JPH10139685A - Adjuvant comprising synthetic bilayer membrane and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an adjuvant suitable for improving antibody production in animal, by using a synthetic bilayer membrane of a specific structure. SOLUTION: This adjuvant comprises a synthetic bilayer membrane formed from an amphipathic compound having a hydrophobic part and a cationic hydrophilic part. Preferably, the synthetic bilayer membrane together with an antigen such as a water-soluble protein is dissolved in a buffer solution or a physiological saline solution to immunize an animal. The membrane is obtained, for example, by a method for dissolving an amphipathic compound of the formula ((m) is 12 or 14; (n) is 2, 4, 6 or 8) in chloroform, etc., removing the solvent by evaporation and forming a thin film.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel adjuvant and a method for using the same, and more particularly, to an adjuvant suitable for improving antibody production in animals.

[0002]

2. Description of the Related Art In recent years, with the increasing number of patients with food allergies and hay fever, these immunity-related illnesses have been highlighted and have attracted public attention. Research themes related to immune response are also increasing in academic societies. In such studies, it is essential to use a laboratory animal to produce a specific antibody, and therefore, an effective adjuvant is required.

[0003] In various fields other than immunological research, molecular recognition has become an important technique, and simple production of specific antibodies capable of capturing substances at the molecular level has been demanded. In fields other than these immunological studies, specific antibodies to substances that are particularly difficult to produce antibodies are often required, and the potential demand for new adjuvants is great.

[0004] Adjuvants, which are reagents for improving the antibody-producing ability of experimental animals and the like, have been devised for a long time with various mechanisms. Incomplete Freund's adjuvant (FIA), a basic adjuvant, is a W / O emulsion of an aqueous solution of an antigen and liquid paraffin, and the antigen solution is wrapped in liquid paraffin, whereby the antigen is rapidly degraded in the experimental animal body And enhances antibody production by continuously providing antigen stimulation for a long period of time. At present, Freund's complete adjuvant (FCA), which is most commonly used, is a mixture of FIA and killed Mycobacterium tuberculosis. In addition to the same function as FIA, various biomass contained in killed Mycobacterium tuberculosis is included. Molecules stimulate the immune system of experimental animals and increase antibody production. These emulsion-based adjuvants have a long track record and are preferably used as adjuvants.Emulsion formation requires more than one hour, and has high viscosity.
Injection into experimental animals requires unique know-how and is difficult to handle. FCA has the drawbacks that it contains dead bacteria of Mycobacterium tuberculosis and may increase the production of unnecessary antibodies.

[0005] In addition, aluminum hydroxide is often used. This is a powdery fine particle and is insoluble in water.
This aluminum hydroxide is dispersed in the antigen solution, incubated for about 30 minutes, and used for injection. During this time, the antigen in the solution is adsorbed on the aluminum hydroxide surface, thereby preventing the antigen from being rapidly degraded in the body of the experimental animal. FIA
Compared to FCA and FCA, antigen presenting cells have more opportunities to come in contact with antigen, are easier to handle, and contain no impurities. However, depending on the surface charge and molecular weight of the target, the target may not be adsorbed on the aluminum hydroxide.

[0006]

Means for Solving the Problems and Effects of the Invention The inventor of the present invention has conducted various studies to solve the above-mentioned problems, and has found no use of a synthetic bilayer membrane. A new type of adjuvant was obtained.

A synthetic bilayer membrane is obtained by dispersing a synthetic amphipathic compound having a hydrophobic part and a hydrophilic part in water, and has been confirmed to have a structure similar to a bilayer membrane of a biological lipid. I have. This synthetic bilayer has a high self-assembly property, and chemical synthesis using this self-assembly property as a template has been realized.

The present inventor has previously developed a gene transfer reagent utilizing the properties of a synthetic bilayer membrane (Japanese Patent No. 1984).
737, JP-A-7-20429). The present inventors have now surprisingly found that when an animal is immunized with a synthetic bilayer having such a specific structure together with an antigenic substance, the ability to produce a specific antibody against the antigen is extremely improved,
The present invention has been led.

That is, the present invention provides an adjuvant for animal immunization comprising a synthetic bilayer formed from an amphiphilic compound having a hydrophobic part and a cationic hydrophilic part.

[0010] The adjuvant of the present invention can be basically applied to all uses for immunizing animals, such as veterinary vaccines.
Especially suitable for getting.

When the adjuvant of the present invention is used, the ability to produce antibodies during animal immunization is significantly improved. Thus, in another aspect, the present invention provides a method of using such an adjuvant, comprising forming a synthetic bilayer from an amphipathic compound having a hydrophobic portion and a cationic hydrophilic portion, using a buffer or physiological solution with an antigen. Disclosed is a method for improving antibody-producing ability, comprising immunizing an animal by dissolving the antibody in saline.

As the antigen, basically any substance having an anionic (anionic) charge can be applied, but a water-soluble protein is particularly suitable. When an animal is immunized with the adjuvant of the present invention together with an appropriate antigen, the production of antibodies against other substances is suppressed as compared with the case where a conventionally used adjuvant is used, and a specific antibody of interest is produced using a conventional adjuvant. Is found to be equal to or higher than that of the case of using, particularly, it has been found that production of total IgG can be suppressed and production of IgG specific to an antigen can be improved.

The reason why a specific antibody is efficiently produced by using the adjuvant of the present invention containing a synthetic bilayer membrane is not completely understood, but is not completely understood. It is thought that the molecular membrane forms a stable complex with an anionic antigen such as a protein, and efficiently delivers the target substance to the antigen-presenting cell.

As a technique similar to the present invention, an adjuvant using a liposome composed of a lipid bilayer membrane is also found, but in such a bilayer membrane, the hydrophilic group on the surface is a neutral group composed of a cation and an anion. Yes, it is not possible to form a complex with a protein or the like by itself, and some contrivance for imparting cationicity is required. On the other hand, when the adjuvant of the present invention comprising a synthetic bilayer membrane compound is used, a bilayer membrane structure is formed in which the cationic hydrophilic part is outside and the hydrophobic parts face each other in the animal body. It is considered that a complex with the antigen substance is directly formed inside or outside the membrane via the cationic hydrophilic part. The adjuvant of the present invention is
It contains no impurities, enhances the ability to produce specific antibodies in this regard, and is easy to handle.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The compound forming a synthetic bilayer used in the adjuvant of the present invention is an amphiphilic compound having a hydrophobic part and a cationic hydrophilic part. Can be mentioned.

[0016]

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[0017]

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[0018]

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[0019]

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[0020]

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These compounds can be synthesized by devising a known synthesis method. In general, a desired amphiphilic compound can be obtained by bonding a precursor compound having a hydrophobic part such as an alkyl chain, a hydrophilic part such as trimethylammonium, and a spacer part connecting both of these via an amide bond or the like. Is obtained.

FIG. 1 shows one example of such a synthesis method, focusing on the synthesis of the compound (A) (m =
12). That is, a compound such as glutamic acid is used as a linking center, and an alcohol having a carbon chain length of 12 to 14 (1 in the figure) is attached to two carboxyl groups in the molecule.
2) is ester-bonded, and HCl is added to the obtained compound to obtain a precursor compound constituting a hydrophobic part (Scheme 1). On the other hand, the compound has a carbon chain length of 2 to 8 (n = 2 to 2).
After introducing the dibromo compound of 8), the carboxyl group formed by removing the ethyl group is thionylated to obtain a precursor compound that constitutes the hydrophilic portion (Scheme 2). A compound (or a compound having a carbon chain length of 2 to 8)
An amphiphilic compound can be synthesized by amide-bonding a compound having a halogen at the carbon terminus of the resulting compound and substituting the halogen at the carbon chain terminus of the resulting compound with trimethylammonium (Scheme 3).

In order to use an adjuvant composed of a synthetic bilayer according to the present invention, a synthetic bilayer is usually formed from the above-mentioned amphiphilic compound at the time of immunization of an animal, and this is combined with an antigen in a buffer or physiological solution. Animals are immunized by dissolving them in saline. However, it is also possible to form a synthetic bilayer from an amphipathic compound in advance, dissolve and store it in an appropriate buffer or physiological saline, and dissolve and administer and immunize the antigen upon use. . Therefore, the term “adjuvant containing a synthetic bilayer membrane” used herein refers not only to a synthetic bilayer membrane used as an adjuvant, but also to a buffer solution or physiological saline in addition to the synthetic bilayer membrane. An adjuvant composition that optionally contains other substances (eg, a film stabilizer such as cholesterol) shall be referred to.

The formation of the synthetic bilayer film is carried out, for example, by dissolving the above-mentioned amphiphilic compound in a suitable solvent (eg, chloroform) and then evaporating and removing the solvent to form a thin film, or It is carried out by a method in which the solvent compound is dissolved in distilled water and uniformly dispersed by an ultrasonic generator.

In practicing the present invention, various buffers or physiological saline can be used according to the purpose. The most commonly used buffer is phosphate buffer (PBS)
It is.

The present invention can be used to immunize a wide range of substances as antigens. Any one having an anionic charge can be applied, but a water-soluble protein is particularly preferred. Examples thereof include ovalbumin (OVA), β-lactoglobulin (βLg), ovomucoid, bovine serum albumin and the like. These are high molecular compounds (generally having a molecular weight of 5,0
00 or more), but it is also possible to immunize a low molecular weight compound by binding to a high molecular weight protein.

The adjuvant of the present invention can be applied to animal vaccines and the like, but is particularly suitable for producing and obtaining specific antibodies using experimental animals. Experimental animals used include rats, mice, rabbits, goats,
Guinea pigs and the like. In addition, dogs, sheep,
Horses and cats are also used.

The specific antibody efficiently obtained according to the present invention can be used in various research fields, and produces various immunoassay reagents, antibody drugs for prevention or treatment of humans or animals, and antibody catalysts. Can be used to At this time, it can also be prepared as a monoclonal antibody according to a conventional method.

[0029]

The present invention will be described in more detail with reference to the following Examples, but it should be understood that the present invention is by no means restricted to such specific Examples.

An adjuvant containing a synthetic bilayer formed from the amphiphilic compound according to the present invention, and, for comparison, the ability to produce antibodies in laboratory animals without adjuvant and with a conventionally known adjuvant Was tested. Examples of the amphiphilic compound include the compounds of the above-mentioned general formula (A) in which m = 12 and n = 2 (hereinafter referred to as 12GP2) and those in which m = 14 and n = 2 (hereinafter referred to as 14GP2). ) Was used.

Example 1 Sample Preparation Without adjuvant: 50m containing 150 mM NaCl containing ovalbumin (Wako Pure Chemical) (hereinafter OVA)
M was dissolved in a sodium phosphate buffer (pH 7.5) (hereinafter, PBS) to 10 μg / ml. 2. Aluminum hydroxide adjuvant: Aluminum hydroxide was dispersed in 10 μg / ml of OVA dissolved in PBS to 10 mg / m
l. After dispersing aluminum hydroxide, 3
Incubate with shaking at 36 ° C for 0 minutes.
3. FCA adjuvant: dissolve OVA in PBS and add 20
μg / ml and commercially available FCA were mixed at a ratio of 1: 1 and subjected to ultrasonic wave to form a W / O emulsion. 4.1
2GP2 adjuvant: 12GP2 was dissolved in chloroform, put in an eggplant flask, and the pressure was reduced while rotating with an evaporator to evaporate the chloroform. OVA was dissolved in PBS to a concentration of 10 μg / ml, which was then added to a thin layer of 12GP2 on the wall surface of the eggplant flask, so that the concentration of 12GP2 was 1.3 mM. This was subjected to ultrasonic waves to form micelles of a bilayer film.
5.14GP2 adjuvant: 14GP2 was dissolved in chloroform, put in an eggplant flask, and the pressure was reduced while rotating with an evaporator to evaporate the chloroform. OVA was dissolved in PBS to a concentration of 10 μg / ml, which was then added to the 14 GP2 layered into a thin film on the wall surface of the eggplant flask, so that the concentration of 12GP2 was 1.3 mM. This was subjected to ultrasonic waves to form micelles of a bilayer film.

Example 2: Implantation into rats and collection of serum
And 1ml increments each injected into preparative intraperitoneally SD rats 4 weeks old samples of the 5. OVA that is an antigen is 1 per individual
The dose is 10 μg per administration. Four rats were used per sample and the rats were hit four times every 10 days. Rats were bled 2 days prior to the first shot and 4 days after each shot from the tail tip of the rats. 4 for rats
After 0 day, the animals were sacrificed and blood was collected as much as possible. The collected blood was centrifuged at 3000 rpm for 15 minutes, separated into blood cells and serum, and the OVA-specific antibody (IgG) in the serum was measured.

Example 3: OVA-specific antibody (I
gG) Measurement of concentration OVA was dissolved (50 μg / ml) in 0.05 M carbonate buffer (pH 9.5), 100 μl was added to each well of a 96-well microplate, and incubated at 37 ° C. for 1 hour. 50% Na phosphate buffer (pH 7.5) containing 0.01% Tween 20 (hereinafter referred to as P
Washed three times with BS Tween 20). Next, add Blo to each well.
300 μl of ckAce (Dainippon Pharmaceutical) diluted 4 times with distilled water was added, incubated at 4 ° C. overnight, and then
The plate was washed three times with BS Tween 20 in the same manner. A rat serum sample was appropriately diluted with BlockAce diluted 20-fold with distilled water, and 50 μl of the diluted sample was added to each well, and incubated at 37 ° C. for 1 hour.
Washed three times with ween 20. Then, peroxdase-labeled goat anti-rat IgG (CAPPEL) diluted 2000-fold with BlockAce diluted 20-fold with distilled water was added to each well at 100 μl.
1 hour, incubated at 37 ° C. for 1 hour, and washed similarly three times. Add 0.003% hydrogen peroxide, 0.3mg /
0.1 M citrate buffer containing 0.1 ml ABTS (pH 4.
100) was added to each well and incubated at 37 ° C. for 15 minutes to develop color. Then, 100 μl of a 1.5% oxalic acid aqueous solution was added to each well to stop the reaction.
The absorbance at 4 nm was measured. FIG. 2 shows the results of this example for the five samples shown in Example 1. From this example, 12GP2 is equivalent to aluminum hydroxide,
14GP2 was found to have the same ability to enhance antibody production as FCA.

Example 4 Concentration of Total Antibody (Total IgG) in Serum
Goat anti-rat I at each measurement 0.05M carbonate buffer (pH 9.5)
gG (CAPPEL) was diluted 1000-fold, added to each well of a 96-well microplate at 100 μl, incubated at 37 ° C. for 1 hour, and then washed three times with PBS Tween 20. Next, 300 μl of BlockAce (Dainippon Pharmaceutical) diluted 4-fold with distilled water was added to each well, incubated at 4 ° C. overnight, and then washed similarly with PBS Tween 20 three times. Rat serum samples were prepared with distilled water for 20 minutes.
The mixture was appropriately diluted with a 1: 2 diluted BlockAce, and 50 μl of the diluted sample was added to each well, incubated at 37 ° C. for 1 hour, and similarly washed three times with PBS Tween 20. And with BlockAce diluted 20 times with distilled water, 200
Peroxdase-labeled goat anti-rat IgG (CA
PPEL) was added to each well (100 μl), incubated at 37 ° C. for 1 hour, and washed similarly three times. 0.0
0.1 M citrate buffer (pH 4.0) containing 03% hydrogen peroxide and 0.3 mg / ml ABTS was added to each well.
After incubating at 37 ° C. for 15 minutes to develop color, 1.5% oxalic acid aqueous solution was added to each well in a volume of 1%.
The reaction was stopped by adding 00 μl, and the absorbance at 414 nm was measured. FIG. 3 shows the results of the present example for the five samples shown in Example 1. From this example, 12 GP
It became clear that both 2,14GP2 reduced the ability to produce total IgG.

[Brief description of the drawings]

FIG. 1 schematically shows an example of a method for synthesizing an amphiphilic compound used in the present invention.

FIG. 2 is a graph showing changes in OVA-specific IgG concentrations in rat serum obtained using various adjuvants.

FIG. 3 is a graph showing changes in total IgG concentration in rat serum obtained using various adjuvants.

Claims (4)

[Claims] 1. An adjuvant for animal immunization comprising a synthetic bilayer formed from an amphiphilic compound having a hydrophobic part and a cationic hydrophilic part. 2. The adjuvant according to claim 1, which is used for antibody production by a laboratory animal. 3. A method comprising forming a synthetic bilayer from an amphipathic compound having a hydrophobic part and a cationic hydrophilic part, dissolving it in a buffer or physiological saline together with an antigen, and immunizing an animal. A method for improving the ability to produce antibodies. 4. The method according to claim 3, wherein the antigen is a water-soluble protein.