JP2003212898A - Method for producing yolk antibody - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a mass-production method of a yolk antibody present in yolk of an egg, in detail, to provide a method comprising hyperimmunizing a layer by using a virus, a microorganism, a protein or the like as an antigen, extracting a lipid from the yolk of the egg with an organic solvent to provide delipidated yolk residue, and readily purifying the yolk antibody in high purity and high recovery from the obtained yolk residue. <P>SOLUTION: The solution of a water-soluble protein of the yolk containing 0.1-10 (v/v)% organic solvent and extracted from the delipidated yolk residue obtained by extracting the lipid component of the yolk with the organic solvent, with a salt solution is brought into contact with an anion exchanger column, and unadsorbed proteins are washed and removed by using a salt solution. The yolk antibody adsorbed on the column is eluted and collected from the resultant column by using a salt solution. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a one-step mass production method of egg yolk antibody present in egg yolk of chicken eggs. More specifically, the virus, microorganisms, proteins, etc. recognized as foreign substances by the laying hens are used as antigens to hyperimmunize the laying hens, and then the defatted egg yolk residue obtained by extracting lipids from the hen's egg yolks with an organic solvent is used. The present invention relates to a method for easily purifying an antibody with high purity and high recovery rate.

[0002]

2. Description of the Related Art As a water-soluble proteins contained in egg yolk, alpha-Ribechin, beta-Ribechin and γ- Ribechin are known, respectively, parent birds serum albumin, alpha ₂ -
It has been proved to be immunologically equivalent to glycoprotein and γ-globulin (egg yolk antibody). Therefore, γ
-Since the yolk antibody that is ribetine (hereinafter referred to as the yolk antibody) has a function as a specific antibody (IgY),
It is expected to be applied as a functional food material, a functional cosmetic material, a drug, and a research reagent.

Egg yolk contains 50% water, 30% lipid and protein.
Since it is a complex emulsion consisting of 20% and most of lipids exist as lipoproteins bound to proteins, the first step in purifying egg yolk antibody is to first remove lipids and lipoproteins from egg yolk and A solution needs to be prepared. As a method for removing lipids and lipoproteins, for example, a method using an organic solvent, various polysaccharides or polyethylene glycol has been reported (Polson, A. et al., Immunol. Commun. 9: 49).
5-514 (1980), Jensenius, JC, J. Immunol. Method
s 46: 63-68 (1981), Hatta, H. et al., Agric. Biol.
Chem. 54: 2531-2535 (1990), JP 64-38098 A, Aulisio, CG, Proc. Soc. Exp. Biol. 126: 312.
-315 (1981), Bade, H., J. Immunol. Methods 72: 421
-426 (1984)).

There are several known methods for purifying an egg yolk antibody from an egg yolk water-soluble protein solution obtained by the above method, but a method in which an ion exchange chromatography method and a salting out method are combined is generally used. .

[0005]

However, in the above-mentioned conventional methods, a plurality of salting-out operations (at least three times or more) are required to purify the egg yolk antibody with high purity, which is very troublesome. There was also a problem that the recovery rate of egg yolk antibody was low.

Further, a method of separating and purifying egg yolk antibody using an ultrafiltration membrane has been tried, but the recovery rate is low and it has not been put to practical use.

Therefore, an object of the present invention is to provide a method for efficiently separating and purifying an egg yolk antibody from an egg yolk water-soluble protein fraction containing an organic solvent obtained from a defatted egg yolk residue with an organic solvent by a simple operation. To do.

[0008]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to achieve the above object, and as a result, purified an egg yolk antibody from an egg yolk water-soluble protein fraction obtained from a defatted egg yolk residue remaining after extraction with an organic solvent. In doing so, they found that the yolk antibody could be efficiently purified by performing column chromatography using an egg yolk water-soluble protein solution having a specific concentration of an organic solvent and a salt concentration, and completed the present invention.

That is, in the method for producing an egg yolk antibody of the present invention, 0.1 to 10 (v / v) obtained by extracting the lipid component of egg yolk with an organic solvent to extract the remaining defatted egg yolk residue with a saline solution is used.
% Yolk water-soluble protein solution containing organic solvent is contacted with anion exchanger column, non-adsorbed protein is washed and removed with salt solution, and then yolk antibody adsorbed on the anion exchanger is eluted and recovered with salt solution. It is characterized by

In the above-mentioned invention, it is preferable to use an egg yolk containing a specific antibody against the antigen obtained from a laying hen which has been hyperimmunized by immunizing with the antigen.

In the above invention, the concentration of the salt solution for extracting the egg yolk water-soluble protein solution from the defatted egg yolk residue is preferably 0.03M to 0.1M.

In the above invention, the concentration of the salt solution for washing and removing the non-adsorbed protein is preferably 0.0 to 0.01M.

In the above invention, the concentration of the salt solution for eluting and collecting the egg yolk antibody is preferably 0.15 M or more.

According to the present invention, the egg yolk antibody present in the egg yolk of chicken eggs can be produced in large quantities with high purity.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The method for producing an egg yolk antibody of the present invention will be described below for each step.

(Removal of Egg Yolk Lipid Component) First, defatted egg yolk powder is prepared by defatting egg yolk powder (or raw egg yolk) with an organic solvent according to a conventional method. The purity of the organic solvent used for degreasing is 95 (v / v) in order to efficiently separate lipids.
v)% or more is preferable, and 99 (v / v)% or more is more preferable.

The type of the organic solvent used in the present invention is not particularly limited, but those usable in foods and cosmetics such as ethanol, acetone and hexane are preferable. In particular, ethanol is more preferable because it can remain in foods and has no problems such as protein denaturation. Commercially available defatted egg yolk can be used, but it is preferable to use defatted egg yolk defatted by a method such as defatting with ethanol or supercritical extraction.

(Preparation of Egg Yolk Water-Soluble Protein Solution) The egg yolk water-soluble protein solution can be prepared, for example, as follows.

To the defatted egg yolk part obtained above, a buffer solution is added in an amount of 10 to 20 times the amount of the defatted egg yolk part, and 30 minutes to 1
After stirring for a long time to extract the egg yolk water-soluble protein, solid-liquid separation is performed to obtain an egg yolk water-soluble protein solution.

In the present invention, the egg yolk used as a raw material can be used without particular limitation as long as it is prepared from eggs of birds (chicken, turkey, duck, etc.). Further, it may be prepared from eggs laid by a bird that is artificially immunized with a specific antigen (bacteria, virus, protein, enzyme, hormone, polysaccharide, etc.).

As the buffer solution, for example, a phosphate buffer solution, a physiological condition phosphate buffer solution (PBS), a Tris buffer solution or the like can be used. The pH of the buffer solution is 6.
0-11.0 is preferable, 7.0-10.0 is more preferable, and 8.0 is especially preferable.

In the present invention, it is preferable to perform extraction by using a salt solution added to the above buffer solution.

The types of salts used in the present invention include NaCl and KC.
l, CaCl_Two, MgCl_Two, NaH_TwoPO_Four, Na _TwoHPO_Four, KH_TwoP
O_Four, K_TwoHPO_Four, Na_TwoSO_Four, K_TwoSO_Four, (NH_Four)_TwoSO_Four, And
And MgSO_FourUse one or more selected from the group consisting of
Is preferred. Especially, from the viewpoint of cost and taste, NaCl is
preferable. Salt for extracting egg yolk water-soluble protein fraction
The salt concentration of the similar solution is preferably 0.03 to 0.1 M,
04-0.08M is more preferable, 0.04-0.05
M is particularly preferred. Egg yolk antibody by adding salt
Contaminant proteins are selectively washed away when purifying the column.
The egg yolk antibody can be easily eluted and collected.

The egg yolk aqueous protein solution must contain an organic solvent. By containing the organic solvent, when the egg yolk antibody is subjected to column purification, most of the contaminating proteins are not adsorbed and are removed by washing, and the highly purified egg yolk antibody can be easily eluted and recovered at a high recovery rate. The concentration of the organic solvent is preferably 0.1 to 10 (v / v)%, more preferably 0.1 to 5 (v / v)%.

(Column Chromatography) The egg yolk aqueous protein solution prepared as described above is subjected to anion exchanger column chromatography to purify the egg yolk antibody.

That is, after the egg yolk antibody contained in the egg yolk water-soluble protein solution is adsorbed on the anion exchanger column, a solution having a salt concentration of 0.1 M or less is preferably flowed to let out non-adsorbed contaminant proteins. And salt concentration 0.
A yolk antibody adsorbed on the column is eluted by flowing a solution having a salt concentration of 15 M or more, more preferably 0.3 M. The obtained eluate contains mainly yolk antibody, but some α
-Since ribetin is also contained, a pure egg yolk antibody can be easily obtained by performing a salting-out operation once by a conventional method.

The carrier of the anion exchanger used in the present invention is not particularly limited, and examples thereof include Sepharose, Sephadex, cellulose and the like.

The dissociative group bound to the carrier of the anion exchanger is not particularly limited, and examples thereof include diethylaminoethyl (DEA).
E), tetramethylammonium (QA), sulfopropyl (SP), carboxymethyl (CM) and the like. The egg yolk water-soluble protein solution is preferably clarified by filtration or the like before being subjected to column chromatography. Further, the pH may be adjusted, preferably pH 6.0 to 11.0, and pH 7.0 to 1
0.0 is more preferable, and pH 8.0 is particularly preferable. p
The H is preferably prepared using a buffer solution having a buffering capacity on the alkaline side, and for example, a phosphate buffer solution can be used.

[0029]

EXAMPLES The present invention will be described in more detail below with reference to comparative examples and examples, but the present invention is not limited to these examples.

The egg yolk antibody activity recovery in the examples was determined by measuring the antibody titer by the ELISA method. That is, the antigen was coated (0.5 μg /
Each sample was added to the wells (96 wells) and allowed to stand at 37 ° C. for 2 hours to allow the antigen to react with the yolk antibody, and then each well was thoroughly washed with PBS-Tween.

Next, PB of anti-chicken IgG-alkaline phosphatase conjugate (manufactured by Zymet)
The S-Tween diluted solution was added, and the mixture was allowed to stand at 37 ° C. for 1 hour to react the specific antibody that had reacted with the antigen with the above conjugate.

Each well was thoroughly washed with PBS-Tween, 0.1% P-phenyldisodium phosphate solution (dissolved in 0.1M sodium carbonate buffer (pH 9.6)) was added as a substrate, and 37 After carrying out an enzymatic reaction at 30 ° C. for 30 minutes, the reaction was stopped by adding a 2M sodium hydroxide solution, and the absorbance of each well at a wavelength of 405 nm was measured by a plate reader. As a control, PBS-Tween was used instead of the sample. Three wells were used for each sample.

Then, the absorbance of the control was subtracted from the average value of the absorbance of each sample, and the value obtained by multiplying the mass of each sample (total absorbance) was taken as the total antibody activity of each sample, and the total antibody activity of the egg yolk powder was 100%. The activity recovery rate of the egg yolk antibody was determined as

In the following examples, specific antibody-containing egg yolk powder (hereinafter, simply referred to as egg yolk powder) is Salmonella Enterit.
Eggs obtained from chickens produced in the blood with anti-SE antibody obtained by immunizing chicken pectoral muscles with (D.
T. Fraser et al .; Immuno. , Volume 26, 3
(P. 47, 1962), egg yolk separated from egg white was freeze-dried to obtain a starting material.

Example 1 (Study of egg yolk antibody activity recovery rate in egg yolk water-soluble protein solution) 3 times amount of ethanol was added to egg yolk powder, and the mixture was stirred at room temperature for 30 minutes to 1 hour, and then filtered with a filter paper. The defatted egg yolk part was collected. A 10-fold amount of 20 mM sodium phosphate buffer (containing 0.05 M NaCl, pH 8) was added to the mass of the obtained defatted egg yolk powder (including ethanol), and the mixture was stirred at room temperature for 1 hour and then centrifuged (8000 rpm). , 15
Minutes, 5 ° C.) and the supernatant was collected to prepare an egg yolk water-soluble protein solution (Sample 1).

Egg yolk water-soluble protein solutions (Samples 2 to 4) were prepared in the same manner by using defatted egg yolk prepared by repeating the above defatting operation 2, 3, and 4 times, respectively.

On the other hand, an egg yolk water-soluble protein solution was prepared by a method using λ-carrageenan as a comparative example (conventional method). That is, the egg yolk powder was dissolved in the same amount of deionized water, and then 3 times the amount of deionized water was added and homogenized with a homomixer to prepare an egg yolk liquid. Next, λ-carrageenan (1% of the yolk weight) was added to 6 times the amount of deionized water of the yolk, and the mixture was homogenized with a homomixer. The above yolk liquid was slowly added to this λ-carrageenan liquid while stirring with a spatula, and the mixture was stirred until a fine lump remained, and left at room temperature for 30 minutes. This is centrifuged (8000 rpm, 15 minutes, 5
After removing the precipitate (lipoprotein), the supernatant was filtered with filter paper to obtain an egg yolk water-soluble protein solution (Sample 5). The egg yolk antibody activity recovery rate of each of the obtained egg yolk water-soluble protein solutions was measured by the ELISA method, and the egg yolk antibody activity recovery rate of each egg yolk water-soluble protein solution was examined.
The results are shown in Table 1.

[0038]

[Table 1]

From Table 1, Samples 1 to 4 prepared from defatted egg yolk defatted with ethanol have a higher egg yolk antibody activity recovery rate than Sample 5 prepared using λ-carrageenan, regardless of the number of delipidations. It can be seen that the egg yolk antibody can be efficiently extracted without loss of activity.

Example 2 (Study on desorption condition of egg yolk antibody to column) Purified egg yolk antibody freeze-dried powder was dissolved in 20 mM sodium phosphate buffer, and ethanol and / or NaCl was further added, and Table 2 below was added. The following six types of samples were prepared, and the ethanol concentration and salt concentration, and the adsorbability of the yolk antibody to the column and the elution from the column were examined.

[0041]

[Table 2]

Column chromatography was carried out under the following conditions. Column: Mono Q column (Pharmarcia anion exchanger prepack column) Device: FPLC Pharmacia detection: Absorbance 280 nm Buffer: Solution A: 20 mM sodium phosphate buffer (pH)
8.0) Solution B: 20 mM sodium phosphate buffer containing 1 M NaCl (pH 8.0)

Each of the above samples was applied to a column to adsorb the egg yolk antibody to the column, and then gradient elution was performed using the above buffer solution to a NaCl concentration of 0 to 0.5M. The results are shown in FIGS. 1 and 2.
The diagonal lines in the figure indicate the salt concentration of gradient elution.

From FIG. 1, regarding the salt concentration in the solution and the column adsorptivity of the egg yolk antibody, no peak was detected before the gradient elution if the salt concentration was 0.10 M or less (Samples 6 and 7). It can be seen that the column adsorptivity is not affected at all. On the other hand, at a salt concentration of 0.15 M or higher (Samples 8 and 9), it can be seen that in Sample 9 having a higher salt concentration, the peak of egg yolk antibody that is not adsorbed to the column and passes through increases.

From FIG. 2, the ethanol content in the solution and the egg yolk antibody's column adsorptivity showed that the ethanol content was 5%.
(V / v)% (Sample 10) had no effect on the column adsorption of egg yolk antibody, but the ethanol content was 10%.
It can be seen that at (v / v)% (Sample 11), a small amount of egg yolk antibody that is not adsorbed on the column and passes through before the gradient elution is confirmed. Further, it can be seen that the adsorption of the yolk antibody on the column is not inhibited by the interaction between the salt and ethanol.

Further, as shown by the intersections of the oblique lines showing the gradient salt concentration and the peaks of the yolk antibody in FIGS. 1 and 2, it can be seen that the yolk antibody is eluted at a salt concentration of 0.3M.

Example 3 (Study of ethanol concentration in extract and washing property of column non-adsorbed fraction) An egg yolk water-soluble protein solution was prepared in the same manner as in Example 1. That is, defatted egg yolk was defatted three times with ethanol, and defatted egg yolk containing the remaining ethanol was extracted with 20 mM sodium phosphate buffer (pH 8.0) containing 0.05 M NaCl to obtain 5 (v An egg yolk water-soluble protein solution (Sample 12) containing / v)% ethanol was prepared. Similarly, 10 (v / v)
An egg yolk aqueous protein solution containing 10% ethanol (Sample 13) was prepared

Further, after completely removing ethanol from the defatted egg yolk, an ethanol-free egg yolk water-soluble protein solution (Sample 14) was prepared in the same manner.

Each of the above samples was treated with 0.05M NaCl.
After being applied to a DEAE-column (trade name “FPLC”, manufactured by Pharmacia, hereinafter simply referred to as column) equilibrated with a 20 mM sodium phosphate buffer (pH 8.0) containing the yolk antibody, the yolk antibody was adsorbed to the column. According to the stepwise elution method, 20 mM sodium phosphate buffer (pH 8.0) containing 0.05 M NaCl was applied to the column,
The non-adsorbed fraction was collected.

Then, the recovered non-adsorbed fraction was subjected to gel filtration to calculate each protein content. The results are shown in Table 3.

[0051]

[Table 3]

From Table 3, it can be seen that no egg yolk antibody was detected in the column-non-adsorbed fraction of Sample 12 containing 5% of ethanol, and all of them were adsorbed to the column.

On the other hand, sample 1 containing 10% ethanol
3. Egg yolk antibody was detected in the column-non-adsorbed fraction of sample 14 containing no ethanol, which shows that it was not completely adsorbed on the column.

Furthermore, sample 12 containing ethanol
Also, since β-ribetin was mainly detected in the column non-adsorbed fraction of Sample 13, it was found that the adsorption of β-ribetin on the column was selectively inhibited by containing ethanol.

Example 4 (Study on Salt Concentration in Extract and Washability of Fraction Not Adsorbed on Column) An egg yolk water-soluble protein solution was prepared in the same manner as in Example 1. That is, defatted egg yolk was defatted three times with ethanol to obtain defatted egg yolk, and the defatted egg yolk containing the remaining ethanol was extracted with 0.05 mM NaCl-containing 20 mM sodium phosphate buffer (pH 8.0) to obtain 5 (v / V)% ethanol-containing egg yolk water-soluble protein solution (Sample 15) was prepared.

In addition, 20 mM sodium phosphate buffer (pH 8.0) or 20 mM containing 0.15 M NaCl
Extraction was performed in the same manner using a sodium phosphate buffer solution (pH 8.0), and egg yolk water-soluble protein solutions (Samples 16 and 1) each containing 5 (v / v)% ethanol.
7) was prepared.

Each of the above samples was subjected to column chromatography in the same manner as in Example 3, non-adsorbed fractions were collected and subjected to gel filtration to calculate the protein content. The results are shown in Table 4.

[0058]

[Table 4]

From Table 4, it can be seen that the yolk antibody was not detected at all in the column non-adsorbed fraction of Sample 15 containing 0.05 M salt, and all were adsorbed to the column.

On the other hand, sample 16, 0.1 containing no salt
Γ-Rivetin was detected in the column-non-adsorbed fraction of Sample 17 containing 5 M salt, which shows that it was not completely adsorbed on the column.

Example 5 (Elution of egg yolk antibody adsorbed on column) The above samples 12, 13 and 14 were each subjected to column chromatography to adsorb the egg yolk antibody to the column.
A 20 mM sodium phosphate buffer solution (pH 8.0) containing 0.3 M NaCl was flown in an amount 5 times the column volume to elute the yolk antibody, and each was collected.

The obtained eluates (column-adsorbed protein fractions) were subjected to gel filtration, and the results of calculating the respective protein contents in the eluates are shown in Table 5.

[0063]

[Table 5]

From Table 5, sample 1 containing ethanol
It can be seen that the eluates of Nos. 2 and 13 mainly contain the yolk antibody.

On the other hand, sample 14 containing no ethanol
The yolk antibody content of the eluate was low, and there were many contaminating proteins, indicating that the yolk antibody was lost during the process.

Example 6 (Comparison of recovery rate of antibody activity with conventional purification method) The above sample 12 was treated with 20m of 0.05M NaCl.
The non-adsorbed fraction was washed with M sodium phosphate buffer (pH 8.0), and then 20 mM sodium phosphate buffer (pH 8.0) containing 0.3 M NaCl was flown in an amount 5 times the column volume to remove yolk antibody. After elution, the column eluate of the present invention (Sample 18) was collected.

On the other hand, by a method (conventional method) in which the ion exchange chromatography method and the salting-out method were combined, the sample 12 was subjected to column chromatography in the same manner as described above to elute the protein adsorbed on the column. That is, the sample 12 was applied to a column equilibrated with a 20 mM sodium phosphate buffer solution, thoroughly washed with a 20 mM sodium phosphate buffer solution (pH 8.0), and then 20
The protein adsorbed on the column was eluted using 0 mM sodium phosphate buffer (pH 8.0).

Then, 15% (W /
Sodium sulfate was dissolved so as to become V), and the salted-out product thus formed was collected by centrifugation (10,000 rpm × 10 minutes) and collected. This salted-out product was dissolved in a 20 mM sodium phosphate buffer (pH 8.0), and the same salting-out operation was repeated twice. Then, the final salted-out product was dissolved in 10 mM sodium phosphate buffer (pH 8.0) and dialyzed against the same buffer to obtain a dialysate (Sample 19).

Also, an ultrafiltration membrane (molecular weight cutoff of 100 kD
a) (conventional method), the above sample 12 is separated from the egg yolk antibody having a molecular weight of 180 kDa, α-ribetin having a molecular weight of 90 kDa, β-ribetin having a molecular weight of 45 kDa, and other low-molecular contaminating proteins, and retentate is concentrated 5 times. At that point, concentration was completed, and this concentrated liquid (Sample 2
0) was recovered.

With the egg yolk antibody activity of sample 12 as 100%, the total antibody activity of the column eluate of the present invention (sample 18) and samples 19 and 20 was measured.

As a result, it was found that the yolk antibody activity recovery rate of the column eluate (Sample 18) according to the method of the present invention was as high as 91.5%, and almost all the yolk antibody activity in the extract can be recovered. .

On the other hand, the egg yolk antibody activity recovery rate of sample 19 was as low as 76.1%, and it was inferred that the loss was caused by repeating the salting out operation. In addition, the egg yolk antibody activity recovery rate of Sample 20 was as low as 53.4%, which suggests that the separation by the ultrafiltration membrane is insufficient.

Example 7 (Comparison of activity recovery rate of egg yolk antibody from starting material before purification) Assuming that the total antibody activity of egg yolk powder as a starting material is 100%, the column eluate (invention) (Sample 18) and The total antibody activity of Sample 19 and Sample 20 was measured, and the yolk antibody activity recovery rate in each purified solution was determined. The results are shown in Table 6.

[0074]

[Table 6]

It can be seen from Table 6 that the yolk antibody activity recovery rate of the column eluate (Sample 18), which is a purified fraction of the yolk antibody according to the present invention, is as high as 80% or more, and the yolk antibody can be purified without waste.

On the other hand, the egg yolk antibody activity recovery rates of sample 19 and sample 20, which are purified fractions of egg yolk antibody by the conventional method, were as low as 69.6% and 48.8%, respectively. It is presumed that loss or inactivation due to degeneration occurred.

Example 8 Although not particularly limited, the egg yolk antibody can be purified on an industrial scale from the production method of the present invention by the one-step column production method as shown in FIG.

[0078]

As described above, according to the present invention,
From the defatted egg yolk residue remaining after extracting the lipid component of the egg yolk with an organic solvent, extract the yolk water-soluble protein with a buffer containing a salt within a specific range of the amount that leaves the organic solvent within a specific range, By subjecting the obtained egg yolk water-soluble protein fraction to anion exchange chromatography as it is, a fraction mainly containing an egg yolk antibody can be selectively obtained by a one-step column operation.

Therefore, the separation operation of each component from the egg yolk dry powder to the purification of the egg yolk antibody can be carried out continuously, and the purification of the egg yolk antibody from the egg yolk water-soluble protein fraction, which has hitherto been the biggest problem, can be performed in one step. Completed in steps, high purity egg yolk antibodies can be mass produced on an industrial scale.

[Brief description of drawings]

FIG. 1 is a column chromatography diagram showing the influence of the adsorbability of an egg yolk antibody to a column and the elution from the column depending on the salt concentration contained in each sample solution in Example 2.

FIG. 2 is a column chromatography diagram showing the influence of the adsorbability of a yolk antibody to a column and the elution from the column depending on the salt concentration and the ethanol concentration contained in each sample solution in Example 2.

FIG. 3 is a diagram simply showing an actual process of the one-step manufacturing method of the present invention in Example 8.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Hajime Hatta             35 Imakumano Kitahiyoshi-cho, Higashiyama-ku, Kyoto-shi, Kyoto Prefecture               Kyoto Women's University Faculty of Home Economics F-term (reference) 4H045 AA11 AA20 DA75 EA20 EA50                       FA71 GA01 GA23

Claims (5)

[Claims] 1. A 0.1 obtained by extracting a lipid component of egg yolk with an organic solvent and extracting with a salt solution from the defatted egg yolk residue left.
Egg yolk antibody adsorbed on the anion exchanger after contacting an anion exchanger column with an egg yolk water-soluble protein solution containing 10 to 10 (V / V)% organic solvent and washing and removing non-adsorbed protein with a salt solution. A method for producing an egg yolk antibody, comprising: 2. The method for producing an egg yolk antibody according to claim 1, wherein the egg yolk is an egg yolk containing a specific antibody against the antigen obtained from a laying hen that has been immunized with the antigen to hyperimmunize. 3. The concentration of the salt solution for extracting the egg yolk water-soluble protein solution from the defatted egg yolk residue is 0.03M to 0.1M.
The method for producing an egg yolk antibody according to claim 1, which is 4. The method for producing an egg yolk antibody according to claim 1, wherein the concentration of the salt solution for washing and removing the non-adsorbed protein is 0.0 to 0.01M. 5. The method for producing an egg yolk antibody according to claim 1, wherein the concentration of the salt solution for eluting and collecting the egg yolk antibody is 0.15 M or more.