JPH075640B2 - Protein immobilization method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for immobilizing and recovering a protein by utilizing the reaction between an anti-hapten monoclonal antibody and a protein-hapten conjugate.

[Conventional technology]

If the protein can be specifically immobilized on the carrier, the target protein can be efficiently obtained by recovering the immobilized protein. Further, by immobilizing a protein, if the protein is an enzyme, it can be repeatedly used as an immobilized enzyme.

For immobilizing proteins, there is a method of using an antibody that utilizes a group-specific adsorbent that acts on a functional group.

Is an amino group (-NH _2), carboxyl group (-COOH), a a hydroxyl group (-OH), a is a fixing method utilizing the binding between side chains of amino acids constituting such a thiol group (-SH), a protein . Amino acids having these functional groups are commonly contained in proteins. Therefore, there is a drawback that the target protein cannot be specifically fixed. The group-specific adsorbent of is a substance that recognizes a structure common to certain proteins, and is an immunoglobulin (I: Immunoglobulin G)
The protein A specifically binds to the Fc portion, the lectin specifically binds to the sugar chain of a glycoprotein, and concanavalin A. However, group-specific adsorbents are found only in a small number of limited proteins, and when one subclass (eg IgG1) is purified from a mixed sample of IgG subclasses (IgG1, IgG2, etc.), etc. Requires a more specific immobilization method. Is a method of immobilizing a protein using an antibody prepared using a protein as an antigen, or conversely, a method of immobilizing an antibody protein using an antigen. This method is superior to that in that the target protein can be specifically immobilized and the method is versatile. That is, by using a monoclonal antibody, the target protein can be immobilized very specifically. In addition, it is possible to obtain an antibody against all proteins, which is a highly versatile method.

[Problems to be Solved by the Invention]

Among the above-mentioned conventional techniques, the method using a monoclonal antibody is widely used, but it takes several months or more to produce a monoclonal antibody,
Sensitization conditions such as differences in immunogenicity depending on the type of antigen and individual differences in animals to be immunized must be taken into consideration, and monoclonal antibodies with various affinity for the antigen are generated. There was a problem in that cross-reactivity with multiple antigens must also be considered.

It is an object of the present invention to provide a method for immobilizing a protein that eliminates the above-mentioned drawbacks of the conventional techniques.

[Means for Solving the Problems]

In order to achieve the above-mentioned object, the present invention is characterized in that an anti-hapten monoclonal antibody is produced, a protein to which a hapten is bound is reacted with an immobilized anti-hapten monoclonal antibody, and the protein is immobilized or recovered. is there.

A hapten is a small molecule that becomes an antigen for the first time when bound to a polymer. Dinitrophenyl (DN
P: Dinitrophenyl), Trinitrophenyl (TNP: Trinitr
ophenyl), nitrophenyl (NP: Nitrophenyl), steroid hormones and the like. The hapten used in the present invention is preferably one that easily binds to a protein. For example, DNP is an amino group less than N, ε-amino group of lysine, hydroxyl group of tyrosine, imidazole group of histidine,
Easily binds to the thiol group of cysteine.

In the present invention, it is not necessary to make monoclonal antibodies against individual proteins. That is, if the hapten is bound to the protein, the protein / hapten conjugate can be immobilized using one kind of anti-hapten monoclonal antibody.

A monoclonal antibody is a single antibody selected from a myriad of antibodies in vivo. The technology for producing monoclonal antibodies was first reported in 1975 by Koehler and Milstein. Monoclonal antibodies have contributed greatly to the field of biotechnology by increasing the usefulness of antibodies. At present, monoclonal antibodies are used in a wide range of fields such as medicine and biology. [Gee. Koehler and Sea. Milstein, Nature (London). 256,495 (1975). ] A general method for obtaining a monoclonal antibody will be described below. A substance (antigen) that becomes a foreign substance to animals is parenterally administered to mammals such as mice and rats. That is, the antigen is injected into a vein, muscle or abdominal cavity.
In this way, the proliferation of lymphocyte B cells that produce antibodies that specifically react with the antigen can be promoted. next,
The spleen of a mammal immunized with the antigen is removed. The spleen is an organ rich in lymphocyte B cells. Therefore, a large amount of antibody-producing cells can be obtained by culturing spleen cells.
However, B cells cannot be maintained long in vitro. Therefore, it is fused with a myeloma cell having high proliferative ability to obtain a fused cell called a hybridoma. Since the hybridoma has both B cell and myeloma genes, it can continue to grow while producing antibodies. Cell fusion is performed using polyethylene glycol, HJV virus, or electric current. Myeloma is an enzyme required for nucleic acid synthesis, hypoxanthine-guanine-
Phosphoribosyl transferase or thymidine kinase is deleted. Although cell fusion occurs in a random combination, when the fused cells are cultured in a medium called Hat medium, only hybridomas that have acquired the enzyme gene of B cells can survive.

Screening is performed to select a hybridoma that produces an antibody that recognizes the target antigen (primary screening). For the screening, an assay method called enzyme-linked immunosorbent assay (ELISA) is applied. In this method, the presence or absence of an antigen-antibody reaction is detected by the color development of the substrate.

Multiple types of antibodies react with one antigen. Therefore, multiple types of hybridomas are obtained by the screening. Therefore, the hybridoma obtained in the primary screening was diluted to one cell and grown,
Use as a single cell population (clone). The operation of cloning a hybridoma is called cloning. Then, screening is performed again by ELISA to select hybridoma clones that produce the desired antibody.

The clones obtained here are examined for antibody-producing ability, antibody affinity with antigen, antibody cross-reactivity, etc., and the target hybridoma is cultured, and the culture supernatant is recovered. The desired monoclonal antibody can be obtained by purifying the antibody from the culture supernatant. The antibody is purified by ultrafiltration, ammonium sulfate precipitation,
It is carried out by combining known methods such as dialysis and chromatography.

In order to produce an anti-hapten monoclonal antibody, first, the hapten was added to bovine serum albumin (BSA: Bovine Serum Album
in), keyhole limpet hemocyanin (Keyhole Li
It is bound to macromolecules such as mpet hemocyanin) and ovalbumin (OVA). If a protein is used as the polymer that binds to the hapten, a conjugate can be easily produced.

Mammals such as mice and rats are immunized with the prepared protein-hapten conjugate as an antigen. Immunization is carried out according to a general method, for example, by injecting the antigen intraperitoneally and then 10
Do several injections every day to 2 weeks. As a result, proliferation of lymphocyte B cells that produce anti-hapten antibody can be promoted.

Next, the spleen of the mammal is extracted and fused with myeloma to prepare a hybridoma. To select anti-hapten antibody-producing hybridomas,
Perform primary screening by ISA. The obtained anti-hapten antibody-producing hybridoma is subjected to secondary screening to obtain a clone of the anti-hapten monoclonal antibody-producing hybridoma.

The cloned anti-hapten antibody-producing hybridoma is cultured, and the culture supernatant is collected. An anti-hapten monoclonal antibody can be obtained by purifying the antibody from the culture supernatant. The antibody is purified by combining known methods such as ultrafiltration, ammonium sulfate salting-out, dialysis, and chromatography.

To immobilize the protein-hapten conjugate, the hapten monoclonal antibody must first be immobilized on a carrier. As a method of immobilizing the antibody, a method of using a secondary antibody that utilizes a group-specific adsorbent that utilizes the functional group of the side chain of the antibody is considered.

Is a method of utilizing an amino group, a carboxyl group, a hydroxyl group, a thiol group, etc., which are side chains of amino acids constituting an antibody molecule, for binding. For example, when glutaraldehyde is used as a cross-linking agent, an antibody can be immobilized on a glass surface. For immobilizing an antibody (particularly IgG), protein A that specifically binds to IgG is insolubilized. IgG can be immobilized by using a binding carrier.

Is a method in which a secondary antibody that binds to an anti-hapten monoclonal antibody is immobilized by the method described above, and then the anti-hapten monoclonal antibody is immobilized via the immobilized secondary antibody.

By the above method, the anti-hapten monoclonal antibody,
If immobilized on a carrier such as gel or glass surface, utilizing the antigen-antibody reaction with the immobilized anti-hapten monoclonal antibody,
The protein-hapten conjugate can be immobilized.

The immobilized protein-hapten conjugate has a pH of 1 to 4
It can be recovered by lowering it to a certain degree and dissociating the hapten from the anti-hapten monoclonal antibody.

[Action]

It is an effective means to use a monoclonal antibody to immobilize or recover a target protein. However, it is not easy to produce the desired monoclonal antibody. Therefore, if a protein-hapten conjugate is prepared and an immobilized anti-hapten monoclonal antibody is used,
The target protein can be immobilized without producing a monoclonal antibody against each protein.

〔Example〕

Hereinafter, embodiments of the present invention will be described with reference to FIG. 1, but the present invention is not limited to these embodiments.

FIG. 1 shows immobilization of anti-DNP antibody 2 on glass beads /, binding of papain 4-DNP3 conjugate thereto, and immobilization of papain 4. The carrier for immobilizing the anti-DNP antibody may be a glass substrate, a plastic substrate, or a polymer resin. Also, other enzymes may be used in place of papain.

The present Example is characterized in that an immobilized enzyme can be easily produced via an anti-hapten monoclonal antibody by binding a hapten such as DNP to the enzyme.

Furthermore, the enzyme can be reused, which is advantageous when using an expensive enzyme.

(1) Preparation of anti-DNP monoclonal antibody a. Preparation of BSA-DNP conjugate 1-fluoro-2,4-dinitrobenzene (FDNB: 1-fluoro)
-2,4-dinitrobenzene) 10 ml and dioxane 2 ml are mixed,
0.75% BSA dissolved in bicarbonate buffer (pH 9.0)
(20 ml) added to the solution and incubated for 24 hours at room temperature in the dark. The reaction solution was subjected to ultrafiltration, and the retentate was replaced with 0.1 M phosphate buffer (pH 7.4) by ultrafiltration, which was used as an antigen. The protein concentration in the antigen was 6 mg / ml. BSA and FDNB obtained from the maximum absorption value of DNP (360 nm)
The reaction efficiency of was 79%.

b. Immunization of mice with BSA-DNP conjugate BSA-DNP conjugate (10
0 μg and 40 μg after the second time, together with 90 μg of Freund's complete adjuvant, 3 ml of 0.1 M phosphate buffer (pH
7.4), and mix 500 μl of this mixture with mouse (BALB
/ c, 9 weeks old), and was intraperitoneally administered, and thereafter, the treatment was continued 3 to 7 times in a 2-week cycle.

c. Preparation of anti-DNP antibody-producing hybridoma The spleen of a mouse immunized with the BSA-DNP conjugate was extracted, fused with myeloma using polyethylene glycol (molecular weight 1500), and hybridoma was selected in a hat medium. The hybridomas obtained here contained DNP, BSA and BSA and D.
It contains three types of cells that produce antibodies that simultaneously recognize NP. Therefore, when screening anti-DNP antibody-producing cells, ELISA was performed using three types of antigens, BSA-DNP conjugate, BSA and poly-L-lysine-DNP conjugate, and BS
Those secreting antibodies positive for the A-DNP conjugate and poly-L-lysine-DNP conjugate and negative for BSA were selected as anti-DNP antibody-producing hybridomas. Subsequently, cloning and screening were performed to obtain 5 clones of anti-DNP monoclonal antibody-producing hybridoma. Table 1 shows the cell lines and the types of antibodies produced.

d. Purification of anti-DNP monoclonal antibody The anti-DNP antibody-producing hybridoma (4-38-10 strain) was cultured in a serum-free medium, and the culture supernatant 1 was sterilized by filtration, followed by ultrafiltration and ammonium sulfate precipitation. . Further, the anti-DNP monoclonal antibody was purified using a hydroxyapatite column (manufactured by Bio-Rad). As a result, a 517 μg / ml IgG solution was obtained.

(2) Immobilization of papain a. Preparation of papain-DNP conjugate 0.38% papain (20 ml) dissolved in 0.1M bicarbonate buffer (pH 9.0) was mixed with FDNB to obtain papain-DNP conjugate. It was After removing unreacted FDNB of the reaction solution by ultrafiltration, 0.1M phosphate buffer (pH 7.4) was added to the retentate, and ultrafiltration was performed again to obtain a solution containing papain-DNP conjugate. Obtained.

b. Preparation of immobilized papain The surface of glass beads (φ6.4 mm) was aminosilanized using 36 mM N-β (aminoethyl) -γ-aminopropyltrimethoxysilane solution (manufactured by Chisso). After washing, it was treated with a 2.5% glutaraldehyde solution, and the anti-DNP monoclonal antibody solution was reacted in 0.1 M phosphate buffer (pH 7.4) to obtain an antibody conjugate. Daddy Pain here-DNP
Immobilized papain was prepared by adding the conjugate and incubating for 1 hour at 37 ° C. in the same manner in 0.1 M phosphate buffer (pH 7.4).

FIG. 2 schematically shows digestion of mouse IgG with immobilized papain obtained in the above-mentioned example. Mouse IgG5
Is decomposed into Fab6 and Fc7 by the immobilized papain shown in FIG. A concrete example of this is as follows.

Mouse IgG was digested with papain according to the method of Porter.
1 mg of mouse IgG was mixed with glass beads (bonding 10 μg of papain) to which papain-DNP conjugate was immobilized, and mixed with 10 ml of 10 mM.
Cysteine-2 mM EDTA-0.1 M phosphate buffer (pH 7.0)
The reaction was carried out at 37 ° C. overnight. Then, the reaction was stopped by adding iodoacetamide to a final concentration of 10 mM.
Mouse IgG was decomposed into Fab and Fc by the action of papain. The decomposition of Fab and Fc was performed using a hydroxyapatite column (manufactured by Bio-Rad). "R Earl Porter, Biochemical Journal. 73,119.
(1959). The papain-immobilized glass beads were washed and then stored in 0.1 M phosphate buffer (pH 7.4) at 4 ° C.

〔The invention's effect〕

According to the present invention, by binding a hapten to a specific protein, the protein can be easily immobilized using an anti-hapten monoclonal antibody, which has an effect of facilitating utilization and recovery of the protein function.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing the anti-DNP antibody according to one embodiment of the present invention immobilized on glass beads, and FIG. 2 shows the enzymatic reaction by immobilized papain according to one embodiment of the present invention. It is a schematic diagram. 1 ... Glass beads, 2 ... anti-DNO monoclonal antibody, 3 ... DNP, 4 ... papain, 5 ... mouse IgG, 6
... Fab, 7 ... Fc.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Norio Shimizu 2520, Akanuma, Hatoyama-cho, Hiki-gun, Saitama, Ltd. Inside the Basic Research Laboratory, Hitachi, Ltd. (56) References JP-B-57-28274 (JP, B1)

Claims (4)

[Claims] 1. A step of chemically binding a hapten to a protein and thereafter obtaining a protein solution from which unreacted hapten has been removed, a step of chemically binding an anti-hapten monoclonal body to a carrier, and the protein solution. Contacting the carrier, to carry out an antigen-antibody reaction between the hapten and the antibody,
A method for immobilizing a protein, comprising the step of immobilizing the protein on the carrier. 2. A dinitrophenyl (DNP: Dinitrophenyl)
The method for immobilizing a protein according to claim 1, wherein is a hapten. 3. A method of immobilizing a protein for recovering a protein-hapten conjugate using an anti-hapten monoclonal antibody immobilized on a carrier. 4. The third aspect of the present invention, wherein DNP is a hapten.
The method for protein immobilization according to the item.