution under such elution conditions causes a problem that even the adsorbed target protein is denatured, and therefore it is preferable to use a ligand that can be eluted under mild conditions such as pH 3 to 5. Also, the elution conditions
It is known that under severe conditions of pH 2 or less, the ligand is likely to be detached from the carrier.Therefore, when used under such conditions, a small amount of the released ligand is mixed in the eluate, while the affinity adsorbent There is a problem of shortening the life of the.

As a ligand, in particular, the above-mentioned mammalian IgG is expensive in terms of cost and generally has severe elution conditions, and thus it is preferable in the art to use an antibody that can be supplied as an alternative to the elution conditions with ease and at low cost. Has been requested.

The object of the present invention is exactly at this point, and it is to provide a chicken egg antibody-binding insoluble carrier using an egg egg antibody as a ligand in affinity chromatography and a method for purifying a substance using the same.

[Means for Solving the Problems]

The inventors of the present invention have conducted extensive studies to solve the above-mentioned problems, and as a result, in affinity chromatography using a chicken egg antibody-bound insoluble carrier bound with an egg egg antibody as a ligand, it is possible to elute the adsorbed substance under mild elution conditions. They have found the present invention and completed the present invention.

That is, the gist of the present invention is (1) a chicken egg antibody-binding insoluble carrier, which is bound with an egg egg antibody as a ligand, (2) an affinity chromatograph using the chicken egg antibody-binding insoluble carrier according to (1) above. (3) a method for purifying a substance by chromatography, and (3) a method for adsorbing and removing impurities by affinity chromatography using the chicken egg antibody-bound insoluble carrier according to (1) above.

The chicken egg antibody used in the present invention is prepared from eggs produced by chickens that have been previously immunized with the antigen.

There is no particular limitation on the chicken to be immunized, but from the viewpoint of mass productivity of the antibody, it is particularly preferable to use an egg species such as a white leghorn system, Rhode Island red system, lateral plymouth rock system, and New Hampshire system. preferable.

The immunization method is not particularly limited as long as it can immunize the chicken, such as subcutaneous injection, intramuscular injection, and intraperitoneal administration.

Examples of the antigen include various proteins or viruses that are required to be separated and purified, and are not particularly limited. For example, as the protein, C-reactive protein, bovine serum albumin (BSA), various complement components, IgG, I
Blood components such as gA, IgM, IgE, IgD, hemoglobin, tissue plasminogen activator (TPA), urine components such as urinary kallikrein and urinary trypsin inhibitor, cancer marker antigens such as α-fetoprotein, carcinoembryonic antigen And cytokines such as interleukin, α, β, γ-interferon, and various other substances such as protein A.
Examples of the virus include rotavirus, influenza virus, AIDS virus, poliovirus and the like.

The dose of the antigen may be appropriately selected so that the desired antibody titer is obtained and the chicken is not adversely affected.

Usually, after the first immunization, if it is repeatedly administered once a week for about 3 to 5 times, an antibody that specifically reacts with the antigen is obtained in chicken eggs.

After that, it is advisable to perform booster immunization every 1 to 4 months for the purpose of maintaining the antibody titer.

In addition, if necessary, immunization may be carried out with an adjuvant such as FCA (Freund's complete adjuvant) or FIA (Freund's incomplete adjuvant).

Thus, usually, one month or more after the first immunization, an antibody having a sufficient antibody titer can be prepared from chicken eggs.

Specific antibody titers in chicken eggs are determined by enzyme-linked immunosorbent assay (ELISA),
It can be measured using a radioimmunoassay, a microtiter method, etc., and the transition of the antibody titer can be traced by measuring the antibody titer at intervals of about 2 weeks after immunization.

The chicken egg antibody in the present invention can be obtained by extracting and separating immunoglobulin contained in the yolk of the egg immunized as described above.

This extraction is carried out by precipitating lipoproteins using, for example, textran sulfate, polyethylene glycol (PEG), agar, carrageenan, furceleran, pectin, xanthan gum, alginic acid, alginates, alginic acid derivatives and the like. The method of separating and purifying from the water (Journal of Immunological Methods, 46, 63-68,
1981 / Immunological Communication, 9 (5), 475-493,1
980 / Japanese Patent Laid-Open No. 63-215699 / Japanese Patent Laid-Open No. 64-38098), and various known methods commonly used for extraction and separation of immunoglobulins, such as extraction methods using propanol, chloroform, etc. , Carrageenan, xanthan gum, pectin and the like are preferably used.

The egg yolk antibody in the present invention is, for example, from an egg of a chicken immunized as described above, an egg yolk is separated from an egg yolk liquid, and then yolk lipoproteins are removed from the yolk liquid using yolk lipoprotein by ion exchange chromatography, A purified chicken egg antibody purified by a known protein purification method such as hydrophobic chromatography, affinity chromatography, gel filtration, salting out with sodium sulfate, salting out with ammonium chloride is used.

The insoluble carrier used in the present invention is not particularly limited as long as it can be used in affinity chromatography. Usually, cellulose gel, dextran gel, agarose gel, polyacrylamide gel, porous glass and the like are used.

Rabbit IgG is a method for binding the egg yolk antibody and the insoluble carrier.
As in the case of binding goat IgG as a ligand,
It can be carried out by a conventional method. That is, when using cellulose, dextran, agarose as a carrier,
A method of activating a carrier by using a cyanogen halide such as cyanogen bromide and binding an antibody, a method of activating a carrier by using periodic acid and binding an antibody, 1,4-bis- (2,3-epoxy) A method of activating a carrier using an epoxide such as propoxy) -butane and binding an antibody, and a method of introducing an active group into a carrier using a crosslinking reagent such as a halogenated triazine to bind an antibody, and polyacrylamide as a carrier. When using, the carrier is an aminoethyl derivative,
Change to hydrazide derivative or carboxyl derivative,
It is carried out by a method of binding an antibody.

As a method for purifying the substance of the present invention, an egg egg antibody to the target substance is prepared by the above method, and affinity chromatography is performed by a conventional method using the egg egg antibody bound to an insoluble carrier as a ligand. The elution treatment of the target substance adsorbed on the ligand can usually be carried out under mild conditions of about pH 3-5.

For example, when anti-mouse IgG chicken egg antibody obtained by immunizing a chicken with mouse IgG as an antigen and bound to an insoluble carrier as a ligand is used and affinity chromatography is performed, mouse serum as a sample is used as a column hair. Mouse IgG in mouse serum by plying
Is adsorbed to the anti-mouse IgG chicken egg antibody which is a ligand, and then the target substance mouse IgG can be recovered under mild elution conditions of pH 4.

Alternatively, when anti-rotavirus hen egg antibody obtained by immunizing a chicken with rotavirus as an antigen and bound to an insoluble carrier as a ligand is used and affinity chromatography is performed, the sample is infected and propagated in cultured cells. By subjecting the crude rotavirus to column hair ply, only the rotavirus is adsorbed to the anti-rotavirus chicken egg antibody which is a ligand, and then the target substance rotavirus can be recovered under mild elution conditions of pH 4.

In addition, if the chicken egg antibody-bound insoluble carrier of the present invention is used, it can also be used for removing impurities. That is, if the chicken egg antibody is against impurities, the impurities are collected in the eluted fraction by performing affinity chromatography, and the target substance can be collected in the flow-through fraction.

For example, fetal calf serum is often used in tissue culture of animal cells, but it is necessary to actively remove fetal calf serum as an impurity in the purification of the target substance. Further, for example, when purifying a monoclonal antibody, it is necessary to actively remove impurities derived from ascites of the mouse in which the hybridoma was cultured. In such a case, the impurities can be effectively removed by preparing an egg egg antibody against these impurities and performing affinity chromatography.

Further, in the present invention, the target substance can be purified to a higher degree of purity by combining the purification process using the above-described method for purifying the target substance and the removal process using the method for removing impurities.

[Operation / Effect] When affinity chromatography is carried out using the egg yolk antibody-bound insoluble carrier of the present invention, the elution conditions are the conventional method as compared with the case of using rabbit IgG or yag IgG which has been conventionally used as a ligand. In contrast, the pH is 2 or less, while the pH is mild to 3 to 5, which has an excellent effect that the outflowing target substance is not likely to be denatured. Further, in the conventional method in which the elution conditions are severe, the problem that the ligand is dislodged from the insoluble carrier, so-called leakage of the ligand occurs, and a trace amount of the ligand is mixed in the eluate, and thus the life of the affinity adsorbent is shortened. Known to have problems. However, in the present invention, since the elution conditions are mild, there is no risk of leakage of the ligand, and there is an effect that the life of the affinity adsorbent is long.

Furthermore, as shown in the Examples described below, the antibody production amount per laying chicken is 13.7 times as high as that of one rabbit, and therefore, it is possible to stably and inexpensively supply antibodies. It also has some advantages.

〔Example〕

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

Example 1 <Immunization of Laying Hens and Rabbits> As the animals to be immunized, white leghorn laying hens and rabbits (STD Jw) were used. Purified mouse IgG (Cappel) was used as the antigen. Immunization was carried out by emulsifying an antigen solution (1 mg / ml) with an equal amount of Freund's complete adjuvant and intramuscularly injecting 1 mg of the antigen once per laying chicken and one rabbit. Immunization was repeated 4 times at biweekly intervals including the first immunization.

For the laying hen, after that, immunize once every four months,
The chicken eggs were collected for one year. As a result, an average of 210 chicken eggs that could be used for the purification of anti-mouse IgG egg antibody per bird per year were obtained.

For rabbits, one week after the final immunization (4th day of immunization), whole blood was collected according to a conventional method and the serum thereof was separated. An average of 52 ml of antiserum was obtained per rabbit which could be used to purify rabbit IgG.

As a control, non-immunized laying hens produced an average of 214 control hens per bird per year. In addition, an average of 50 ml per rabbit is obtained by collecting whole blood.
Control sera were obtained.

Test Example 1 <Measurement of Specific Antibody Titer in Egg Yolk and Rabbit Serum> The specific antibody titer against mouse IgG was measured by ELISA. Mice used as antigens on Nunc ELISA 96-well plates
IgG was coated according to a conventional method. Ovalbumin was used for blocking. Egg yolk and rabbit serum diluted 1000-fold with PBS-Tween (physiological phosphate buffer containing 0.5% Tween20, pH 7.2) were added to mouse IgG-coated plates (100 μl / well), and left at room temperature for 2 hours.
Then, after washing each well with PBS-Tween, an anti-hen egg antibody (IgY) rabbit IgG conjugated with alkaline phosphatase and an anti-rabbit IgG goat IgG (Zymet) were used as secondary antibodies and added to the corresponding wells ( 100 μl /
Well). After allowing to stand at room temperature for 2 hours, place each well in PBS-Twe
After washing with en, an enzymatic reaction (room temperature: 15 minutes) was performed using p-nitrophenyl phosphate (Sigma) as a substrate, and the absorbance at a wavelength of 405 nm was measured by a plate reader. Changes in mouse IgG-specific antibody titers in egg yolk and rabbit serum after the first immunization are shown in FIG.

Example 2 <Purification of anti-mouse IgG chicken egg antibody and rabbit IgG> (1) Purification of chicken egg antibody (IgY) Egg yolk was separated from the egg and homogenized by adding deionized water in an amount equal to that of the egg yolk. This 2-fold diluted egg yolk was mixed with a 2-fold amount of an aqueous solution of λ-carrageenan (1.5 mg / ml) to aggregate egg yolk lipoprotein. After leaving it at room temperature for 30 minutes, the lipoprotein was removed as a precipitate by centrifugation (10,000 × g, 10 minutes). The supernatant was suction-filtered with a filter paper (Advantech Toyo No. 2 paper filter). Disodium phosphate was dissolved in the filtrate to 20 mM, and 3N hydrochloric acid was added to adjust the pH to 8.0. This solution was applied to a DEAE Sephacel column previously equilibrated with 20 mM phosphate buffer pH 8.0, and non-adsorbed substances were thoroughly washed with the same buffer. Elution of the adsorbed protein from the column was performed with 200 mM phosphate buffer pH 8.0. Sodium sulfate was dissolved in the eluate so as to be 15% (W / V), and the resulting insoluble matter (salted out product) was collected by centrifugation (10,000 xg, 10 minutes). The salted-out product was dissolved in 20 mM phosphate buffer pH 8.0, and the same sodium sulfate salting-out operation was repeated twice. Final salted out product is 10 mM
It was dissolved in phosphate buffer pH 8.0 and dialyzed against the same buffer. The solution after dialysis was lyophilized after passing through a 0.45 μm membrane filter.

As a result of the above operation, one egg (15 g of yolk) with an increased titer of specific antibody to mouse IgG was used as the protein amount of 6
5 mg of IgY was obtained.

(2) Purification of rabbit serum antibody (IgG) Rabbit IgG was purified by a conventional method. To rabbit serum, an equal amount of 10 mM phosphate buffer pH 7.0 (containing 0.15 NaCl) was added, and further an equal amount of saturated sodium sulfate solution to the serum was added,
33% ammonium sulfate salting out was performed. Centrifuge the salted out product (10.000 x
g, 10 minutes), 50 mM phosphate buffer pH 8.0 (0.1
Dissolved in 5M NaCl). Add this solution to Superose 12 pre
An IgG fraction was obtained by gel filtration using p.grade. The IgG fraction was dialyzed against 20 mM Tris-HCl buffer pH 8.3.
Then, DEAE ion-exchange chromatography (MonoQ, Pharmacia) was performed according to a conventional method to adsorb IgG to 20 m.
Gradient elution was performed with M Tris-HCl buffer pH 8.3, 0 to 0.2 M NaCl to obtain a purified IgG fraction. This was dialyzed against 10 mM phosphate buffer pH 8.0 and then freeze-dried.

By the above operation, 10.3 mg of rabbit IgG was obtained as the protein amount per 1 ml of rabbit serum immunized with mouse IgG.

Test Example 2 <Antibody Purity Assay> The antibody purity assay is carried out by the Pharmacia FPLC system,
Gel filtration analysis (Superose 12HR 10/30) and SDS-electrophoresis by Remley et al.

The IgY and rabbit IgG obtained in Example 2 both have a single peak in gel filtration analysis, and only the H chain and L chain of the antibody were confirmed by SDS-electrophoresis analysis. It was confirmed that the product was purified to a pure product.

Test Example 3 <Measurement of amount of mouse IgG-specific binding antibody> The ratio of the mouse IgG-specific binding antibody contained in IgY and rabbit IgG obtained in Example 2 was determined by affinity chromatography using mouse IgG as a ligand. It was measured by separating the adsorption fraction and the adsorption fraction and measuring the amount of each protein.

The mouse IgG used as a ligand is purified mouse IgG manufactured by Kappel that was used as an immunogen. As the gel, cyanodiene bromide (CN-Br) -activated Sepharose 4B (Pharmacia) was used, and 10 mg of mouse IgG was bound per 1 ml of the gel according to the manual of Pharmacia.

IgY and rabbit IgG were added to 50 mM phosphate buffer (0.15 M NaCl
Dissolved) at pH 8.0. Each was applied to a mouse IgG-Sepharose 4B (1 ml gel) column, non-adsorbed antibody was washed with 50 mM phosphate buffer (containing 0.5 M NaCl) pH 8.0, and then 0.1 M sodium carbonate solution (containing 0.5 M NaCl) pH 1
The adsorbed antibody was eluted at 1.3. Apply antibody to column,
The amounts of non-adsorbed antibody and adsorbed antibody were calculated using the absorbance at a wavelength of 280 nm as the amount of protein, and the following results (Table-1)
Got As controls, IgY and rabbit IgG were purified from the yolk of non-immunized hens and the blood of non-immunized rabbits, and the same operations as above were performed.

The amount of antibody calculated from the absorbance at a wavelength of 280 nm was calculated as follows: ▲ E ^1% _{280 nm} ▼ = 14.8 for IgY, ▲ for rabbit IgG.
An extinction coefficient of E ^1% _{280 nm} ▼ = 13.5 was used.

From the above results, 28% of the total amount of antibody was detected in the egg yolk antibody (IgY) of the laying chicken immunized with mouse IgG as an antigen, and 52% of the total amount of the antibody was 52% in the mouse serum antibody (IgG).
It was shown to be a specific binding antibody.

Here, comparing the amount of mouse IgG-specific binding antibody produced per hen and one rabbit, 21
From 0 chicken eggs, 3150 ml of yolk was obtained, from which 13,650 mg of anti-mouse IgG chicken egg antibody was purified. The mouse IgG-specific binding antibody contained therein was 3,822 mg.

On the other hand, in the case of rabbits, an average of 52 ml of antiserum was obtained, and from this, 536 mg of anti-mouse IgG rabbit IgG was purified. The mouse IgG-specific binding antibody contained therein is 279 mg.

Therefore, it was shown that the production amount of the mouse IgG-specific binding antibody per hen that was immunized with mouse IgG was 13,7 times that of the conventional method using rabbits.

Example 3 (1) Anti-mouse IgG chicken egg antibody (IgY) -Sepharose 4B
Preparation of Pharmacia Cyanophane bromide activated Sepharose 4B (10.0 g) was swollen in 1 mM HCl, and thoroughly washed on a glass filter (3G). Then, 330 mg of the anti-mouse IgG chicken egg antibody (IgY) obtained in Example 2 was dissolved in the coupling buffer as a ligand, which was thoroughly washed with the coupling buffer (0.1 M NaHCO ₃ , 0.5 M NaCl buffer pH 8.3). , The gel suspension, and the coupling reaction was performed at room temperature for 2 hours. The ratio of gel suspension to ligand solution was 1: 2. Gel blocking reagent (0.2M
It was transferred to glycine (pH 8.0) and unreacted active groups were blocked at room temperature for 2 hours. The gel was then loaded onto a glass filter with coupling buffer, 0.1M acetate buffer (0.
It was thoroughly washed with pH 4.0 (including 5M NaCl) and the coupling buffer in this order. As a result, 33 ml of anti-mouse IgG chicken egg antibody (IgY) -Sepharose 4B gel was obtained with a ligand binding rate of 98%.

(2) Preparation of anti-mouse IgG rabbit IgG-Sepharose 4B Using the anti-mouse IgG rabbit IgG obtained in Example 2 as a ligand, the same operation as above was performed, and the anti-mouse IgG rabbit IgG was obtained at a ligand binding rate of 97.5%. -Sepharose 4B gel
33 ml was obtained.

Test Example 4 <Comparison of elution conditions of immunoaffinity column> Each Sepharose 4B gel 10 prepared in Example 3
ml was packed in a column (φ1.6 cm × 5 cm) and equilibrated with 50 mM phosphate buffer pH 8.0 (containing 0.5 M NaCl).

As a sample, purified mouse IgG (Cappel) was dissolved in equilibration buffer (10 mg / ml) and 500 μl was applied to each column.
I applied.

After washing the column with equilibration buffer, the elution buffer was first 0.1M acetate buffer pH 4.0 (containing 0.5M NaCl) and then glycine / hydrochloric acid buffer pH 2.0 (0.5M Na).
(Including Cl), two-step stepwise elution of pH 4.0 and pH 2.0 was performed. Elution was performed in the direction opposite to the direction of applying the sample.

The eluted fraction was fractionated (1 ml / fraction) with a fraction collector, and the absorbance at a wavelength of 280 nm was measured (Fig. 2). Table 2 shows the results of the recovery rates of mouse IgG in pH 4 and pH 2 elution determined from the absorbance at a wavelength of 280 nm.

Incidentally, by the enzyme immunoassay, the elution fraction of each column, as a result of detecting the antibodies used as ligands (rabbit IgG and IgY), the ligand antibody was not detected in the elution at pH 4.0, Both ligand antibodies were detected in the elution at pH 2.0.

From the above results, rabbit IgG that has been conventionally used as a ligand in immunoaffinity chromatography
Therefore, it was found that the elution condition of pH 2.0 or less is required for elution of the adsorbed substance, but affinity chromatography using IgY as a ligand revealed that the adsorbed substance can be eluted and recovered under a relatively mild elution condition of pH 4.0. .

Example 4 <Immunoaffinity chromatography purification of mouse serum IgG> Mouse serum (5 ml) was salted out with ammonium sulfate at 33% saturation. The salted out product was dissolved in 50 mM phosphate buffer (containing 0.5 M NaCl) pH 8.0 and dialyzed sufficiently against the same buffer to give a crude mouse IgG sample. 20 ml of the anti-mouse IgG chicken egg antibody (IgY) -Sepharose 4B gel prepared in Example 3 was packed in a column, and 50 mM phosphate buffer pH 8.0 (0.5
Equilibrated with M NaCl). To this column crude mouse IgG
The sample was applied and washed thoroughly with equilibration buffer.
Then, the adsorbed protein was eluted with 0.1 M acetate buffer pH 4.0 (including 0.5 M NaCl) to obtain 34 mg of mouse IgG.

The recovery rate of mouse IgG was 72% by the enzyme immunoassay method (sandwich method). In terms of its purity, the gel filtration analysis of Test Example 2 showed a single peak, and only the H chain and I chain of IgG were confirmed by SDS-electrophoresis of Remley.

Example 5 <IgG Purification from Mouse Hybridoma Culture Supernatant> A mouse hybridoma producing a human superoxide dismutase monoclonal antibody (mouse IgG ₁ class) was cultured in RPMI medium containing 10% fetal calf serum. 1000 ml of the culture supernatant was subjected to buffer exchange with 50 mM phosphate buffer pH 8.0 (including 0.5 M NaCl) using an ultrafiltration membrane having a molecular weight cut off of 10,000. This solution was applied to the anti-mouse IgG chicken egg antibody-Sepharose 4B column used in Example 4,
The non-adsorbed protein was thoroughly washed with the equilibration buffer. Next, the adsorbed protein was eluted with 0.1 M acetate buffer pH 4.0 (containing 0.5 M NaCl) to recover 10 mg of human superoxide dismutase monoclonal antibody. Mouse IgG ₁ recovery rate is 76% by enzyme immunoassay (sandwich method)
Met. As for the purity, a single peak was confirmed by gel filtration analysis, and only the H chain and L chain of IgG were confirmed by SDS-electrophoresis of Remley.

Example 6 <IgG Purification from Mouse Hybridoma Peritoneal Culture Fluid> A mouse hybridoma producing a human superoxide dismutase monoclonal antibody (mouse IgG ₁ ) was prepared as follows.
After culturing in RPMI1640 medium containing 10% fetal calf serum, cells were collected by centrifugation, washed with MEM medium, and then suspended in PBS. BALB / c mice (male,
1 to 2 × 10 ⁷ cells were injected per mouse intraperitoneally for 6 weeks). A laparotomy was performed about 10 days after the injection of the hybridoma to obtain 5 ml of ascites per mouse. The collected ascites was centrifuged (300
Ammonium sulfate fractionation (0-50% saturation) was performed after 0 rpm, 20 min), and the precipitate obtained by centrifugation was treated with 50 mM phosphate buffer (pH 8.0,
After dissolving in 0.5NaCl), it was thoroughly dialyzed against the same buffer. The dialyzed solution was applied to the anti-mouse IgG chicken egg antibody-Sepharose 4B column used in Example 4, and the non-adsorbed protein was thoroughly washed with the equilibration buffer. Then, the adsorbed protein was eluted with 0.1 M acetate buffer (pH 4.0, containing 0.5 M NaCl) to recover 50 mg of human superoxide dismutase monoclonal antibody. Mouse IgG recovery rate is E
It was 68% by LISA (sandwich method). In addition, the purity is a single peak in gel filtration analysis and SD of REMULY.
Even in S-electrophoresis, only the H chain and L chain of IgG were confirmed.

Example 7 <Preparation of Anti-Human Rotavirus Chicken Egg Antibody-Sepharose 4B> Egg egg antibody (IgY) was purified from the egg yolk of laying hens immunized with human rotavirus Mo strain (serotype 3) in the same manner as in Example 2. did. This IgY was treated in the same manner as in Example 3 with cyanogen bromide-activated-Sepharose 4B gel at 1 / ml.
Anti-human rotavirus chicken egg antibody-Sepharose 4B was prepared by binding 0 mg.

[Brief description of drawings]

FIG. 1 is a diagram showing changes in mouse IgG-specific antibody titers in egg yolk and rabbit serum. Fig. 2 shows the wavelength 28 of the fraction eluted with the immunoaffinity column.
It is the figure which showed the light absorbency in 0 nm.

Front page continuation (72) Inventor Shigemitsu Akabori Shinmachi 9-5 Yokkaichi-shi, Mie Taiyo Kagaku Co., Ltd. (72) Inventor Kim Takehisa 9-5 Akahori Shinmachi, Yokkaichi-shi Mie Taiyo Kagaku Co., Ltd. (72) Inventor Takehiko Yamamoto 3-4-2-2, Tsuruyamadai, Izumi City, Osaka Prefecture

Claims (7)

[Claims] A chicken egg antibody-binding insoluble carrier, which is bound with an egg egg antibody as a ligand. 2. A chicken egg antibody-bound insoluble carrier, wherein the chicken egg antibody of claim 1 is prepared from eggs produced by a chicken that has been previously immunized with an antigen. 3. A chicken egg antibody-bound insoluble carrier in which the antigen according to claim (2) is a protein. 4. A chicken egg antibody-bound insoluble carrier in which the antigen according to claim (2) is a virus. 5. Claim (1), (2), (3) or (4)
A method for purifying a substance by affinity chromatography using the chicken egg antibody-bound insoluble carrier as described above. 6. Claim (1), (2), (3) or (4)
A method for adsorbing and removing impurities by affinity chromatography using the chicken egg antibody-bound insoluble carrier as described above. 7. The affinity chromatography according to claim (5) or (6), wherein the adsorbed substance is eluted under elution conditions of pH 3 to 5. Method.