JP2000081435A - Protein stabilizer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To stabilize protein of a free antigen/antibody or a labeled antigen/ antibody by using poly-β-cyclodextrin and/or its derivative as a protein stabilizer. SOLUTION: As poly-β-cyclodextrin, a commercially available one or a synthesized one prepared by using epichlorohydrin is usable. A poly-β-cyclodextrin derivative is prepared by cross-linking with epichlorohydrin after proper substitution of β-cyclodextrin by an alkyl group, a hydroxyalkly group, an acyl group, or the like, or alternatively, replacing a free hydroxyl group in poly-β- cyclodextrin by an alkyl group, a hydroxyalkyl group, an acyl group, or the like. A concentration of β-cyclodextrin in the reagent or a standard solution is set to be about 0.1-8 w/v%. In this way, protein such as immunoglobulin A, immunoglobulin E, immunoglobulin G, and the like can be stabilized, and a free/labeled antigen/antibody can be also stabilized.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention
The present invention relates to a stabilizing agent for a protein existing in a solution such as a reagent or a standard solution used in the field of a diagnostic agent and a method for stabilizing a protein using the same.

[0002]

2. Description of the Related Art In recent years, in the field of clinical examinations and diagnostics, measurement methods utilizing an immune reaction have been widely used, and are widely used for analyzing trace biological components.
On the other hand, as a method for stabilizing a protein such as a free antigen, an antibody or a label thereof, which is used for a reagent or a standard solution constituting the measurement method, for example, (1) albumin, (2) Polyhydric alcohols such as glycerol, polyethylene glycol and sucrose; (3) amino acids such as glutamic acid and lysine; (4) reducing agents such as glutathione and dithiothreitol; (5) metal chelating agents such as EDTA; and (6) citric acid (7) Heavy metal salt, (8) Substrate or coenzyme, etc. in a solution
Volume 1, Protein I Isolation / Purification / Properties, p. However, with the advancement of measurement technology, it has become possible to measure proteins with high sensitivity.Accordingly, it has been necessary to store proteins having a small amount of activity for a long period of time without inactivating them. There is a problem that the target protein cannot be sufficiently stabilized only by addition.

[0003]

In view of the above situation, an object of the present invention is to provide a stabilizing agent for stabilizing a protein such as a free antigen, an antibody or a label thereof in a solution. An object of the present invention is to provide a method for stabilizing a protein in a solution using the same.

[0004]

DISCLOSURE OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and has been made under the following circumstances: "(1) a protein stabilizer comprising poly-β-cyclodextrin or / and a derivative thereof; . (2) A method for stabilizing a protein, comprising coexisting poly-β-cyclodextrin or / and a derivative thereof in a solution containing the protein. (3) A composition comprising poly-β-cyclodextrin or a derivative thereof, and a protein. ].

That is, the present inventors have conducted intensive studies for a compound capable of stabilizing a protein present in a solution such as a reagent or a standard solution. The abbreviation of β-cyclodextrins of the present invention) has been found to be able to achieve the above object, and the present invention has been completed.

The poly-β-cyclodextrin according to the present invention includes, for example, those having the following structure obtained by crosslinking β-cyclodextrin with epichlorohydrin to form a polymer.

[0007]

Embedded image

(Where X represents a β-cyclodextrin residue, i = 0 to 3, j = 1 to 5, k = 2 to 50)

As these poly-β-cyclodextrins, water-soluble ones are desirable.
Those having a range of 50,000 and in which j in the above general formula is 1 or more and less than 3 are preferable. The more preferable value of k is 3 to 3.
0.

The poly-β-cyclodextrin derivative according to the present invention is preferably a water-soluble one.
The hydroxyl group of -β-cyclodextrin is, for example, an alkyl group such as a methyl group or an ethyl group, for example, a 2-hydroxyethyl group,
2-hydroxypropyl group, 3-hydroxypropyl group, 2,
A hydroxyalkyl group such as a 3-dihydroxypropyl group,
For example, an acyl group such as an acetyl group, a carboxyalkyl group such as a carboxymethyl group, for example, a glucosyl group,
Those substituted with a sugar residue such as a maltosyl group,
Those which are water-soluble are preferred specific examples.

The poly-β-cyclodextrin according to the present invention may be a commercially available product or a product synthesized according to a conventional method using epichlorohydrin as appropriate.

The poly-β-cyclodextrin derivative according to the present invention can be prepared by, for example, first substituting β-cyclodextrin with a group as described above and then crosslinking with epichlorohydrin. It can be obtained by a method in which a free hydroxyl group is substituted with a group as described above. As a method for substituting the hydroxyl group of β-cyclodextrin or poly-β-cyclodextrin with the above-mentioned group, for example, U.S. Pat.No. 3,453,258, U.S. Pat.No. 3,453,259, Polymer Journal, Vol.13 ,
No. 8, p. 777-781 (1981), JP-A-61-266401, JP-A-63-122701, JP-A-62-243602, Carbohyd
rate Polymer, Vol.5, p343-349 (1985), JP-A-57-130914
And those described in Japanese Patent Application Publication No.

[0013] The concentration of the stabilizer of the present invention is not particularly limited as long as it can stabilize the protein present in a solution such as a reagent or a standard solution. Normal concentration of β-cyclodextrins
It is added so as to be 0.1 to 8 w / v%, preferably 1 to 4 w / v%.

The β-cyclodextrins of the present invention may be used alone or in an appropriate combination of two or more.

The proteins which can be stabilized by the stabilizer of the present invention include, for example, immunoglobulin A (IgA), immunoglobulin E (IgE), immunoglobulin G (IgG), β ₂ -microglobulin, albumin, ferritin Serum proteins, such as
For example, enzyme proteins such as amylase, alkaline phosphatase, and γ-glutamyltransferase (γ-GTP), such as rubella virus, herpes virus, hepatitis virus,
Antigens derived from clinically noted viruses such as ATL virus and AIDS virus (for example, HBe antigen, HBc antigen,
HBs antigen) and antibodies against these antigens.

The antigens and antibodies which can be stabilized by the β-cyclodextrins according to the present invention may be free or labeled with various labeling substances such as enzymes, fluorescent substances and radioactive substances.

The present invention can be in the form of a composition obtained by dissolving the β-cyclodextrins according to the present invention in a solution such as the above-mentioned protein-containing aqueous solution or buffer solution. Until it is used, it can be stored in various forms, such as in a state of being subjected to a freezing treatment or a freeze-drying treatment or a state of a solution. Then, at the time of use, these compositions may be used.

In order to stabilize proteins present in solutions such as reagents and standard solutions using the β-cyclodextrins according to the present invention, free antigens, antibodies or labeled products thereof are contained. The β-cyclodextrins according to the present invention may be added and dissolved in a solution such as a reagent or a standard solution. Further, the composition containing the β-cyclodextrins and the protein according to the present invention as described above may be dissolved in water or a suitable buffer.

The buffer which can be used in the composition of the present invention includes, for example, Tris buffer, phosphate buffer,
A good buffer such as a veronal buffer, a borate buffer such as a 2-morpholinoethanesulfonic acid (MES) buffer, and a 3-morpholinopropanesulfonic acid (MOPS) buffer; Buffers commonly used in the fields of enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA) and the like can be mentioned.

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

[0021]

EXAMPLES Example 1 (1) Preparation of Anti-HBe Monoclonal Antibodies After immunization (two times) of BALB / c mice (female) with Freund's complete adjuvant with recombinant HBe antigen (manufactured by Oriental Yeast), spleen cells isolated And myeloma cells (FO) were fused by a conventional method using polyethylene glycol (for example, the method described in JP-A-5-244983). Thereafter, anti-HBe monoclonal antibody-producing hybridomas were selected by a conventional method, and cultured to obtain anti-HBe monoclonal antibodies. The obtained anti-HBe
A portion of the monoclonal antibody was labeled with peroxidase (POD, Boehringer Mannheim) by a conventional method (Eiji Ishikawa, "Enzyme Labeling Method", Gakkai Shuppan Center, 1991, p.62) to prepare a labeled antibody. .

(2) Preparation of Protein Stabilizing Buffer A 50 mM 3-morpholinopropanesulfonic acid (MOPS) buffer (containing 2% BSA, pH 7.5) was added to the poly-β-protein of the present invention.
Cyclodextrin (Wako Pure Chemical Industries, Ltd. sales code No.
168-15082; average molecular weight 14700, molecular weight range 3000-5000
0] was dissolved to a concentration of 2% (w / v) to prepare a buffer solution containing a stabilizer. For comparison, a buffer prepared by adding EDTA, N-acetylcysteine, hydroxypropyl-β-cyclodextrin or hydroxypropyl-γ-cyclodextrin at a predetermined concentration to a 50 mM MOPS buffer (containing 2% BSA, pH 7.5). A liquid was also prepared.

(3) Preparation of Recombinant HBe Antigen Sample Recombinant HBe antigen (manufactured by Oriental Yeast) was dissolved at 4 ng / ml in the buffer prepared in (2) above and stored at 37 ° C. for 10 days. Was used as a recombinant HBe antigen sample.

(4) Examination of protein stabilizing buffer Polystyrene balls (manufactured by Immunochemical Co.)
Anti-HBe monoclonal antibody (unlabeled POD) obtained at 20μg / ml
Was immersed in a 50 mM MOPS buffer (pH 7.5) containing, and allowed to stand at 4 ° C. overnight. 50 mM MOPS buffer containing 1% BSA (pH
After washing 3 times in 7.5), immerse in the same buffer solution and further 4 ° C
And left overnight to prepare an anti-HBe monoclonal antibody-immobilized ball. 150 μl of various recombinant HBe antigen samples prepared in the above (3) were dispensed into a polypropylene tube,
Anti-HBe monoclonal antibody immobilized ball 1 prepared above
Individuals were added and reacted at 37 ° C. for 7 minutes. Next, the ball was washed three times with 1 ml of physiological saline, and then a 50 mM 2-morpholinoethanesulfonic acid (MES) buffer (containing 2% BSA, pH 6) containing 2.5 μg Ab / ml of the POD-labeled antibody obtained in the above (1) was used. .5) 150μ
l was added and reacted at 37 ° C. for 7 minutes. After washing the ball three times with 1 ml of physiological saline, 200 μl of 50 mM Tris buffer (pH 8.5) containing 5 mM luminol and 0.02% hydrogen peroxide was added, and a chemiluminescence meter (Bertol, Oatlmat LB95) was added.
The amount of chemiluminescence was measured in 3). Recombinant HBe
The antigen activity remaining rate (%) was determined in the same manner as described above except that a 50 mM MOPS buffer (containing 2% BSA, pH 7.5) containing 4 ng / ml of the recombinant HBe antigen newly prepared on the measurement day was used. The figure shows the ratio of the amount of luminescence obtained for the recombinant HBe antigen sample when the amount of chemiluminescence obtained by measurement is 100%. Table 1 shows the results.

[0025]

From the results shown in Table 1, it can be seen that when the poly-β-cyclodextrin according to the present invention was added, the residual ratio was almost 100%, in other words, the protein in the sample was stabilized.

[0027]

As described above, the present invention relates to a stabilizing agent for stabilizing a protein present in a solution such as a reagent or a standard solution without affecting an immune reaction, and a stabilizing method using the same. And by utilizing the present invention,
Compared with the conventional method, a small amount of an active protein can be stored for a long period of time without inactivating it.

Claims (10)

[Claims] 1. A protein stabilizer comprising poly-β-cyclodextrin or a derivative thereof. 2. The protein according to claim 1, wherein the protein is an antibody or an antigen.
The stabilizer according to 1. 3. The stabilizer according to claim 2, wherein the antigen is an HBe antigen. 4. A method for stabilizing a protein, comprising coexisting poly-β-cyclodextrin or a derivative thereof in a solution containing the protein. 5. The protein according to claim 4, wherein the protein is an antibody or an antigen.
The method of stabilization described in 1. 6. The method according to claim 5, wherein the antigen is an HBe antigen. 7. A composition comprising poly-β-cyclodextrin or a derivative thereof and a protein. 8. The composition according to claim 7, further comprising a buffer. 9. The protein according to claim 7, wherein the protein is an antibody or an antigen.
Or the composition according to 8. 10. The composition according to claim 9, wherein the antigen is an HBe antigen. [0001]