JPH11209397A - Production of soluble gamma-livetin complex - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject complex with raised immunoreactivity, usable as e.g. an examination medicine with high cohesiveness, by subjecting a γ-livetin solution to irreversible reaction followed by removing insoluble components to facilitate making the intermolecularcrosslink at an antigen-binding site. SOLUTION: This soluble γ-livetin complex with raised immunoreactivity is obtained by the following procedure: as a specific antigen, a solution containing Streptococcus mutans MT8148 microbial cells is prepared; laying hens are immunized with the solution, yolk is separated from the resulting eggs and homogenized into a yolk liquid, to which water is added to effect homogenization, a 0.15% λ-carrageenan aqueous solution is added to the homogenized liquid followed by agitation and then filtration to form a yolk water-soluble protein solution, to which sodium sulfate is added, the resulting precipitate is collected and purified to afford anti-Streptococcus mutans γ-livetin-contg. powder; subsequently, water is added to the powder followed by heating the resultant solution at 60-80 deg.C for 10-120 min at a γ-livetin concentration of 1 to <50 mg/mL to perform an irreversible reaction followed by removing insolubles from the reaction product.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a soluble γ-libetine complex. More specifically, the present invention relates to a conventional method for producing a soluble γ-libetin complex having enhanced immunoreactivity.

[0002]

2. Description of the Related Art Conventional γ-libetin has one antigen binding site.
Since there is only one site, it is difficult to form an intermolecular cross-link, and when used as a test agent, there is a drawback that a satisfactory precipitation reaction cannot be obtained depending on the type of the antigen to be measured. As a method for facilitating cross-linking, addition of 1.5 M sodium chloride is disclosed (Otani, Bulletin of the Japan Society of Animal Science, Hokuriku Branch, 57, p16-25 (1988)). This method utilizes the property that in the presence of 1.5 M sodium chloride, γ-libetine is present as a dimer and becomes tetravalent, making it easy to form a lattice and making it easy to determine a sedimentation reaction. Since a large amount of sodium chloride is required, there is a disadvantage that it is difficult to determine a sedimentation reaction depending on the type of the antigen to be measured.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for producing a soluble γ-libetin complex having high agglutinating reactivity.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, a soluble complex having high aggregation reactivity was obtained by subjecting γ-libetin to an irreversible reaction. And found that the present invention was completed. That is, the present invention is a method for producing a soluble γ-libetin complex containing γ-libetin as a main component, characterized by using an irreversible reaction.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The irreversible reaction in the present invention refers to a reaction that forms a covalent bond between γ-libetin or between γ-libetin and a contaminating protein. This refers to a covalent bond reaction. Although not particularly limited, a method for determining a covalent bond includes, for example, the SDS-PAGE method. The soluble γ-libetin complex of the present invention is a protein complex having a molecular weight of 360 kDa or more measured by this method. Polymers that are dimers or salted-out products of γ-libetin generated under high salt concentration are hydrophobic bonds or ionic bonds and SD
S-PAGE exists as a monomer and is different from the soluble γ-libetin complex of the present invention. Irreversible reaction method in the present invention is not particularly limited, heating reaction method, ultra-high pressure reaction method, a reaction method using a reducing agent, and the like.
Preferably, a heating reaction method is used. The heating conditions in the heating reaction method are selected so that the activity of γ-libetin remains at 90% or more. Specifically, the γ-libetin-containing solution is 60 to
The heating is preferably performed at 80 ° C. for 10 to 120 minutes, and the concentration of the γ-libetine-containing solution at that time is preferably 1 mg / ml to less than 50 mg / ml. More preferably, a concentration of 5 mg / ml to 30 mg / ml is preferred. When the concentration is lower than 1 mg / ml, it is difficult to form a soluble complex, and when the concentration is higher than 50 mg / ml, the proportion of aggregates in which γ-libetin is insoluble increases.

The pH of the γ-libetine-containing solution before the heat treatment in the present invention is preferably from pH 5.0 to pH 11.0, more preferably from pH 7.0 to pH 9.0. If the pH is inclined toward an acidic side from pH 5.0 or toward an alkaline side from pH 11.0, the activity of γ-libetine may be inactivated. In the present invention, the main component is γ-
The proportion of γ-libetin in the rivetine complex is 50%
Say that occupies the above. In the present invention, “soluble” means solubility in water. The soluble γ-libetin complex of the present invention may be further purified and used, and the purification method is not particularly limited, but includes membrane separation, salting out, chromatography, ultracentrifugation and the like. In particular, a membrane separation method and a salting-out method can be suitably used.

[0007] The membrane separation method is not particularly limited, but is performed by using one or more types of membranes selected from ultrafiltration membranes, microfiltration membranes, reverse osmosis membranes, or filter papers, filter plates, and glass filters. In particular, an ultrafiltration membrane can be suitably used. The salt used in the salting out method is NaCl,
KCl, CaCl ₂ , MgCl ₂ , NaH ₂ PO ₄ , N
a ₂ HPO ₄ , Na ₂ SO ₄ , (NH ₄ ) ₂ SO ₄ , Mg
One or two or more salts selected from SO _{4. The} concentration varies depending on the type of each salt, but is preferably 2 to 12%, more preferably 5 to 10%, and most preferably 8 to 10%. is there. Hereinafter, the present invention will be described in detail with reference to examples. However, these do not limit the invention.

[0008]

Example 1 Preparation of Anti-S. Mutans γ-Libetin-Containing Egg Yolk Water-Soluble Protein Solution Streptococcus mutans was used as a specific antigen.
A bacterial solution (CFU 1) containing MT8148 (hereinafter referred to as S. mutans) cells
0 ⁸ ) was prepared and laying hens were immunized. The yolk was separated from the obtained eggs and the homogenized solution was used as a yolk solution. 1 kg of water was added to 1 kg of the obtained egg yolk liquid to homogenize, and 0.1 g of the mixture was added thereto.
4 kg of a 15% aqueous solution of λ-carrageenan was added and stirred. After standing, suction filtration was performed to separate the filtrate. The filtrate obtained at this time was used as an egg yolk water-soluble protein solution.

Example 2 Preparation of Powder Containing Anti-S. Mutans γ-Libetin 150 g of sodium sulfate was added little by little to 1 L of the egg yolk water-soluble protein solution obtained in Example 1, and the mixture was allowed to stand for 30 minutes and centrifuged (at room temperature). , 8000 rpm for 15 minutes). The supernatant is discarded, and the precipitate is added with 10 mM Na ₂ HPO ₄ buffer 2
Add 00 ml and dissolve. The salting out method was performed again using the obtained solution in the same manner as described above. This solution was added to 10 mM N
Perform dialysis overnight using a ₂ HPO ₄ buffer. The obtained solution was freeze-dried to obtain a specific γ-libetine-containing powder. The obtained powder was confirmed by gel filtration ("Protein I", edited by The Biochemical Society of Japan, Chapter 11, Tokyo Kagaku Dojin (1990)) to have a ratio of γ-libetin to all proteins of 90% or more. .

Example 3 Preparation of soluble anti-S. Mutans γ-libetin complex The anti-S. Mutans γ-libetin-containing powder obtained in Example 2 was prepared to a concentration of 10 mg / ml using pure water. 200 ml of the prepared solution was sealed in a heat-resistant container and heated in a thermostat at 70 ° C. for 30 minutes to obtain a mixed solution of a soluble complex and γ-libetine alone. The mixed solution was subjected to diafiltration by a membrane separation method (Nihon Pole: 500 kDa cut off) to separate γ-libetin monomer, and the soluble γ-libetin complex was dried with a freeze dryer to obtain a powder. Obtained.

Test Example 1 Confirmation of Irreversible Reaction of Specific γ-Libetin Complex The powder obtained in Example 3 was electrophoresed by SDS-PAGE.
The binding mode of this complex was confirmed. The used polyacrylamide gel was subjected to electrophoresis using a gel (AE-6000 NPG-310L: ATTO Co., Ltd.) (fraction molecular weight: 40 to 500 kDa).
The electrophoresis sample was 1 mg of the powder obtained in Example 3 or 4.
/ Ml and applied to the gel by 5 μl of this solution. As a result, the sample obtained in Example 3 or 4 hardly migrated and stopped at the upper end portion. This confirmed that this sample was a protein having 500 kDa or more, which had a covalent bond and did not return to the original monomer.

Test Example 2 Comparison of recovery rate of specific antibody activity The specific antibody titer of the specific γ-libetin complex powder obtained in Example 3 against S. mutans was determined by enzyme immunoassay (hereinafter referred to as ELISA method). And the specific antibody activity recovery rates were compared. Each sample solution was added at a rate of 100 μl / well to an ELISA plate (96-well, manufactured by Nunc) having S. mutans used as an antigen coated on a solid phase.
The reaction between the antigen and the specific antibody was performed by allowing the mixture to stand at 25 ° C. for 2 hours. After each well was sufficiently washed with PBS-Tween, a PBS-Tween diluted solution (2000-fold) of anti-chicken IgG rabbit IgG-alkaline phosphatase conjugate (Zymet) was added at a ratio of 100 μl / well. By leaving the mixture at 25 ° C. for 1 hour, the specific antibody reacted with the antigen was reacted with the conjugate. After washing each well with PBS-Tween, 0.1% P-
Phenyl disodium phosphate solution (dissolved in 0.1 M sodium carbonate buffer pH 9.6)
The mixture was added at a rate of μl / well, and an enzyme reaction was performed at 25 ° C. for 15 minutes. To stop the reaction, add 2M sodium hydroxide solution
This was performed by adding 50 μl / well, and the absorbance of each well at 405 nm was measured using a plate reader. As a control, PBS-Tween was used instead of the sample. Four wells were used for each sample. The absorbance of the control was subtracted from the average value of the absorbance of each sample, and the value obtained by multiplying each sample weight (total absorbance) was defined as the total antibody activity of each sample. As a result of measuring the total antibody activity, the specific γ-libetin powder obtained in Example 2 and the specific γ-libetin complex powder obtained in Example 3 were compared. As a result, the specific γ-libetin complex powder obtained an antibody activity recovery rate of about 90%.

Example 4 Application of Soluble Specific γ-Libetin Complex to SRID Method As a means for quantifying the amount of S. mutans bacteria present in human oral cavity, SRID (one-way radiation immunodiffusion) method (G. Mancini et al. ., Im
munochemistry, 2, 235 (1965)) was used. Human volunteers were recruited and the mouth was washed with pure water. This mouthwash was used as a measurement sample. As a plate for measurement, the anti-S. Mutans γ-libetin obtained in Example 2 was used for 1 plate.
2% agar plate (plate A), plate A
(Plate B) containing 1.5 M of sodium chloride added thereto and the soluble antibody obtained in Example 4.
An attempt was made to determine the amount of bacteria from the size of the sedimentation ring formed on plate (plate C) prepared in the same manner as plate A using the S. mutans γ-libetin complex. As a result, plate A,
The sedimentation ring formed on B was unclear, and the amount of bacteria could not be determined. On the other hand, the sedimentation ring formed on plate C was clear.
The value was multiplied by the volume of the solution to obtain the amount of S. mutans bacteria. As a result, it has become possible to measure antigens, which were difficult to measure by the conventional SRID method because the sedimentation rings were not clear. Embodiments of the present invention and the target product are as follows.

(1) A method for producing a soluble γ-libetin complex containing γ-libetin as a main component, characterized by using an irreversible reaction. (2) The method for producing a soluble γ-libetin complex according to the above 1, wherein the irreversible reaction is heat treatment. (3) Heating reaction is performed at 60 to 80 ° C for 10 to 120 minutes, γ-
2. The method for producing a soluble γ-libetin complex according to the above 1, wherein the concentration of the livetin solution is 1 to less than 50 mg / ml, and insoluble components are removed from the reaction product. (4) The γ according to the above 1, wherein the irreversible reaction is an ultra-high pressure treatment.
-A method for producing the rivetine complex.

(5) The method for producing a γ-libetin complex as described in (1) above, wherein the irreversible reaction uses a reducing agent. (6) The method for producing a γ-libetin complex according to the above 1, wherein the soluble γ-libetin complex of dimer or more is purified using a membrane. (7) The method for producing a γ-libetin complex according to (1) above, wherein the membrane is an ultrafiltration membrane, a microfiltration membrane, or a reverse osmosis membrane. (8) The method for producing a γ-libetin complex according to the above 1, wherein the soluble γ-libetin complex of dimer or more is purified using a salt.

(9) The salt is NaCl, KCl, CaC
l ₂ , MgCl ₂ , NaH ₂ PO ₄ , Na ₂ HPO ₄ ,
The γ-molecule according to claim 4, wherein one or more selected from the group consisting of Na ₂ SO ₄ , (NH ₄ ) ₂ SO ₄ and MgSO ₄ is purified by adding 10% or less. Libetin complex and its separation method. (10) The method for producing a γ-libetin complex according to the above 1, wherein the soluble γ-libetin complex of dimer or more is purified by chromatography.

[0017]

According to the present invention, the conventional γ-libetin is converted into a soluble complex by using an irreversible reaction.
It has the effect of dramatically increasing the functions of Libetin, and further expands the application range of γ-Libetin and greatly contributes to industry.

Claims (3)

[Claims] 1. γ characterized by using an irreversible reaction
-A method for producing a soluble γ-libetin complex containing rivetine as a main component. 2. The method for producing a soluble γ-libetin complex according to claim 1, wherein the irreversible reaction is a heating reaction. 3. A heating reaction at 60 to 80 ° C. and 10 to 120.
2. The method for producing a soluble γ-libetin complex according to claim 1, wherein the γ-libetin solution concentration is less than 1 to 50 mg / ml, and insoluble components are removed from the reaction product.