JPH08277297A - Immunogen and immunization with the immunogen - Google Patents

Abstract

PURPOSE: To obtain an immunogen enabling to efficiently produce an antibody specific to an antigen on the immunological production of the antibody by binding the antigen to an antigen-binding carrier comprising albumin, thyroglobulin, etc., originated from an animal to be immunized with the antigen. CONSTITUTION: An antigen consisting of e.g. a peptide comprising the N-terminal 10 amino acid residues of interleukin 8 having 77 amino acid residues is reacted with a carrier (e.g. mouse serum albumin) for binding to the antigen, such as albumin or thyroglobulin, originated from a mouse, guinea pig, rabbit or goat in the presence of a condensing agent such as a water-soluble carbodiimide at 4 deg.C for a night. The reaction solution is dialyzed with a phosphate buffer solution at 4 deg.C to obtain the objective carrier-bound immunogen, which enables to efficiently obtain an antibody specific to the antigen, when the antibody is produced with the immunogen.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunogen and an immunization method using the immunogen. More specifically, it relates to a carrier-bound immunogen comprising an antigen and an antigen-binding carrier that is a substance derived from an immunized animal, and an immunization method using the immunogen.

[0002]

2. Description of the Related Art Antibodies obtained from animals immunized with a target antigen are widely used in the fields of missile therapy using the antibodies and in vitro diagnosis using immunoassays. In recent years, a method of chemically synthesizing only an epitope portion specific to a target antigen and using this as an immunogen to obtain a highly specific antibody has been developed. When producing these antibodies, if the molecular weight of the target antigen is small, or if the antigen is a peptide with only an epitope part, etc., KLH, albumin, thyroglobulin, etc. are used to enhance the antigenicity to the immunized animal. A method using a carrier-bound immunogen in which the carrier of (1) is bound to the antigen is widely used.

However, when an antibody is obtained using a carrier-bound immunogen, an antibody against the carrier portion is often obtained, and it has been difficult to efficiently obtain an antibody against the target antigen.

[0004]

Therefore, an object of the present invention is to provide an immunogen that efficiently obtains an antibody specific to the antigen when the antibody is obtained using a carrier-bound immunogen and the immunogen. It is to provide the immunization method used.

[0005]

Means for Solving the Problems As a result of intensive studies to solve the conventional problems, the present inventors have found that an antibody using a carrier-bound immunogen composed of an antigen and an antigen-bound carrier which is a substance derived from an immunized animal. In the acquisition method, it was found that the use of a substance derived from the immunized animal as the carrier enables efficient acquisition of an antibody specific to the target antigen, and the present invention has been completed.

That is, the present invention provides a carrier-bound immunogen comprising an antigen and an antigen-binding carrier which is a substance derived from an immunized animal, and an immunization method using the immunogen.
It is intended to provide a method for efficiently obtaining an antibody specific to a target antigen.

The present invention will be described in detail below.

The immunogen of the present invention means a carrier-bound antigen in which an antigen of interest is bound to a carrier which is a substance derived from an immunized animal.

[0009] The antigen of the present invention generally means an antigen having a small molecular weight that is difficult to exhibit antigenicity by itself, and includes, for example, a hapten, a peptide and the like, specifically, 77-amino acid residue interleukin 8 ( Examples include AVLPRSAKEL (hereinafter abbreviated as LU-10), which is a peptide consisting of amino acids at the N-terminal 10 residues of IL-8 (77). These antigens may be purified from an antigen-containing substance or may be produced by a gene recombination technique, a chemical synthesis technique or the like.

The carrier of the present invention is used as an immunogen by binding to an antigen when producing an antibody against the target antigen, and includes, for example, thyroglobulin, bovine serum albumin (abbreviated as BSA hereinafter), mouse serum. Albumin (hereinafter abbreviated as MSA) and the like can be mentioned.

These carriers are used to immunize an animal as an immunogen by binding with an antigen by a chemical binding method such as a carbodiimide method or a glutaraldehyde method, and the carrier-bound antigen alone or with an adjuvant.

When immunizing with the immunogen of the present invention, a commonly used immunizing method can be used as it is. For example, an in-vivo immunization method, an in vitro immunization method and the like can be mentioned.

A further preferred condition of the present invention is that the antigen is LU.
-10 is the one in which MSA is used as the carrier for the substance derived from the immunized animal. The present inventors obtained hybridomas D77 and E677 that produce monoclonal antibodies that specifically react with LU-10 using LU-10-bound MSA as an immunogen. Also, these hybridomas produce monoclonal antibodies D77 and E677, respectively. Note that these hybridoma D77 and E677 cells were deposited at the Institute of Biotechnology and Industrial Science, and the deposit numbers are FERM P-14665 and FERM P-14, respectively.
666.

[0014]

The present invention will be described in more detail with reference to Reference Examples and Examples.

Example 1 Preparation of LU-10 LU-10 was chemically synthesized with a peptide synthesizer (Model AB1-430A) manufactured by Applied Systems. After LU-10 was purified by HPLC on a reverse phase column, it was confirmed by Edman's method that the amino acid sequence was correct.

Example 2 Preparation of MSA-Bound LU-10 MSA was added to 0.5 mg of LU-10 prepared in Example 1 and reacted with a water-soluble carbodiimide at 4 ° C. overnight to give an MSA-bonded LU.
-10 (hereinafter referred to as LU-MSA) was prepared, and this was used as a phosphate buffered saline solution (NaCl 8g, KCl 0.2g, Na ₂ HPO ₄ 1.15g, KH ₂ P).
It was dialyzed against O ₄ 0.2 g / l) (hereinafter abbreviated as PBS) at 4 ° C. Reference Example 1 Preparation of Thyroglobulin-bound LU-10 In the same manner as in Example 2, porcine-derived thyroglobulin (hereinafter abbreviated as TG) and LU-10 were bound (hereinafter abbreviated as LU-TG).

Reference Example 2 Preparation of silica-bound LU-10 In the same manner as in Example 2, silica (hereinafter abbreviated as SLC) and LU were used.
-10 was bound (hereinafter abbreviated as LU-SLC).

Example 3 Immunization with LU-MSA The LU-MSA prepared in Example 2 was used as an immunogen for immunization. 100 μl of LU-MSA (0.5 mg / ml) was mixed with 100 μl of Freund's complete adjuvant, and BALB / C mice (6 to 8 weeks old) (f
As an initial immunization, 3 animals were subcutaneously injected at 3 sites. The secondary immunization was performed 2 weeks after the initial immunization, and the final immunization was performed 3 days before the cell collection. At this time, 70 μl of LU-MSA was mixed with an equal amount of incomplete Freund's adjuvant, and the whole amount was subcutaneously injected.

Reference Example 3 Immunization of LU-10, LU-TG, LU-SLC LU-10 1 mg / ml prepared in Example 1, LU-SLC prepared in Reference Examples 1 and 2 10 μg / ml, LU- Using TG 36 mg / ml, LU-10 was added to one BALB / C mouse (female) 6 to 8 weeks old, and LU-SLC was 6 to 8 weeks old BALB / in the same manner as in Example 3. LU-TG is 6-8 weeks old BALB / C mice in 2 C mice (female).
(female) 4 animals were immunized respectively.

Example 4 Preparation of LU10-specific monoclonal antibody-producing hybridoma (1) Cell fusion of spleen cells Spleen cells isolated from the mouse immunized in Example 3 were treated with RP.
Suspended in MI-1640 medium. Add 0.17M NH ₄ Cl 5ml,
After allowing to stand for 10 minutes at 0 ° C to hemolyze, it was centrifuged at 1200 rpm for 3 minutes and washed with RPMI-1640 medium. The PAI strain is used as the myeloma cells to be fused, and 3 days before cell fusion, 10 ml of cells in the logarithmic growth phase are used in PRMI-1640 medium containing 10% fetal calf serum (hereinafter abbreviated as FCS) (hereinafter abbreviated as PRMI-1640-FCS). ) After diluting with 90 ml, it was cultured in a 75 cm ² flask. For cell fusion, PEG method and electrofusion method were used.

(2) Preparation and screening of hybridoma Hybridomas were cultured in RPMI-1640-FCS and HAT selection medium and screened by the following method. 1 μ
The hybridoma culture supernatant was dispensed into a well to which 100 μl of g / ml LU-10 was adsorbed, and reacted for 1 hour. After washing with PBS, peroxidase-labeled anti-mouse immunoglobulin goat
IgG (Organon Technical Co., Ltd. 3211-0081) (hereinafter HR
The reaction was carried out for 1 hour with a P-labeled anti-mouse IgG antibody).
After washing well with PBS, 0.4 mg / ml o-phenylenediamine solution was added, and after reacting at room temperature for 10 minutes, the absorbance at 492 nm was measured. 1728 for 18 plates of hybridoma
As a result of dispensing into wells and screening, 9 clones of hybridomas that specifically react with LU-10 were obtained. Two of these clones are D77 and E677. Note that these hybridoma D77 and E677 cells were deposited at the Institute of Biotechnology and Industrial Science, and the deposit numbers are FERM P-14665 and FERMP-146, respectively.
66.

Reference Example 4 Preparation of LU-10-Specific Monoclonal Antibody-Producing Hybridoma A hybridoma producing an LU-10-specific antibody in the same manner as in Example 4 using the mouse immunized in Reference Example 3. Was checked for. LU-10 has 3 plates of 288 well hybridomas, LU-SLC has 12 plates of 1152.
21 wells of hybridoma for LU-TG 2
Although 016 wells of the hybridoma were screened, no hybridoma producing an antibody that reacts with LU-10 was obtained. Although about 20% of the hybridomas obtained from mice immunized with LU-TG reacted with LU-TG,
All had specificity for TG.

[0023]

INDUSTRIAL APPLICABILITY According to the present invention, an immunogen for efficiently obtaining an antigen-specific antibody is provided, and it becomes possible to efficiently obtain a desired antibody by immunization with the immunogen.

Claims (7)

[Claims] 1. A carrier-bound immunogen comprising an antigen and an antigen-bound carrier which is a substance derived from an immunized animal. 2. The immunogen according to claim 1, wherein the carrier is a substance derived from mouse, guinea pig, rabbit or goat. 3. The immunogen according to claim 1, wherein the carrier is albumin. 4. The immunogen according to claim 1 or 2, wherein the carrier is thyroglobulin. 5. The immunogen according to claim 3, wherein the carrier is mouse serum albumin. 6. The immunogen according to claim 5, wherein the antigen is a peptide consisting of N-terminal 10 amino acid residues of 77 amino acid residue interleukin 8. 7. An immunization method using the immunogen according to claim 1.