JPH0930986A - Immunization of bovid animal - Google Patents

Abstract

PROBLEM TO BE SOLVED: To reduce a pain given to an animal, not to leave a lesion in dressed carcass, to raise an antibody titer equally to intramuscular administration and to obtain a large amount of an antibody by administering an antigen to a dewlap hypodermic of a bovid animal when the bovid animal is immunized. SOLUTION: An antigen is administered to a dewlap hypodermic of a bovid animal, preferably at a part rich in fat tissue when the antigen is administered to the bovid animal to obtain an antibody from its milk or blood. Human skin, hair, a protein extracted from hair, etc., are preferable as the antigen when the prepared antibody is used as a cosmetic or a medicine and applied to skin and hair. A dose of the antigen is 10<5> to 10<11> viruses and bacteria per animal per time in the case of a virus or a bacterium and is preferably 0.01-200mg in the case of the organism component. Bovine, sheep, goat, etc., are preferable as the bovid animal. The administration is preferably carried out by an injection. The antigen is optionally used with an adjuvant and may be microcapsulated with liposome, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing an antibody that is widely used in the fields of medicine, food, cosmetics and the like.

[0002]

2. Description of the Related Art Conventionally, in order to increase the antibody titer in producing an antibody, many studies have been conducted on the administration site of the antigen, the administration time, the time and frequency of booster immunization, the age of the immunized animal, and the like. Therefore, regarding the administration site, it is generally said that intramuscular injection into the muscles of the buttocks and subcutaneous administration to the neck, hips, back and the like are effective. However, since there is no thoracic drop in animals other than bovines, it is customary to administer it to the above-mentioned sites when trying to immunize a bovine animal without the idea of administering it to the thoracic appendage. However, since the antibody titer was actually obtained to some extent, no attempt was made to darely administer it to another site.

However, with intramuscular injection, as a result of observation of swelling and induration, inflammation was observed deep inside the carcass,
In addition, lesion tissue such as swelling and induration persists for a long period of time. Since such carcasses are not preferable from the viewpoint of meat hygiene, the carcass (carcass) is eventually subject to defects. In other words, the carcass around it was cut off, and the carcass in the remaining part had a great decrease in commercial value, which was a great loss for dairy farmers.

Similarly, when subcutaneously administered to the neck, hips, back, etc., the muscle tissue immediately underneath the subcutaneous tissue sometimes causes inflammation in the muscle, and swelling and induration restrict movement. However, there was a drawback that it was painful for animals.

[0005]

SUMMARY OF THE INVENTION Therefore, the object of the present invention is to provide bovine animals with an antibody titer which does not affect carcass and causes less pain to animals, and is equivalent to intramuscular administration. It is to provide a method of immunizing bovine animals that can be obtained.

[0006]

The above object is characterized in that, when an antigen is administered to a bovine and an antibody is obtained from its milk or blood, the antigen is administered subcutaneously to the thoracic thoracic of the bovine. It is achieved by the method of immunizing bovine animals.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the contents of the present invention will be described in detail. In the present invention, the thoracic epidermis to which an antibody is administered is a fold of the skin that hangs from the neck to the forelimbs, and is a part filled with adipose tissue. Among them, it is preferable to administer to a portion having more adipose tissue.

The antigen used in the present invention includes
Examples include antigens used to obtain useful antibodies generally used for medical, food, cosmetics, and the like. For example, microorganisms such as Escherichia coli, dental caries, and rotavirus, toxins thereof, or extract components thereof, Clostridium, etc. Examples include protozoa or their extracted components, and biological components such as proteins extracted from human skin, body hair or hair, or nails, wool, feathers, etc., and the obtained antibodies are used as cosmetics or pharmaceuticals as skin or hair. In the case of application to humans, human skin, body hair, or hair extract protein and the like are preferable in consideration of species differences and the like.

The dose of the antigen is such that a desired antibody titer can be obtained.
In addition, an amount that does not adversely affect animals may be appropriately selected and used. For example, in the case of once a virus and bacteria, it is 10 ⁵ to 10
^In the case of ¹¹ biogenic components, 0.01 to 200 mg is preferable.

Freund's complete adjuvant (FCA) and Freund's incomplete adjuvant (FIA) as needed
Adjuvants such as may be used in combination with the antigen. The antigen may be microencapsulated with liposomes or the like.

The dose of FCA may be appropriately selected and used so that it does not adversely affect the animals. For example, one dose is preferably 0.05 to 50 ml.

Upon immunization, an antigen and an FCA adjuvant may be mixed and used. For example, an antigen solution is dissolved in physiological saline, and the solution and FCA are mixed to prepare a water-in-oil type (hereinafter W / O type). Abbreviated as ").

The mixing ratio of the antigen solution and FCA when used as an emulsion can be appropriately selected from the mixing ratio when FCA is generally used. For example, the mixing ratio of 1: 1 by volume can be used. .

The bovine animals used for immunization in the present invention include all bovine animals, but cows, sheep, goats and the like are preferable.

The immunization of the present invention is carried out by administering an antigen solution or an antigen suspension subcutaneously to the thoracic thoracic of a bovine animal by injection. At this time, it may be appropriately administered together with an adjuvant such as FCA, FIA or alumina.

In the present invention, the following known method may be used to collect the antibody from the serum or milk of the immunized bovine animal.

There are many reports on a method for separating immunoglobulin from serum or plasma of bovine animals. For example,
The Journal of Biological Chemistry
(TheJournal of Biological Chemistry), Volume 234 (1959
Year), 2645 pages / Transfusion
, Volume 6 (1966), Page 146 / Biochemica et Biophysica Acta,
Volume 214 (1970), page 107, etc., can be implemented according to these. As an industrially suitable method, mammalian serum or plasma is diluted 2-3 times with physiological saline,
A crude immunoglobulin is obtained by a stepwise ammonium sulfate fractionation method and, if necessary, an ion exchanger (eg, an anion exchanger such as DEAE cellulose and DEAE Sephadex, or a cation such as CM cellulose and CM Sephadex). (Exchanger). Furthermore, gel filtration fractionation can be performed if necessary.

[0018] The immunoglobulin can be obtained from milk according to a known method [Archieves of Biochemistry].
and Biopysics, 108, 230, (1964) / The
Journal of Immunology, 103, 334, (1969)
/ Biochimica Et Bio-physica Acta, 181,381,
(1969) / Journal of Dairy Science, 55, 15
1, (1972) / Journal of immunology, 118,4
61, (1977)].

For example, casein is removed from defatted milk by acid treatment, and whey is fractionated to obtain a crude purified immunoglobulin. If necessary, salting out operation or anion exchanger (for example, DEAE-cellulose, DEAE-sepharose,
Purification by using an ion exchange chromatography operation using QAE-Sephadex) or a gel filtration chromatography operation using a gel filtration carrier (for example, Sepharose S-300, Sephadex G-200) alone or in combination. You can also Further, a carrier to which keratin is bound (for example, Affigel 15,
The antibody can also be specifically purified by performing an affinity chromatography operation using CH-Sepharose 4B).

The antibody obtained as described above is preferably a purified antibody, but may be an unpurified antibody, which is provided as an aqueous solution or a dried product obtained by operations such as freeze-drying and spray-drying.

The antibody titer (antibody titer) can be measured by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay or the like. If necessary, the transition of the antibody titer can be traced by this, and can be used as a guide for the timing of booster immunization or the timing of milk collection.

The antibody produced by the present invention is shampoo,
In addition to cosmetics such as rinse, lotion and cream, it can be widely used in various medical and food fields.

[0023]

INDUSTRIAL APPLICABILITY According to the method of the present invention, in immunization of a bovine animal used for obtaining a large amount of antibody, the method is equivalent to other administration methods such as intramuscular injection without leaving lesions such as swelling and induration on carcass. The antibody titer can be increased, and the antibody can be collected easily and in large quantities. Also, because it is administered to adipose tissue,
It causes less pain to the immunized cattle.

[0024]

The present invention will be described in more detail with reference to the following examples. Prior to that, the cow used for immunization and the method for measuring the antibody titer will be described.

(Test cow) 12 Holstein dairy cows 6
Heads were placed in 2 sections, and the administration site of the antigen was changed for immunization.

(Measurement of antibody titer) The antibody titer in blood was measured by ELI.
It was measured by the SA method. A 96-well plate was coated with the antigen, and a sandwich method was performed using a peroxidase-labeled goat anti-bovine IgG · F (ab ′) ₂ antibody (HAF-413 manufactured by EY Laboratories) as a _secondary antibody.

(Preparation of antigen) 5 g of normal male hair and 5 g of normal female hair were mixed and washed with a 2% sodium polyoxyethylene lauryl sulfate (3 EO) aqueous solution, and then 8 M urea, 0.2 M 2-Mercaptoethanol-containing 0.2 M Tris-hydrochloric acid buffer solution (pH 9.2) (2.5 liters) was added, and the mixture was stirred for 1 hour under nitrogen bubbling at 50 ° C., which was ground using a Teflon homogenizer. This operation was repeated, and the insoluble matter was removed by centrifugation at 10,000 xg for 30 minutes to extract the hair keratin antigen. To this, a solution of 200 g of iodoacetic acid (dissolved in 760 ml of a solution prepared by dissolving 400 g of Tris in advance) was added, and the mixture was stirred and reacted at room temperature for 1 hour. The reaction was stopped by adding 7 ml of 2-mercaptoethanol, and dialyzed against sufficient water.
An insoluble matter was removed through a μm filter to obtain a hair keratin antigen solution (6 liters). Furthermore, 4 parts of this solution was added to 0.
1 part of 5M sodium acetate buffer (pH 4.2) was added (acetic acid was adjusted to pH 4.2), and hair keratin was isoelectrically precipitated. Centrifuge at 1000 xg for 10 minutes,
The supernatant was removed and the precipitate was collected. It was dissolved in physiological saline, sterilized through a 0.2 μm filter, and further concentrated with an ultrafiltration membrane to obtain purified hair keratin antigen (2.6 g as protein).

Examples 1-6, Comparative Examples 1-6 (immunization of cattle) The purified hair keratin antigen solution described above was adjusted to a protein concentration of 20 mg / ml with physiological saline, and the solution and FCA were adjusted to 1: 1. The mixture was mixed at a volume ratio of 1 to prepare a water-in-oil type (hereinafter abbreviated as W / O type) emulsion. Of the 12 test cows described above, 6 of the obtained emulsions were subcutaneously administered to the thoracic epidermis (Examples 1 to 6), and the remaining 6 were administered to the hip muscle (Comparative Examples 1 to 6) (2). 0.5 ml: antigen amount 50 m
g / head). One month later, and one more month later,
Each of the emulsions prepared by FIA and containing the same amount of antigen as the previous time was administered to the same site as that at the time of the first immunization, that is, subcutaneously to the thoracic epidermis or to the muscle of the buttocks for immunization.

Test Example 1 (Measurement of antibody titer) The antibody titer in blood before immunization and one week after the final immunization was measured by ELI.
It was measured by SA.

[0030]

[Table 1]

As can be seen from Table 1, the subcutaneous titration of the thoracic gland also gave an antibody titer equal to or higher than that of the conventionally preferred administration to the hip muscle. In addition, as a result of anatomical findings 2 weeks after the final immunization, inflammation was found in the flesh due to intramuscular injection into the muscle of the buttocks, which was a target of defects, but in the case of subcutaneous injection into the appendix, inflammation in adipose tissue. It was not covered by defects.

Claims (1)

[Claims] 1. A method for immunizing a bovine animal, which comprises administering the antigen subcutaneously to the thoracic thoracic of a bovine animal when the antibody is obtained from the milk or blood of the bovine animal.