JP7294012B2 - Protein Stabilizer, Reagent Containing Stabilized Protein, and Method for Stabilizing Protein Contained in Reagent - Google Patents

Description

The present invention relates to a protein stabilizer capable of stabilizing a protein contained in a reagent, a reagent containing the stabilized protein, and a method for stabilizing the protein contained in the reagent.

Biochemical measurements such as enzyme immunoassay, turbidimetric immunoassay, latex agglutination, immunochromatography, nucleic acid analysis, and immunostaining are widely used in the fields of clinical examination, in vitro diagnosis, companion diagnosis, and the like. In recent years, food analysis and environmental analysis applying these biochemical measurement techniques have also been widely performed.
Accuracy is an essential requirement in the above biochemical measurements.
Therefore, it is important to stably maintain the physiological activities of proteins such as enzymes, antibodies, and labeled antibodies contained in reagents for biochemical measurements for a long period of time.
Accuracy also requires constant input of reagents. In biochemical measurements, reagents are often used in the form of solutions. It is important that the reagent solution is drained well and does not remain in the pipetting instrument when the reagent solution is collected in step 1 and pipetted into a container to be used in the next step.

Many of the proteins contained in reagents are easily denatured and deactivated due to various factors, for example, self-aggregation during refrigeration storage in a solution state. Therefore, the stability required for the protein contained in the reagent is specifically the stability of the protein-containing reagent, particularly the reagent in solution, when stored in a refrigerator. "Stability when refrigerated" is stability in consideration of long-term storage of reagent solutions or on-board storage of reagents in automated analyzers.

As a method of stabilizing a solution in which proteins are dissolved, a method of adding bovine serum albumin (hereinafter sometimes abbreviated as "BSA") to a reagent solution is generally known.
However, it cannot be said that the effect of stabilizing the protein by the above method is sufficient. Furthermore, the use of BSA poses the risk of infectious diseases such as mad cow disease. In addition, BSA has a problem that the stabilizing effect varies greatly depending on the lot, and a sufficient effect cannot be obtained depending on the lot. In addition, there is a problem that the interaction between BSAs increases the viscosity of the reagent solution, making it difficult to run out of the solution in a piston-type pipette or the like.

Therefore, as alternatives to BSA, Patent Document 1 uses amino acid esters and polyamines, Patent Document 2 uses polymers having phosphorylcholine groups, Non-Patent Document 1 uses amino acids, and Non-Patent Document 2 uses polyethylene glycol. , disclose techniques for stabilizing proteins contained in reagents.
However, none of the above-described methods has been found to have a sufficient protein-stabilizing effect.
Therefore, there has been a demand for a protein stabilizer that can sufficiently stabilize proteins contained in reagents used in biochemical measurements and that does not affect reagent solution depletion.

Japanese Patent Application Laid-Open No. 2004-108850 JP-A-10-045794

K. Shiraki et al.; J. Biochem. (2002) 132 pp. 591-595 J. L. Cleland et al.; J. Biol. Chem. (1992) 267 pp. 13327-13334

An object of the present invention is to contain a protein stabilizer capable of stabilizing proteins such as enzymes, antibodies, and labeled antibodies contained in reagents used in biochemical measurements, and stabilized proteins, and To provide a reagent which is easily drained when it is brought into a solution state.
Another object of the present invention is to provide a method for stabilizing proteins contained in reagents used in biochemical measurements.

As a result of intensive studies in view of the above problems, the present inventors have found that proteoglycan can satisfactorily stabilize the protein contained in the reagent, and furthermore, can improve the drainage of the reagent in a solution state. , completed the present invention.
That is, the present invention relates to the following.
[1] A protein stabilizer comprising a plant proteoglycan purified from gum arabic and having a weight average molecular weight of 900,000 to 3,500,000 .
[2] The protein stabilizer according to [1] , further comprising a copolymer containing a structural unit based on 2- ( meth )acryloyloxyethylphosphorylcholine .
[3] The protein stabilizer according to [1] or [2] , wherein the content of plant proteoglycan is 0.001% by mass to 10% by mass.
[4] The protein according to [ 2 ] or [ 3 ], wherein the content of the copolymer containing structural units based on 2- ( meth ) acryloyloxyethylphosphorylcholine is 0.001% by mass to 10% by mass. stabilizer.
[5] A stabilized protein-containing reagent, which is purified from gum arabic and contains 0.001% by mass of plant proteoglycan having a weight average molecular weight of 900,000 to 3,500,000 A reagent containing ~10% by mass.
[6] The reagent according to [ 5 ], further comprising 0.001% to 10% by mass of a copolymer containing a structural unit based on 2- ( meth )acryloyloxyethylphosphorylcholine .
[7] A protein-containing reagent is added with vegetable proteoglycan purified from gum arabic and having a weight average molecular weight of 900,000 to 3,500,000, and the content of proteoglycan in the reagent is 0.5. A method for stabilizing a protein in a reagent, comprising adding the protein to a concentration of 001% by mass to 10% by mass.
[8] Further, a copolymer containing a structural unit based on 2- ( meth ) acryloyloxyethylphosphorylcholine is added so that the content of the copolymer in the reagent is 0.001% by mass to 10% by mass. The stabilization method of [ 7 ], comprising adding to

The protein stabilizer of the present invention can stabilize proteins contained in reagents used in biochemical measurements. In particular, when the reagent is stored in the form of a solution, proteins such as enzymes, antibodies, and labeled antibodies contained in the reagent can be well stabilized.
Further, according to the present invention, a reagent containing a stabilized protein can be provided. Dispensing utensils such as pipettes are easy to drain.
Furthermore, the present invention can provide a method for stabilizing proteins that can satisfactorily stabilize proteins contained in reagents used in biochemical measurements.

The present invention provides a protein stabilizer (hereinafter also referred to as "stabilizer of the present invention").
The stabilizer of the present invention contains proteoglycans.

The proteoglycan contained in the stabilizer of the present invention is a sugar-protein complex having a special structure, and includes animal and vegetable proteoglycans.
Animal proteoglycans are glycoproteins in which multiple glycosaminoglycans such as chondroitin sulfate and keratan sulfate are covalently bound to one core protein. are widely distributed in the body.
Plant proteoglycans are arabinogalactan and core protein bound in a certain manner, formally called arabinogalactan-protein (AGP), and are found in plant cell walls and sap as an extracellular matrix. exist.
In the present invention, both animal proteoglycans and plant proteoglycans can be used as proteoglycans, but plant proteoglycans are preferably used in consideration of production costs and the recent tendency to prefer plant-based raw materials. .

As the vegetable proteoglycan, those made from gum arabic obtained from Acacia senegal Willdenow belonging to the genus Acacia of the subfamily Albaceae of the family Fabaceae or from plants closely related to the same genus are preferably used. Here, gum arabic is obtained by drying the exudate exuding from the wound of the bark of the plant.
Gum arabic obtained from Acacia senegal Willdenow or its seyal species, Acacia seyal Delile, is used for the purpose of the present invention, because it is possible to obtain proteoglycans having bioactivity equal to or higher than that of animal proteoglycans. The raw material is more preferable, and the raw material is more preferably gum arabic obtained from Acacia senegal Willdenow.

In the present invention, plant proteoglycan, which is preferably used as proteoglycan, is obtained as a purified product obtained by removing mainly arabinogalactan and glycoprotein from gum arabic obtained from plants such as Hevea arabic.
For purposes of the present invention, plant proteoglycans have a weight average molecular weight of 900,000 to 3,500,000 as measured by size exclusion chromatography with an on-line multi-angle light scattering detector and a differential refractive index detector. One is preferably used, and one of 1,000,000 to 3,000,000 is more preferably used.
In addition, it is measured by an aldehyde quantification kit such as Amplite (trademark) Aldehyde Quantitation Kit (colorimetric) (product number: 10051) (manufactured by AAT Bioquest). Those having a total aldehyde content of 0.005 μmol equivalent/g to 2 μmol equivalent/g are preferably used.
In the present invention, the plant proteoglycan may be used in either liquid form such as aqueous solution or dispersion, or solid form such as powder.

For the stabilizer of the present invention, the proteoglycans described above can be used singly or in combination of two or more.
Animal proteoglycans may be extracted and purified from animal tissues such as salmon nasal cartilage, shark fin cartilage and squid head cartilage, and may be used, but commercial products provided by various companies may also be used. can.
In the present invention, the animal proteoglycan may be used in any of liquid forms such as aqueous solutions and dispersions, and solid forms such as powders.
On the other hand, vegetable proteoglycan is preferably purified by subjecting an aqueous solution of gum arabic adjusted to 0.5% by mass to 40% by mass to a porous type I strongly basic anion exchange resin and a strongly acidic cation exchange resin. Although it can be manufactured, it is also possible to use a commercially available product, which is generally available as an aqueous solution having an active ingredient content of about 1% by weight.
Such commercial products of vegetable proteoglycans include, for example, "Phytoproteoglycan (registered trademark)" manufactured by NOF Corporation.

A proteoglycan consists of a sugar chain portion and a protein portion, and the protein portion of the proteoglycan exhibits an adsorption effect on proteins. On the other hand, the sugar chain portion has a much larger radius of gyration than the protein portion, and can prevent self-aggregation between proteins. In addition, since the sugar chain portion of proteoglycan has a branched structure, it has the characteristics of low viscosity and easy penetration into the uneven regions of proteins, despite having a larger molecular weight than general polymer components. Due to these properties, the proteoglycan-containing stabilizer of the present invention prevents protein deactivation and exerts an excellent protein stabilizing effect.

The stabilizer of the present invention may further contain a copolymer containing structural units based on 2-(meth)acryloyloxyethylphosphorylcholine (MPC) (hereinafter sometimes referred to as "MPC copolymer").
Examples of the MPC copolymer that can be contained in the stabilizer of the present invention include copolymers containing a structural unit based on MPC and a structural unit based on a hydrophobic monomer. Examples of units include structural units based on (meth)acrylic acid alkyl esters, (meth)acrylamide derivatives, and the like.

A structural unit based on MPC is represented by the following formula (1). In addition, in this specification, "(meth)acryloyl" means "acryloyl or methacryloyl."

(In the formula, R ¹ represents a hydrogen atom or a methyl group.)

A structural unit based on a (meth)acrylic acid alkyl ester is represented by the following formula (2). Here, "(meth)acrylic acid" means "acrylic acid or methacrylic acid".

(In the formula, R ² represents a hydrogen atom or a methyl group, and R ³ represents an alkyl group having 1 to 24 carbon atoms.)

In formula (2), the alkyl group represented by R ³ is a linear or branched alkyl group having 1 to 24 carbon atoms, and is methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, 2-ethylhexyl, decyl. , dodecyl, tetradecyl, hexadecyl, octadecyl, docosyl and the like.

Therefore, structural units based on the (meth)acrylic acid alkyl ester represented by formula (2) include methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, and (meth)acrylic acid. Based on butyl, 2-ethylhexyl (meth)acrylate, dodecyl (lauryl) (meth)acrylate, tridecyl (meth)acrylate, octadecyl (stearyl) (meth)acrylate, docosyl (behenyl) (meth)acrylate, etc. structural units.
Further, structural units based on (meth)acrylic acid alkyl esters include diethylaminoethyl (meth)acrylate, trifluoroethyl (meth)acrylate, heptadecafluorodecyl (meth)acrylate, and the like. A (meth)acrylic acid alkyl ester in which the alkyl group represented by R ³ is substituted with a dialkylamino group, a halogen atom, or the like, and an alkyl group represented by R ³ is substituted with a hydroxyl group and a quaternary ammonio group Structural units based on 2-hydroxy-3-(meth)acryloyloxypropyltrimethylammonium chloride are also exemplified as structural units based on hydrophobic monomers.

A structural unit based on a (meth)acrylamide derivative is represented by the following formula (3). Here, "(meth)acrylamide" means "acrylamide or methacrylamide".

(In the formula, R ⁴ represents a hydrogen atom or a methyl group, R ⁵ and R ⁶ each independently represent a methyl group or an ethyl group, and m represents an integer of 1 to 3.)

For the purposes of the present invention, copolymers containing structural units based on N,N-dimethylaminoethylacrylamide or N,N-dimethylaminopropylacrylamide as structural units based on (meth)acrylamide derivatives are preferred.

In the present invention, as the MPC copolymer, a copolymer containing one or more of a structural unit based on MPC and a structural unit based on the above hydrophobic monomer can be used.
For purposes of the present invention, structural units based on hydrophobic monomers include structural units based on (meth)acrylic acid alkyl esters, structural units based on 2-hydroxy-3-(meth)acryloyloxypropyltrimethylammonium chloride, and (meth)acrylamide derivative-based structural units.

The ratio of the respective contents of the structural unit based on MPC and the structural unit based on the hydrophobic monomer in the MPC copolymer ([structural unit based on MPC]: [structural unit based on hydrophobic monomer] ) (n ₁ :n ₂ ) is in a molar ratio of 95:5 to 65:35, preferably 90:10 to 70:30, more preferably 85:15 to 75:25.

The weight average molecular weight of the MPC copolymer contained in the stabilizer of the present invention is 10,000 to 5,000,000, preferably 20,000 to 1,000,000.
If the weight-average molecular weight of the MPC copolymer is less than 10,000, the radius of gyration may decrease, and the effect of improving liquid drainage may not be expected. rises sharply, making it difficult to prepare protein stabilizers and reagents containing the stabilizers.
In addition, the weight average molecular weight of the MPC copolymer is measured by gel permeation chromatography (GPC) and is indicated by the pullulan-equivalent molecular weight.

The polymerization form of the MPC copolymer is not particularly limited, and may be a random copolymer or a block copolymer, but a random copolymer is preferred.
Further, the MPC copolymer may be a copolymer containing other structural units in addition to the structural units based on MPC and the structural units based on the hydrophobic monomer.
The other structural units can be appropriately selected from those that can usually be a structural unit of a copolymer as long as they do not affect the effects of the present invention. Other structural units include, for example, alkenyl esters of (meth)acrylic acid such as allyl (meth)acrylate; cyclic alkyl esters of (meth)acrylic acid such as cyclohexyl (meth)acrylate; tetrahydro(meth)acrylate; (Meth) acrylic acid esters having an alkyl group substituted with a heterocycle such as furfuryl; (meth) acrylic acid esters containing an aromatic group such as benzyl (meth) acrylate and phenoxyethyl (meth) acrylate; Esters of (meth)acrylic acid and monoterpene alcohol such as isobornyl (meth)acrylate; glycidyl (meth)acrylate; styrene-based monomers such as styrene, methylstyrene, chloromethylstyrene; methyl vinyl ether, butyl vinyl ether, etc. vinyl ether-based monomers; and vinyl ester-based monomers such as vinyl acetate and vinyl propionate.
These other structural units may contain one or more types, and the content thereof in the MPC copolymer is relative to the total amount of the structural units based on the MPC and the structural units based on the hydrophobic monomer. It is 40 mol % or less, preferably 20 mol % or less.

The MPC copolymer can be produced by a production method known per se.
For example, a monomer mixture containing hydrophobic monomers such as MPC and (meth)acrylic acid alkyl esters, and, if necessary, monomers corresponding to the above other structural units, in the presence of a radical polymerization initiator. , under an atmosphere of an inert gas such as nitrogen, by a known method such as solution polymerization.
The content ratio of each monomer at that time may be a ratio corresponding to the content ratio of each structural unit in the MPC copolymer.

The stabilizer of the present invention may contain one of the above MPC polymers, or may contain two or more of them in combination.
The form of the MPC copolymer contained in the stabilizer of the present invention may be either solid such as powder or liquid such as solution or dispersion.

In the present invention, the above-described proteoglycan or a mixture of proteoglycan and MPC copolymer can be directly used as the stabilizer of the present invention.
In addition, excipients, binders, lubricants, disintegrants, coating agents, bases, solvents, diluents, solubilizers, solubilizers, emulsifiers, dispersants, Suspending agents, stabilizers, thickeners, pH adjusters, buffers, antioxidants, preservatives, preservatives, and other general pharmaceutical additives are added, and known formulation means, such as the Solid forms such as powders, granules, tablets, etc., semi-solid forms such as pastes, solutions, suspensions, and dispersions, according to the methods described in the 17th revision of the Japanese Pharmacopoeia General Rules for Pharmaceutical Preparations [3] It can also be in the form of a liquid, such as a liquid or emulsion.

The stabilizing agent of the present invention contains proteoglycan, or proteoglycan and MPC copolymer, so that the content thereof in the protein-containing reagent described below is the content described below.
When the stabilizer of the present invention contains proteoglycan and MPC copolymer, their content ratio (proteoglycan: MPC copolymer) is preferably 0.001:10 to 10: 0.001, more preferably 0.005:5 to 5:0.005.

The stabilizer of the present invention is added to the protein-containing reagent described below so that the content of proteoglycan or proteoglycan and MPC copolymer will be the content described below.

In biochemical measurements, a protein-containing reagent is often used in the form of a solution. Therefore, from the viewpoint that it can be easily added to, mixed with, or easily mixed with the liquid reagent, The stabilizers of the invention are preferably provided in liquid form.
The liquid stabilizing agent of the present invention can be prepared by adding proteoglycan or proteoglycan and MPC copolymer to water or a buffer solution and uniformly dissolving them.
As water, it is preferable to use pure water such as purified water and deionized water.
As the buffer, buffers commonly used for preparing reagents containing proteins used in biochemical measurements can be used, including phosphate buffers, Tris buffers, Good's buffers, glycine buffers, boric acid buffers. buffers, carbonate buffers and the like. One or more of these buffers can be used.

The content of proteoglycan in the stabilizer of the present invention in liquid form is 0.001% by mass to 10% by mass, and from the viewpoint of the effect on the liquid drainability of the reagent, it is preferably 0.005% by mass to 0.005% by mass. 5% by mass.
If the proteoglycan content is less than 0.001% by mass, a sufficient protein stabilizing effect cannot be exhibited, and if the proteoglycan content exceeds 10% by mass, the viscosity of the stabilizer increases sharply, Handling may become difficult.

The content of the MPC copolymer in the stabilizer of the present invention in a liquid form can be appropriately selected in the range of 0.001% by mass to 10% by mass, and from the viewpoint of the effect on the liquid-removing property of the reagent. , 0.005% by mass to 5% by mass.
If the content of the MPC copolymer is less than 0.001% by mass, it may not be possible to sufficiently improve the liquid drainability of the reagent. may impair the liquid drainability of

Proteins stabilized by the stabilizer of the present invention are not particularly limited, but are peroxidase, alkaline phosphatase, β-D-galactosidase, lipase, DNA polymerase, RNA polymerase, enzymes such as reverse transcriptase, immunoglobulin G, Examples thereof include antibodies such as immunoglobulin E, labeled antibodies such as complexes of the above antibodies and the above enzymes (enzyme-labeled antibodies), and the like.
Among these, the stabilizer of the present invention can be suitably used for stabilizing enzyme-labeled antibodies that are frequently used in enzyme immunoassays.

The stabilizing agent of the present invention is capable of stabilizing the above proteins, such as enzymes, antibodies, labeled antibodies, etc., contained in reagents. , can maintain its physiological activity for a long period of time.
Therefore, the stabilizing agent of the present invention can allow the reagent containing the protein to be stored for a long period of time in the form of a solution.

The present invention also provides a reagent containing the stabilized protein (hereinafter also referred to as "reagent of the present invention").
The reagents of the invention contain proteoglycans.
The proteoglycan contained in the reagent of the present invention is as described above for the stabilizer of the present invention.
The reagent of the present invention may contain one or more of the proteoglycans described above. In addition, proteoglycan may be prepared and added as the stabilizer of the present invention described above.

The content of proteoglycan in the reagent of the present invention is 0.001% by mass to 10% by mass, and preferably 0.005% by mass to 5% by mass from the viewpoint of the protein stabilization effect and the reagent's liquid drainability. be.
If the proteoglycan content is less than 0.001% by mass, a sufficient protein stabilizing effect cannot be exhibited. Handling such as preparation may become difficult.

The reagents of the invention may further contain MPC copolymers.
The MPC copolymer contained in the reagent of the present invention is as described above for the stabilizer of the present invention.
The reagent of the present invention may contain one or more of the MPC copolymers described above. Incidentally, the MPC copolymer may be prepared and added as the stabilizer of the present invention described above.

The content of the MPC copolymer in the reagent of the present invention can be appropriately selected in the range of 0.001% by mass to 10% by mass. It is preferable to select within a range.
If the content of the MPC copolymer is less than 0.001% by mass, the reagent of the present invention may not be sufficiently drained. This may impair the liquid drainability of the reagent of the present invention.

The stabilized protein contained in the reagent of the present invention is as described above as the protein stabilized by the stabilizing agent of the present invention.
The content of the protein in the reagent of the present invention is appropriately set depending on the ^type of protein and the like. It is preferable to adjust according to the purpose.

Furthermore, the reagent of the present invention can contain general additives commonly used in this field as long as they do not impair the features of the present invention.
Such additives include, for example, polyols, polyethers, proteins other than proteins used for the purpose of biochemical measurements, amino acids, inorganic salts, buffers, chelating agents, organic acids, surfactants, biochemical reagents, preservatives. agents, organic solvents, and the like.
Polyols include, for example, glycerol, sucrose, glucose and the like.
Examples of polyethers include polyoxyethylene glycol and the like.
Examples of proteins other than proteins used for biochemical measurement include gelatin and casein.
Examples of amino acids include amino acids such as glycine, alanine, glutamic acid, aspartic acid, lysine and histidine, salts thereof, peptides and the like.
Examples of inorganic salts include sodium salts, potassium salts, magnesium salts, calcium salts, phosphates, sulfates, hydrochlorides and the like.
Examples of buffering agents include trishydroxymethylaminoethane, tris(2-hydroxyethyl)aminomethane and the like.
Chelating agents include, for example, ethylenediaminetetraacetic acid and the like.
Organic acids include, for example, acetic acid, citric acid, and malic acid.
Examples of surfactants include polyoxyethylene alkyl ethers, polyoxyethylene sorbitan monoalkyl esters, alkylbetaines, and the like.
Examples of biochemical reagents include flavins, coenzymes such as colipase, and nucleic acids such as nucleosides and nucleotides.
Examples of antiseptics include sodium azide, paraoxybenzoic acid preparations, dehydroacetic acid preparations, proclin preparations and the like.
Examples of organic solvents include methanol, ethanol, n-propanol, isopropanol, isoamyl alcohol, dimethylformamide, dimethylsulfoxide, tetrahydrofuran, chloroform and phenol.
The reagent of the present invention may contain one or more of these additives as required.

The reagent of the present invention can be prepared by adding other additives as described above to proteins such as proteoglycan, proteoglycan, proteoglycan and MPC copolymer, enzymes, antibodies, labeled antibodies, etc., and preparing powders, It can be in the form of solids such as granules and tablets, semi-solids such as pastes, and liquids such as solutions, suspensions, dispersions, and emulsions.
The reagent of the present invention can also be prepared by adding the above protein to the stabilizer of the present invention.

Since the reagent of the present invention is excellent in stability in a solution state, proteoglycan, or proteoglycan and MPC copolymer, and proteins such as enzymes, antibodies, labeled antibodies, etc. are dissolved in water or a buffer to form a solution. It is preferable to prepare as Alternatively, the stabilizer of the present invention described above and the protein described above may be dissolved in water or a buffer to form a solution.
The reagent of the present invention may be dissolved in water and stored as an aqueous solution, or dissolved in a buffer and stored as a buffer solution.
Pure water such as purified water and deionized water is preferably used as water.
As the buffer, a buffer commonly used in biochemical measurements can be used, and examples thereof include phosphate buffer, Tris buffer, Good's buffer, glycine buffer, borate buffer, carbonate buffer, and the like. mentioned. Moreover, you may use these in mixture of 2 or more types.

When the stabilizing agent of the present invention is provided in a liquid form, a protein such as an enzyme, an antibody, or a labeled antibody is added to the stabilizing agent and dissolved, or the protein is added to an aqueous solution or buffer solution. It can be added as a dissolved solution and mixed to form the reagent of the present invention.
As described above, the final protein content in the reagent of the present invention is appropriately set according to the type of protein, the purpose of use of the reagent of the present invention, and the like. , is added to the adjusted content.

As described above, the reagent of the present invention is excellent in storage stability in a solution state.
Suitable temperatures for protein stabilization are -30°C to 40°C, particularly preferably 0°C to 30°C.
That is, when the reagent of the present invention is stored in the form of a solution within this temperature range, the protein in the reagent can be stably stored for a long period of time and its physiological activity can be maintained.

The present invention further provides a method for stabilizing proteins contained in reagents used in biochemical measurements (hereinafter also referred to as "the method of the present invention").
The method of the invention involves adding proteoglycans to a protein-containing reagent.
In the method of the present invention, the protein contained in the reagent is as described above as the protein to be stabilized in the stabilizing agent of the present invention.
Moreover, in the method of the present invention, the proteoglycan added to the reagent is as described above for the stabilizer of the present invention.
In the method of the present invention, one or more of the above-described proteoglycans can be added. In addition, proteoglycan may be prepared and added as the stabilizer of the present invention described above.
The amount of proteoglycan to be added in the method of the present invention can be such that the content of proteoglycan in the reagent used for biochemical measurement is the content in the reagent of the present invention described above.

The method of the invention further comprises adding an MPC copolymer.
In the method of the present invention, the MPC copolymer further added is as described above for the stabilizer of the present invention.
In the method of the present invention, one or more of the above MPC copolymers can be added. The MPC copolymer may be prepared and added as the stabilizer of the present invention.
The amount of the MPC copolymer to be added in the method of the present invention can be such that the content of the MPC copolymer in the reagent used for biochemical measurements is the content in the reagent of the present invention described above. .

INDUSTRIAL APPLICABILITY According to the method of the present invention, proteins contained in reagents used for biochemical measurements can be stabilized, and the reagents can be stored for a long period of time.
The method of the present invention can be suitably applied when reagents used for biochemical measurements are stored in a solution state such as an aqueous solution or a buffer solution.

EXAMPLES The present invention will be described in more detail below based on examples, but the present invention is not limited to these.

[Examples 1 to 4, Comparative Examples 1 and 2] Protein Stabilizer According to the formulation shown in Table 1, protein stabilizers of Examples 1 to 4 and Comparative Examples 1 and 2 were prepared as follows.
Vegetable proteoglycan, MPC copolymer, and BSA were added to a phosphate buffer (pH = 7.4) so that the contents shown in Table 1, respectively, were dissolved by stirring until uniform, and the protein was stabilized. agent.
As the vegetable proteoglycan, "Phytoproteoglycan" (manufactured by NOF Corporation) (weight average molecular weight = about 1,200,000, total aldehyde content = 1.8 μmol equivalent / g) was used, and as the MPC copolymer used an MPC/butyl methacrylate copolymer (copolymer composition ratio of MPC and butyl methacrylate [MPC/butyl methacrylate] (molar ratio) = 80/20, weight average molecular weight = 600,000).
As BSA, "A2153" (manufactured by Sigma-Aldrich) was used.

[Test Example 1] Evaluation of Protein Stabilizing Effect The protein stabilizing agents of Examples 1 to 4 and Comparative Examples 1 and 2 were evaluated for their protein stabilizing effect during refrigerated storage as follows.
Horseradish-derived peroxidase was added to a phosphate buffer (pH=7.4) to a concentration of 2 μg/mL, and dissolved by stirring until homogeneous to prepare a protein solution. 50 μL of the protein solution was added to 1 mL of each of the protein stabilizers of the above Examples and Comparative Examples, and the mixture was stirred until uniform to prepare a solution for evaluation.
The evaluation solution was stored at 4° C. for 1 day, 4 days, 8 days, 13 days, 21 days, and 28 days, 10 μL was added to each 96-well plate, and ABTS (2,2′-azobis (3 -ethylbenzothiazoline-6-sulfonic acid) ammonium salt) solution (manufactured by SeraCare Lifescience) was added and shaken at room temperature for 30 minutes. After shaking, 100 μL of 1 mass % sodium dodecyl sulfate solution was added to stop the reaction. The absorbance at 410 nm was measured to evaluate the protein stabilizing effect.
That is, immediately after preparing the evaluation solution and after storing for 1 day, 4 days, 8 days, 13 days, 21 days, and 28 days, the absorbance of the evaluation solution was measured under the following measurement conditions. The enzymatic activity residual rate (%) was calculated by Equation 1).
The protein stabilizing effect is evaluated by the residual enzyme activity rate, and the higher the residual enzyme activity rate, the higher the protein stabilizing effect. The number of days during which 85% or more of the enzyme activity residual rate was maintained was defined as the high activity maintenance period (days).
A solution obtained by similarly mixing the protein solution with a phosphate buffer (pH=7.4) was used as a control, and the protein stabilizing effect was similarly evaluated.
The evaluation results are also shown in Table 1.
<Measurement conditions>
Measuring equipment: Plate reader manufactured by DS Pharma Biomedical Co., Ltd. Measuring temperature: Room temperature Measuring wavelength: 410 nm

[Test Example 2] Evaluation of liquid drainability The liquid drainability of the protein-stabilizing reagent of the present invention was evaluated by the following method. A pipette tip (long tip, manufactured by Watson) was attached to a Pipetman (P1000, manufactured by Gilson), and 1,000 μL of each of the evaluation solution prepared in Test Example 1 and the control was collected, and the entire amount was discharged, and the initial discharge liquid was obtained. was measured. This operation was repeated 60 times without replacing the pipette tip, and the final weight of the 61st discharge liquid was measured. A liquid depletion index was calculated according to the following formula (Formula 2), and the liquid depletion property was evaluated according to the following evaluation criteria. The evaluation results are also shown in Table 1.

<Evaluation Criteria>
Liquid shortage index = 99.95 to 100: ◎
Liquid shortage index = 99.90 or more and less than 99.95: ○
Liquid shortage index = less than 99.90: ×

As shown in Table 1, peroxidase was rapidly deactivated in the control, but each of the protein stabilizers of Examples 1 to 4 containing vegetable proteoglycan was stored under refrigerated storage conditions for a storage period of 28 The high activity of peroxidase was maintained during the day).
In contrast, the protein stabilizer of Comparative Example 2 containing BSA did not exhibit a sufficient effect of stabilizing peroxidase.
In addition, the evaluation solutions containing the protein stabilizers of Examples 1 to 4 of the present invention were all evaluated to be excellent in drainability, but they contained the protein stabilizers of Comparative Examples 1 and 2. All of the evaluation solutions were evaluated to be inferior in liquid drainability.

As described in detail above, according to the present invention, proteins such as enzymes, antibodies, and labeled antibodies contained in reagents used in biochemical assays can be well stabilized even when the reagents are stored in a solution state. It is possible to provide a protein stabilizer capable of
Further, according to the present invention, a protein contained in a reagent used in biochemical measurements can be stably stored in a cold storage state for a long period of time in a solution state, and the reagent can be easily drained in a dispensing instrument such as a pipette. can be done.
Furthermore, the present invention can provide a stabilization method capable of satisfactorily stabilizing proteins contained in reagents used in biochemical measurements.
Therefore, the present invention is useful in fields where the physiological activity of proteins contained in reagents is required to be stably maintained, preferably in fields such as clinical testing, in-vitro diagnostics, companion diagnostics, food analysis, and environmental analysis. It is expected to be applied to biochemical measurements such as immunoassay, immunoturbidimetry, nucleic acid analysis, and immunostaining. In addition to these fields, it is expected to be applied in a wide range of fields such as medical equipment, pharmaceuticals, and biosensing.

Claims (8)

A protein stabilizer comprising a vegetable proteoglycan purified from gum arabic and having a weight average molecular weight of 900,000 to 3,500,000 . 2. The protein stabilizer according to claim 1 , further comprising a copolymer containing a structural unit based on 2- ( meth )acryloyloxyethylphosphorylcholine . 3. The protein stabilizer according to claim 1 , wherein the content of plant proteoglycan is 0.001% by mass to 10% by mass. 4. The protein stabilizer according to claim 2 , wherein the content of the copolymer containing structural units based on 2- ( meth )acryloyloxyethylphosphorylcholine is 0.001% by mass to 10% by mass. 0.001 % to 10% by mass of plant proteoglycan, which is a stabilized protein-containing reagent purified from gum arabic and having a weight average molecular weight of 900,000 to 3,500,000 % containing reagents. 6. The reagent according to claim 5 , further comprising 0.001% by mass to 10% by mass of a copolymer containing a structural unit based on 2- ( meth )acryloyloxyethylphosphorylcholine . A protein-containing reagent is added with a vegetable proteoglycan purified from gum arabic and having a weight average molecular weight of 900,000 to 3,500,000, and the content of the proteoglycan in the reagent is 0.001 mass . A method for stabilizing a protein in a reagent, comprising adding so as to be 10% to 10% by mass. Furthermore, a copolymer containing a structural unit based on 2- ( meth ) acryloyloxyethylphosphorylcholine is added so that the content of the copolymer in the reagent is 0.001% by mass to 10% by mass. 8. The stabilization method of claim 7 , comprising: