WO2005053740A1

WO2005053740A1 - Immune enhancer and enhanced antigen obtained therewith

Info

Publication number: WO2005053740A1
Application number: PCT/JP2004/017927
Authority: WO
Inventors: Yasufumi Murakami; Nobuhiko Hasegawa
Original assignee: Bio Matrix Research Inc.
Priority date: 2003-12-02
Filing date: 2004-12-02
Publication date: 2005-06-16
Also published as: JPWO2005053740A1

Abstract

An immune enhancer that realizes immune with an efficiency superior to that of prior art methods; and an enhanced antigen obtained therewith. There is provided an immune enhancer comprising a styrene-based porous material and/or hydroxyapatite as a main component. It is preferred that the styrene-based porous material be crosslinked-styrene-based porous polymer beads and that the hydroxyapatite material be porous beads. Further, there is provided an enhanced antigen comprising a carrier containing the immune enhancer and an antigen adsorbed on the carrier.

Description

Specification

Immune enhancer and enhanced antigen using the same

Technical field

The present invention relates to an immunopotentiator that enhances the immunogenicity of an antigen and activates an immune reaction artificially performed in a living body, and an enhanced antigen using the same.

Background art

[0002] Monoclonal antibodies have been used for the purpose of detecting, isolating, identifying, and purifying various biological substances, mainly proteins, regardless of academic research or commercial purposes. The production of this monoclonal antibody is currently performed as follows.

[0003] First, an experimental animal (usually a mouse or a rat) is immunized with a target antigen substance to produce an antibody in the animal body, and then antibody-producing cells are removed from the animal body. Thereafter, the antibody-producing cells and myeloma cells are fused to obtain hybridoma cells. Hybridoma cells proliferate semipermanently like cancer cells and secrete the desired antibody into the cell culture medium.

[0004] In the production of this monoclonal antibody, a conventional antigen is mixed with an adjuvant in order to enhance the immunogenicity of the antigenic substance, activate the immune reaction and obtain the desired monoclonal antibody-producing cells in good yield, A method has been adopted that enhances the immune response by injection into the animal body. The use of adjuvants has the effect of increasing the overall immune response locally upon injection and promoting the aggregation of immunocompetent cells. Also, by forming an emulsion of the antigen and the adjuvant, the antigen can be present at the injection site without being decomposed for a long period of time.

[0005] In addition, as a method for enhancing the immunogenicity of an antigen, a method of binding a hapten to a carrier protein or binding a peptide to a protein to prepare an antibody against a specific peptide is known. Being done.

[0006] Further, as a method for enhancing immunogenicity without using an adjuvant, a method using nitrocellulose beads is known as disclosed in Patent Document 1. According to the method disclosed in Patent Document 1, the amount of antigen to be administered is the same as that of a normal adjuvant method. Immunity can be established even with about one-hundredth.

Patent Document 1: Patent No. 2089076

Disclosure of the invention

Problems to be solved by the invention

[0007] Certainly, the method disclosed in Patent Document 1 has made it possible to improve the efficiency of immunization. However, for more detailed analysis of trace biological components and more precise proteome analysis, more efficient immunity is required. There is a need for enhanced immunization techniques. In the method of Patent Document 1, since nitrocellulose is used as a carrier, nitrocellulose itself is often recognized as an epitope when performing Western blotting. It leaves room for.

Accordingly, an object of the present invention is to provide an immunopotentiator capable of immunizing with a higher efficiency than conventional methods, and an enhanced antigen using the same, in view of the above situation.

Means for solving the problem

[0008] As a result of intensive studies conducted by the present inventors to achieve the above-mentioned object, it was found that the use of a porous material made of styrene significantly increased immunogenicity, and completed the present invention. I came to.

That is, the present invention includes the following.

(1) An immunopotentiator containing a styrene-based porous material as a main component.

(2) The immunopotentiator according to (1), wherein the styrene-based porous material is a crosslinked styrene-based porous polymer bead.

(3) An enhanced antigen comprising a carrier containing the immunopotentiator according to (1) or (2) and an antigen adsorbed on the carrier.

(4) An immunization method comprising immunizing an animal with the enhanced antigen according to (3).

(5) An immunopotentiator containing a hydroxyapatite material as a main component.

(6) The immunopotentiator according to (5), wherein the hydroxyapatite material is a porous bead.

(7) An enhanced antigen comprising a carrier containing the immunopotentiator according to (5) or (6) and an antigen adsorbed on the carrier. (8) An immunization method comprising immunizing an animal with the enhanced antigen according to (7).

[0010] According to the immunity enhancer of the present invention, immunization can be performed with an efficiency far exceeding that of the conventional method. Further, according to the enhanced antigen of the present invention, even if the amount of the antigen is very small, it can exhibit excellent immunogenicity and does not react with the trocellulose membrane commonly used in Western plots. Antibodies can be efficiently produced. Therefore, the present invention can greatly contribute to detailed analysis of minute biological components and more precise proteome analysis.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

The immunopotentiator to which the present invention is applied contains a styrene-based porous material and / or a hydroxyapatite material as a main component. The enhanced antigen to which the present invention is applied is composed of a carrier containing an immunopotentiator and an antigen adsorbed on the carrier.

Further, the present invention relates to a method for immunizing an animal with the above-mentioned enhanced antigen.

[0012] i) Immunopotentiator

The present inventors examined the ability as an immunopotentiator when various substances having protein adsorption ability other than nitrocellulose beads were used as a carrier. As a result, they found that when a styrene-based porous material or hydroxyapatite was used as an immunopotentiator, an excellent immunopotentiating effect was exhibited as compared with conventional nitrocellulose-based immunopotentiators.

Here, the styrene-based porous material means a material having styrene and / or a styrene derivative as a main component and being porous. Examples of the styrene derivative include a crosslinked styrene-based polymer and the like. The term “porous” as used herein refers to a structure that extends to the inside of fine continuous pore particles called pores. With this structure, the beads have a large surface area comparable to activated carbon and can efficiently adsorb proteins in an aqueous solution.

The hydroxyapatite used in the present invention is calcium hydroxide phosphate represented by the composition formula Ca5 (OH) (P04) 3, and is known to adsorb high polyvalent anions such as proteins and nucleic acids. Hydroxyapatite as an immunopotentiator has its shape and size. The size and the like are not particularly limited, and for example, commercially available porous beads can be used.

The fact that the styrene-based porous material is the main component in the immunopotentiator means that contaminants other than styrene and / or a styrene derivative may be contained in an amount of about 5 to 10% by weight. However, it is preferable that the immunopotentiator is a substance which does not contain contaminants and has only styrene and / or a styrene derivative.

The expression that hydroxyapatite is the main component in the immunopotentiator means that contaminants other than hydroxyapatite may be contained in an amount of about 5 to 10% by weight. However, it is preferable that the immunopotentiator is a substance which does not contain contaminants and has only hydroxyapatite.

An immunopotentiator containing a styrene-based porous material or hydroxyapatite as a main component is more excellent in antigen-adsorbing ability than nitrocellulose. Further, since the immunopotentiator is not present in the living body, it is recognized as a foreign substance by the immunocompetent cells, and the aggregation of the immunocompetent cells is promoted. For this reason, the antigen adsorbed on the immunopotentiator is easily recognized as a foreign substance. Furthermore, an immunopotentiator containing a styrene-based porous material as a main component and an immunopotentiator containing the above hydroxyapatite material as a main component have low cytotoxicity and do not inhibit the in vivo immune system.

ii) Enhanced antigen

The enhanced antigen to which the present invention is applied is composed of a carrier containing the above-mentioned immunopotentiator and an antigen adsorbed on the carrier. The antigen is not particularly limited as long as it is a substance that causes an antigen-antibody reaction. In particular, a substance that is a trace biological component or a component having low antigenicity and that cannot be obtained by an ordinary method can be used as the antigen. Examples of trace biological components or components with low antigenicity include blood trace components, various lymphokines, various enzymes, various regulatory proteins, various intercellular substrates, various cytoskeleton components, various cell membrane components, and the like. it can. Examples of antigens other than pure proteins include polysaccharides, glycoproteins, protein carbohydrates, glycolipids, lipids, lipoproteins, and polyglycerol phosphate. In addition, it is possible to use components such as pathogenic bacteria and viruses with low immunity as antigens. To adsorb the antigen to the carrier means that the above-mentioned antigen is physically bound to the surface and / or inside of the above-mentioned immunopotentiator, and that the above-mentioned antigen is chemically bonded to the surface of the above-mentioned immunopotentiator. Means The method of adsorbing the antigen to the carrier is not particularly limited, and for example, a method of adding an immunopotentiator and an antigen to a solution and stirring the solution can be adopted.

iii) Usage of enhanced antigen

The above-mentioned enhanced antigen has a very small amount of antigen or weak immunogenicity and has an antigen.Since it can efficiently agglutinate immunocompetent cells, it can efficiently produce an antibody against the target antigen. Can be guided. As an immunization method using the enhanced antigen, a conventionally known method without any particular limitation can be applied. Animals to be immunized include rats, mice, egrets, goats, sheep, higgins, wilds, musters, and birds.

Examples of the use of the above-mentioned enhanced antigen include, for example, (1) use when immunizing an animal to produce various monoclonal or polyclonal antibodies, and (2) use as an animal or human vaccine. can do.

When using an enhanced antigen to produce a monoclonal antibody,

However, since the immunopotentiator has an excellent immunopotentiating effect as compared to the conventional trotrocellulose-based immunopotentiator, it is sufficient if about 0.3-0.3 g of the antigen protein is present. is there. 0.03-0. 0.3 g of antigen protein together with the above-mentioned immunopotentiator, for example, is added to a solution such as a phosphate buffer, and mixed to prepare an enriched antigen for producing monoclonal antibodies. be able to. The prepared enhanced antigen is used to immunize an animal according to a standard method. At this time, according to the enhanced immunity to which the present invention is applied, immunization can be performed with a very small amount of an antigen protein. Specifically, the amount of antigen protein is about 0.03-0. Per animal. As described above, the enriched antigen to which the present invention is applied has a very small amount of antigen that cannot be immunized by the conventional method (i.e., one-thousandth of the conventional adjuvant method, nitrocellulose). Therefore, even if it is less than one tenth of the bead method, immunization can be achieved. Using an antigen that was difficult to obtain, a monoclonal antibody against the antigen can be obtained at low cost. By using the monoclonal antibody obtained in this way, it is possible to confirm the location of the trace in-vivo component in the body, to inhibit the physiological activity of the trace in-vivo component, and to determine the physiological activity of the trace in-vivo component. It becomes possible to purify the inhibitory substance. Therefore, the enhanced antigen to which the present invention is applied is considered to be applied as an academic tool for obtaining a deeper understanding of the physiological activity of a trace amount of a body component. In addition, the enhanced antigen to which the present invention is applied can be applied to the production of application tools in the medical, food, and environmental fields with higher accuracy and a wider range of application.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to the following Examples.

Example 1

[0016] Evaluation of beads by protein adsorption ability

In this example, the protein adsorption ability of various materials was examined. Specifically, “Diaion (trademark)” manufactured by Mitsubishi Chemical Corporation was used as a styrene-based porous material, and “BIO-GELR HTP” manufactured by Bio-Rad Laboratories was used as hydroxyapatite beads. For example, -trocellulose beads disclosed in Japanese Patent No. 2089076 and chitosan beads "Chitopearl (trademark)" manufactured by Fuji Boseki Co., Ltd. were used.

Equal amounts of the various bead pellets and 500 μg Zml of BSA solution were mixed, and incubated at room temperature for 0 to 2 hours with periodic stirring. At each time, the amount of protein in the supernatant was quantified, and the reduced amount was compared with the protein adsorption ability. Figure 1 shows the results. As shown in FIG. 1, "hydroxyapatite" and "Diaion" have been shown to have better protein adsorption capacity than trocellulose beads or chitopearl disclosed in Japanese Patent No. 2089076. Therefore, in the following examples, comparison of the antibody-producing ability was performed with nitrocellulose beads, hydroxyapatite and diamond ions. Example 2

Production of C-terminal enhanced antigen of KIAA1259 and candidate

Powdered trocellulose beads (2 ml), hydroxyapatite (2 ml) and diaion (2 ml) were each suspended in phosphate buffer (2-4 ml) to prepare a pellet. Next, the C-terminal (36 kDa) of KIAA1259 was purified from the recombinant protein expression system of E. coli. KIAA1259 is a human gene product (180 kD) that shows high similarity in amino acid sequence homology to one of 134 helicases found in yeast, and its function is unknown at present. Here, it was used only as a model protein. A DNA fragment encoding the C-terminal of KIAA1259 was inserted into a pET32b plasmid (vector) to obtain an expression construct. Escherichia coli (BL21) was transformed using the expression construct, and the C-terminus of His-tagged KIAA1259, in which protein expression was induced, was sonicated with E. coli, and the cell lysate (cell lysate (cell lysate) was crushed). The KIAA1259 C-terminal was purified by Cobalt-fixed metal affinity chromatography, and the purified KIAA1259 C-terminal 1 or g became 25-50% (vZv) in phosphate buffer. Was mixed with 500 1 of each bead solution suspended as described above. As a result, KIAA1259 was adsorbed to each bead. Next, the beads were recovered by centrifugation and washed with a phosphate buffer. After washing, each bead recovered as a precipitate was suspended in a phosphate buffer solution to a concentration of 25-50% (vZv) to prepare an injection solution.

Example 3

[0018] Immunization treatment

0.5 ml of the injection solution prepared in Example 2 was intraperitoneally injected into 2 to 3 Balb / cByJ Jcl (female) 6 to 8 weeks old. By injecting 0.5 ml of the injection solution per animal, one has immunized beads corresponding to the C-terminus of KIAA1259.

Four, five, six, seven and eight weeks later, booster immunization was performed by the same method using 10 g of KIAA1259 beads corresponding to the C-terminal of each mouse.

Example 4

[0019] Comparison of increase in antibody titer between the nitrocellulose bead method and the method of the present invention

After 6 weeks and 8 weeks, blood was collected from the mouse ocular vein (100-5001), and the blood was coagulated and centrifuged to obtain serum.

Next, the serum was diluted to a dilution of 500-32000 with 1% bovine serum albumin solution. A diluent was prepared. In order to measure the antibody titer in the obtained serum diluent, a 96-well plate for Atsey to which C-terminal of 100 ng of KIAA1259 was adsorbed was prepared. The antibody titer in the serum diluent was measured using this plate. As the measurement method, an enzyme-linked immunosorbent assay was used, which was an indirect method in accordance with a standard method (Cell Engineering Separate Volume Experimental Protocol Series Anti-Peptide Antibody Experimental Protocol) Shinobu Oumi Kunio Tsujimura Masaaki Inagaki. The results are shown in Figure 2 (after 6 weeks) and Figure 3 (after 8 weeks).

As shown in Figs. 2 and 3, compared with the enhanced antigen using nitrocellulose beads as the immunopotentiator, the enhanced antigen using diamond ion, which is a crosslinked styrene-based porous polymer bead, as the immunopotentiator was more effective. Excellent antibody titers could be achieved. Also, with hydroxyapatite, an antibody titer equal to or higher than that of nitrocellulose beads could be obtained. This clearly shows that crosslinked styrene-based porous polymer beads (Diaion) have a superior immunopotentiating effect compared to immunopotentiators consisting of nitrocellulose beads. Was. Also, it was shown that hydroxyapatite also exhibited an immunopotentiating effect equal to or higher than that of an immunopotentiator composed of nitrocellulose beads.

In addition, and chitopearl (see Example 1) having relatively excellent protein adsorption ability, although having almost the same protein adsorption ability as nitrocellulose, an immunopotentiating effect was not recognized.

Brief Description of Drawings

[FIG. 1] FIG. 1 is a characteristic diagram showing the results of comparing the protein adsorption capacities of nitrocellulose beads, chitopearl, hydroxyapatite and diamond (styrene-based porous material).

[Fig. 2] Fig. 2 is a characteristic diagram showing the results of comparing the antibody titers of sera collected 6 weeks after immunization using each bead (nitrocellulose beads, hydroxyapatite, and diamond).

[FIG. 3] FIG. 3 is a characteristic diagram showing the results of immunization using each bead and comparing antibody titers in sera collected 8 weeks later.

Claims

The scope of the claims

[1] An immunopotentiator containing a styrene-based porous material as a main component.

[2] The immunopotentiator according to claim 1, wherein the styrene-based porous material is a crosslinked styrene-based porous polymer bead.

[3] An enhanced antigen comprising a carrier containing the immunopotentiator according to claim 1 and an antigen adsorbed to the carrier.

[4] An immunization method comprising immunizing an animal with the enhanced antigen according to claim 3.

[5] An immunopotentiator containing a hydroxyapatite material as a main component.

6. The immunopotentiator according to claim 5, wherein the hydroxyapatite material is a porous bead.

[7] An enhanced antigen comprising a carrier containing the immunopotentiator according to claim 5 and an antigen adsorbed on the carrier.

[8] An immunization method comprising immunizing an animal with the enhanced antigen according to claim 7.