JP4965302B2 - Antibody stabilization methods - Google Patents

Description

  The present invention relates to a method for stabilizing antibodies. Specifically, the present invention relates to a method for stabilizing an antibody, which can be stabilized for a long time in a liquid state or a dry state by allowing the antibody to coexist with sericin and / or a hydrolyzate thereof, or an equivalent thereof.

  The antibody has been applied to various uses including clinical diagnosis as a reagent for immunological measurement because of its high substrate specificity and simplicity. Specific examples include analytical reagents for molecular biology, analytical reagents for biochemistry, and in vitro diagnostic agents. Antibodies applied to these reagents may be labeled or modified with various compounds. These reagents are provided in a liquid state, a dry state, a state where an antibody is immobilized on a carrier and immersed in a buffer solution, or a dry state, depending on the purpose and application.

  Needless to say, in order to maintain the performance of the antibody-containing composition as described above over a long period, it is important to stably maintain the antibody activity. For example, when the antibody activity in the composition decreases over time, there are measures to add an excess amount of antibody in advance in accordance with the desired effective period of the composition and the rate of decrease in antibody activity. In such a case, such a measure does not provide a fundamental solution to the problem, and it is inevitable that the cost of the antibody increases.

  Therefore, as in the case of compositions containing general proteins, the antibody-containing composition contains salts such as phosphates, hydrochlorides and sulfates, saccharides such as lactose, sucrose and trehalose, polyvalents such as glycerol, ethylene glycol and erythritol. Amino acids such as alcohol, arginine, lysine, glycine, fatty acid esters, surfactants such as polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (10) octylphenyl ether, proteins such as albumin, casein, and gelatin Many methods for stabilizing by coexisting have been proposed (for example, Patent Documents 1 to 7). Among them, bovine serum albumin has been widely used as a stabilizer because of its excellent effect.

However, bovine serum albumin has a limited source and is very expensive, leading to an increase in the cost of the composition. In addition, animal proteins such as bovine serum albumin have a risk of infection by viruses, and safety may not be sufficiently secured. Furthermore, consideration for coloring is also necessary.
In addition, when salts or amino acids are used in a solution, a sufficient amount cannot be added due to low solubility or precipitation during storage, and a desired effect may not be obtained. In addition, when saccharides or amino acids are applied to a diagnostic agent, an unintended reaction may occur between a contaminant substance coexisting in a reagent system or mixed in an antibody and a stabilizer. In addition, polyhydric alcohols and surfactants may interfere with the reaction with the antigen.
Furthermore, many of the conventionally proposed stabilizers have different stabilizing effects depending on the type of antibody, and lack general versatility.

JP-A-60-35263 JP-T 63-500562 JP-A-6-186230 Japanese Patent Laid-Open No. 9-80051 Japanese Patent Laid-Open No. 10-279594 JP-T-2001-503781 JP 2003-215127 A

  The present invention has been made in view of such a current situation, and the object of the present invention is to be widely applicable regardless of whether it is in a liquid state or a dry state, and the antibody can be applied over a long period of time. It is to provide a way to stabilize.

  As a result of various studies to achieve the above object, the present inventors have found that an antibody can be effectively stabilized by allowing the antibody to coexist with sericin and / or a hydrolyzate thereof, or an equivalent thereof. The present invention has been completed.

That is, the present invention is as follows.
(1) A method for stabilizing an antibody, which comprises allowing an antibody to coexist with sericin and / or a hydrolyzate thereof, or an equivalent thereof.
(2) The method for stabilizing an antibody according to (1), wherein the antibody is in a liquid state.
(3) The method for stabilizing an antibody according to (1), wherein the antibody is in a dry state.
(4) The method for stabilizing an antibody according to (2), wherein the antibody is immobilized on a carrier.
(5) The method for stabilizing an antibody according to (3), wherein the antibody is immobilized on a carrier.
(6) Stabilization of antibody according to any one of (1) to (5), wherein sericin and / or a hydrolyzate thereof is derived from natural sericin extracted from silkworm silk or raw silk Method.
(7) The method for stabilizing an antibody according to any one of (1) to (5), wherein the sericin equivalent is obtained by a genetic engineering technique.

According to another aspect of the invention, a composition comprising a stabilized antibody is provided. That is,
(8) The composition wherein the antibody coexists with sericin and / or a hydrolyzate thereof, or an equivalent thereof.
(9) The composition according to (8), wherein the antibody is in a liquid state.
(10) The composition according to (8), wherein the antibody is in a dry state.
(11) The composition according to (9), wherein the antibody is immobilized on a carrier.
(12) The composition according to (10), wherein the antibody is immobilized on a carrier.
(13) The antibody composition according to any one of (8) to (12), wherein sericin and / or a hydrolyzate thereof is derived from natural sericin extracted from silkworm silk or raw silk. .
(14) The antibody composition according to any one of (8) to (12), wherein the sericin equivalent is obtained by a genetic engineering technique.

According to still another aspect of the present invention, a method for producing the composition is provided. That is,
(15) A method for producing a composition, comprising a step of allowing an antibody to coexist with sericin and / or a hydrolyzate thereof, or an equivalent thereof.

According to still another aspect of the present invention, a diagnostic agent and biosensor comprising the composition are provided. That is,
(16) A diagnostic agent comprising the composition according to (8) to (14).
(17) A biosensor comprising the composition according to (8) to (14).

  According to the present invention, by allowing an antibody to coexist with sericin and / or a hydrolyzate thereof, or an equivalent thereof, the stability of the antibody can be effectively improved regardless of the liquid state or the dry state. it can. Therefore, the effectiveness of the composition containing the antibody can be maintained over a long period of time.

The present invention is described in detail below.
The gist of the antibody stabilization method of the present invention is to coexist an antibody with sericin and / or a hydrolyzate thereof, or an equivalent thereof.

  In the present invention, the antibody may be in a liquid state or a dry state. The antibody may be immobilized on a carrier. In this specification, the “liquid state” means that the antibody is present in the liquid, the antibody is completely dissolved in the liquid, the state is dispersed in the liquid like a suspension, the carrier It is also intended to include a state of being fixed in and immersed in a liquid.

In the present invention, “stabilization” means that the residual function retained by an antibody after it has been stored for a certain period of time when the antibody coexists with a substance (a) and when it does not coexist (a) (a) )> (B).
For example, when the antibody is in a liquid state (a) and when it does not coexist (b), the ratio of the residual activity after storage for a certain time at an appropriate temperature is (a)> (b ).
In addition, when the antibody is in a dry state (a) and when it is not coexisting (b), the ratio of the residual activity after storage for a certain time at an appropriate temperature is (a)> (b ).
Furthermore, the residual activity after storage for a certain period of time at an appropriate temperature in the case where an antibody is immobilized on a carrier and a substance is coexisted in a liquid (a) or not (b) The ratio of (a)> (b).
Furthermore, in the case where the antibody is immobilized on a carrier and a certain substance coexists in a dry state (a) and in the case where it does not coexist (b), the residual activity after storage at an appropriate temperature for a certain period of time. The ratio is in a state where (a)> (b).

The evaluation of stabilization can be performed, for example, by measuring the residual activity over time in the case where a certain substance coexists (a) and in the case where it does not coexist (b), and comparing the half lives.
The conditions of “storage at a suitable temperature for a certain period of time” are not particularly limited as long as there is a difference in the residual activity between the above (a) and (b), but preferably the long-term storage stability in a diagnostic agent, etc. The accelerated (severe) test conditions in mind are selected. Specific examples include “storage at 40 ° C. for 2 weeks” and “storage at 50 ° C. for 1 week”. If time permits, storage for 6 months or longer may be selected under refrigerated conditions of 2 ° C. to 10 ° C., which is widely used as a temperature at which diagnostic agents including antibodies are actually stored for a long period of time.

As one embodiment of the present invention, the residual activity rate after storing the antibody at 50 ° C. for 6 days in phosphate buffered saline (pH 7.2 to 7.5) is obtained by allowing sericin to coexist. This is a method that improves compared to the case where sericin is not coexistent.
In another embodiment of the present invention, the residual activity rate after immobilizing an antibody on an assay plate made of polyvinyl chloride and storing it in a dry state at 50 ° C. for 8 days is allowed to coexist with sericin. It is a method of improving compared to the case where the coexistence is not allowed.
In the above embodiment, sericin hydrolyzate or sericin equivalent may be used instead of sericin. Needless to say, the present invention is not limited to the above embodiment.

Here, sericin is a non-crystalline natural protein present in silkworm silk or raw silk, and in this specification, it is particularly meant to be extracted from silkworm silk or raw silk in a non-hydrolyzed state. Sericin has an amino acid sequence disclosed in WO2002 / 086133 pamphlet, and includes a functional peptide consisting of 38 amino acids as a repetitive sequence. The hydrolyzate is a product obtained by hydrolyzing natural product-derived sericin containing the functional peptide with an acid or alkali. Moreover, an equivalent means what was chemically synthesized so that at least 1 or more functional peptide might be included, and what was obtained by the genetic engineering method. Further, in these, amino acid residues were deleted, substituted, inserted or added to the natural amino acid sequence within the range not impairing the stabilizing action of the present invention or for improving the characteristics. It may be a thing. The sericin used for this invention, its hydrolyzate, and its equivalent can be obtained in accordance with the well-known method disclosed by the international publication 2002/086133 pamphlet.
In the examples of the present invention, easily available hydrolysates are used. The molecular weight of the hydrolyzate is not particularly limited as long as it has a stabilizing action, but from the viewpoint of handleability, the weight average molecular weight is in the range of 5,000 to 100,000, particularly 10,000 to 50,000. Some are preferably used.

  What is necessary is just to set suitably the usage-amount of sericin and / or its hydrolyzate, or its equivalent according to the kind of antibody to be used, the concentration of an antibody, the form of the composition containing an antibody, etc. For example, when the composition is in a liquid state, it can be added at a concentration of 0.1 to 200 g / L, preferably 0.1 to 100 g / L, more preferably 0.2 to 20 g / L. At this time, the weight ratio of the antibody to sericin and / or a hydrolyzate thereof or an equivalent thereof is 1: 1 to 1: 10,000,000, preferably 1:10 to 1: 1,000,000, More preferably, it can be set to 1: 100-1: 100,000.

The antibody to be the subject of the present invention is not particularly limited, but is typically an antibody used for clinical diagnosis. Specifically, for example, mouse IgG and immunoglobulin (IgG, IgA, IgM, IgD, IgE), hepatitis B virus, hepatitis C virus, HTLV (adult T cell leukemia virus), HIV (AIDS virus), Examples thereof include antibodies that use pathogens of various infectious diseases such as influenza virus, chlamydia, syphilis treponema, and toxoplasma as antigens. The antibody may be a polyclonal antibody, a monoclonal antibody, or a mixture of monoclonal antibodies. Further, it may be an antibody fragment such as F (ab ′) 2, Fab ′, or Fab fragmented by enzyme treatment or genetic engineering.
Furthermore, the antibody may be labeled with various enzymes such as peroxidase, amylase, catalase, glucose oxidase, glucose dehydrogenase, alkaline phosphatase, β-galactosidase, fluorescent dyes, metal colloids, colored latex particles and the like.
The storage state of the antibody to be the subject of the present invention is not particularly limited, and it may be in a liquid state, a frozen state, or a dry state, and may be stored in any state. It can be used and includes once dried or redissolved or resuspended in liquid.

According to the method for stabilizing an antibody of the present invention, a composition comprising a stabilized antibody is provided. The presence state of the composition may be a liquid state or a dry state, and is not particularly limited. Further, the antibody may be immobilized on a carrier, and the antibody may be immersed in a liquid in this state or may be in a dry state. When the composition is in a liquid state, it includes not only a state in which the antibody is completely dissolved in the liquid but also a state in which the antibody is dispersed in the liquid, such as a suspension.
Such a composition may be processed into a chip shape, a slit shape, or the like. Then, it can be placed in an appropriate container or mounted on an appropriate device, for example, as an analytical reagent for molecular biology, an analytical reagent for biochemistry, an in vitro diagnostic agent, a biosensor, a pharmaceutical, etc. Provided as a kit.

The composition may further contain other substances for the purpose of stabilization and shape improvement.
A composition in a liquid state is prepared by dissolving or suspending at least a target antibody and sericin and / or a hydrolyzate thereof, or an equivalent thereof in a liquid such as water, physiological saline or various aqueous buffers. Can be prepared. At this time, the concentration of the antibody is not particularly limited, but is usually 0.001 to 100 mg / mL, preferably 0.01 to 10 mg / mL. Specific examples of the aqueous buffer include Good buffers such as PIPES, MES, TES, MOPS, and HEPES, phosphate buffers, acetate buffers, borate buffers, citrate buffers, and tris buffers. A buffer known in the field of molecular biology or biochemistry can be mentioned. Such a composition may contain salts such as ammonium sulfate, ammonium phosphate, sodium chloride and potassium chloride. Moreover, surfactants, such as polyhydric alcohols, such as glycerol and ethylene glycol, alkyl glucoside, polyethyleneglycol alkyl ether, fatty-acid alcohol ester, may be included. Furthermore, azide, 1,1′-methylen-bis [3- (1-hydroxymethyl-2,4-diomidazolidin-5-yl) -urea], 2-methyl-3 (2H) -isothiazole-hydrochloride, 5- An antiseptic such as Bromo-5-nitro-1,3-dioxane, 2-hydroxypyridine-N-oxide, 2-chloroacetamide may be included.

  The dry composition can be prepared by drying the liquid composition by a conventional method, for example, freeze-drying or spray-drying. Such compositions include, as general stabilizers and shape improvers, sugars such as glucose, fructose, galactose, mannose, xylose, lactose, sucrose, raffinose, trehalose, cyclodextrin, pullulan, inulin, soluble starch, Sugar alcohols such as glucitol, mannitol, inositol, xylitol, amino acids and amino acid salts such as glycine, alanine, serine, threonine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, histidine, glycylglycine, glycylglycylglycine, etc. Inorganic salts such as peptides, phosphates, borates, sulfates, tris salts, proteins such as gelatin, casein, albumin, polyoxyethylene alkyl ethers, polyethylene glycols May include such surfactants such as alkyl ether, it is sufficient to add at the stage of a liquid state before drying.

  In the liquid and dry compositions, when the antibody is particularly enzyme-labeled, NAD +, NADH, NADP +, NADPH, ATP, ADP, AMP, GTP are used for the purpose of stabilizing the labeled enzyme. , GMP, FAD, FMN, biotin, niacin, cobalamin, coenzymes such as PQQ, metal salts such as sodium, potassium, zinc, magnesium, calcium, lithium, copper, iron, manganese, nitrates, phosphates, sulfates , Salts such as borates and tris salts, thiol compounds, selenium compounds, amino acids and amino acid salts, sugars and glycosides. Further, it may contain various phenol-based and aniline-based Trinder reagents, couplers such as 4-aminoantipyrine, tetrazolium salts, phenazine methosulfate and other electron carriers, leuco-based dyes, and substrates. Furthermore, a nonspecific reaction inhibitor may be included. The non-specific reaction inhibitor is not particularly limited, and can include an antibody of the same type as the labeled antibody and an enzyme of the same type as the labeled antibody. Examples of the same type of antibody include mouse IgG, mouse IgM, high molecular weight mouse IgG polymer, goat IgG, sheep IgG, horse IgG, rat IgG, rabbit IgG and the like. These can be added as body fluids such as animal serum and ascites, but it is desirable to add them after performing virus inactivation treatment, complement inactivation treatment, degreasing treatment, and the like. Examples of the same type of enzyme include peroxidase, amylase, catalase, glucose oxidase, glucose dehydrogenase, alkaline phosphatase, β-galactosidase and the like.

When the antibody is immobilized on a carrier, the carrier used is not particularly limited, and may be appropriately selected from those used for immunoassay. Examples thereof include synthetic polymer compounds such as polystyrene, polyethylene, and polypropylene, inorganic substances such as porous glass, glass beads, and magnetic particles. Examples of the form include tubes, plates, microtiter plates, and fine particles.
Immobilization of the antibody to the carrier can be performed according to a known method. For example, chemical coupling method using so-called coupling agents such as glutaraldehyde, bisdiazobenzidine, tolylene diisocyanate, difluoronitrobenzene, carbodiimides, quinones, chromium chloride, tannic acid, etc., antibody and carrier in water, physiological salt Examples thereof include a physical adsorption method in which contact is made in a liquid such as water or various aqueous buffers. In any method, the antibody is immobilized on a carrier, and then sericin and / or a hydrolyzate thereof, or an equivalent thereof is allowed to coexist.
The composition may contain various additives as in the composition in which the antibody is not immobilized on the carrier.
Thus, a composition in which an antibody is immobilized on a carrier and immersed in a liquid can be prepared.

A composition in which an antibody is immobilized on a carrier and in a dry state can be prepared by drying a composition immersed in the liquid by a conventional method, for example, natural drying, aeration drying, vacuum drying, or freeze drying. it can. At this time, in order to effectively exhibit the stabilizing effect by sericin and / or its hydrolyzate or its equivalent, it is 6 to 24 hours at 4 ° C, preferably 10 to 20 hours, or 25 ° C before drying. It is desirable to take an immersion time of 2 to 8 hours, preferably 4 to 6 hours, or further at 37 ° C. for 10 minutes to 3 hours, preferably 30 minutes to 2 hours.
The composition may contain various additives as in the composition in which the antibody is not immobilized on the carrier.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in more detail, this invention is not limited to a following example.

In Test Examples 1 to 3, sericin hydrolyzate was prepared according to the following method.
[Preparation of sericin hydrolyzate]
1 kg of koji (made by Bombyx mori) was treated in 50 L of 0.2% aqueous sodium carbonate (pH 11-12) at 95 ° C. for 2 hours, and sericin was partially hydrolyzed and extracted. The obtained extract was filtered using a filter having an average pore size of 0.2 μm to remove aggregates, and then the filtrate was desalted with a reverse osmosis membrane to obtain a colorless and transparent purified solution having a concentration of 0.2%. . This purified solution was concentrated using an evaporator until the concentration reached about 2%, and then lyophilized to obtain 100 g of sericin hydrolyzate powder having a purity of 90% or more and an average molecular weight of 20,000. Obtained.

[Test Example 1]
Stabilization test for solution antibody (1) Preparation and storage of solution mouse IgG Sericin hydrolyzate and bovine serum albumin (hereinafter referred to as BSA, manufactured by Nacalai Tesque) were tested as stabilizers. 10 g of each test substance in phosphate buffered saline (hereinafter referred to as PBS, pH 7.2 to 7.5) composed of 136 mM sodium chloride, 8 mM disodium hydrogen phosphate, 3 mM potassium chloride and 1 mM potassium dihydrogen phosphate After dissolving at a / L concentration, the solution was filtered through a 0.22 μm filter to obtain a test substance solution. A 400 μg / mL mouse IgG solution (manufactured by Santa Cruz Biotechnology) was diluted to 50 ng / mL with the above test substance solution, and the storage stability was evaluated. That is, the activity of mouse IgG on the day of preparation (day 0) and on the 6th day after storage at 50 ° C. or 4 ° C. was measured according to the method of (2) to determine the residual activity rate. Each mouse IgG diluted solution was returned to room temperature before measurement and subjected to activity measurement.

(2) Mouse IgG activity measurement method 100 μL of 1 μg / mL goat-derived anti-mouse IgG antibody solution (Chemicon) diluted with PBS was added to each well of a 96-well assay plate (flat bottom type, manufactured by Falcon). And incubated at 37 ° C. for 90 minutes. After the incubation, the solution in each well was removed by decantation. To this, 180 μL of 5% (w / w) skim milk solution prepared in PBS was added and incubated at 37 ° C. for 90 minutes, so that the well surface of the plate not coated with goat-derived anti-mouse IgG antibody was removed. Coated. After the incubation, the solution in each well was removed by decantation, and the plate was washed 3 times with a PBS solution containing 0.1% (w / w) Tween 20 (hereinafter referred to as T-PBS), and further with PBS. Washed once. Thereto, 100 μL of each mouse IgG diluted solution prepared in (1) was added, and an antigen-antibody reaction was carried out by incubating at 37 ° C. for 90 minutes.

  After completion of the incubation, the solution in each well was removed by decantation, and the plate was washed 3 times with T-PBS, and further washed once with PBS. Thereto was added 100 μL of 1.7 μg / mL horseradish peroxidase (hereinafter referred to as HRP) -labeled goat-derived anti-mouse IgG antibody solution (Cappel) diluted with PBS, and incubated at 37 ° C. for 90 minutes. After completion of the incubation, the solution in each well was removed by decantation, and the plate was washed 3 times with T-PBS, and further washed once with PBS. Thereto, 75 μL of 2 mM orthophenylenediamine dihydrochloric acid solution as a substrate for HRP was added and reacted at 25 ° C. for 40 minutes. After completion of the reaction, the degree of color development was measured at a wavelength of 490 nm (control wavelength: 600 nm).

For each test substance, the percentage (%) of the difference in absorbance on the 6th day of storage with respect to the value obtained by subtracting the absorbance in the blank from the absorbance on the day of sample preparation (day 0) (hereinafter referred to as the absorbance difference) is obtained, and the residual activity rate is calculated. did.
The results are shown in Table 1.

(3) Results In the storage at 50 ° C. for 6 days, the residual activity rate of mouse IgG was 0% without addition and 89% with BSA. On the other hand, in the sericin hydrolyzate, it was 84%, showing a value similar to BSA.
From these results, it was shown that the sericin hydrolyzate has excellent antibody stability equivalent to that of BSA with respect to the solution antibody.

[Test Example 2]
Stabilization test for dry immobilized antibody-Mouse IgG quantification system-
(1) Preparation and storage of dried immobilized goat-derived anti-mouse IgG antibody plate As stabilizers, sericin hydrolyzate, BSA (manufactured by Nacalai Tesque), skim milk (manufactured by Nacalai Tesque) and protein hydrolyzate from wheat (Manufactured by Quest International). Each test substance was dissolved in PBS to a concentration of 1 g / L and then filtered through a 0.22 μm filter to obtain a test substance solution.
100 μL of 1 μg / mL goat-derived anti-mouse IgG antibody solution (Chemicon) diluted with PBS was added to each well of a 96-well assay plate (flat bottom type, manufactured by Falcon) and incubated at 37 ° C. for 90 minutes. After the incubation, the solution in each well was removed by decantation. Thereto, 180 μL of the test substance solution was added and incubated at 37 ° C. for 90 minutes to coat the well surface of the portion of the plate not coated with the goat-derived anti-mouse IgG antibody, and the immobilized goat-derived anti-mouse IgG An antibody plate was used (day 0). Then, it dried in the 50 degreeC incubator and evaluated the storage stability. That is, the activity of the immobilized goat-derived anti-mouse IgG antibody on the day of preparation (day 0) and on day 11 after storage at 50 ° C. was measured according to the method of (2) to determine the residual activity rate.

(2) Method for measuring activity of immobilized goat-derived anti-mouse IgG antibody Among the immobilized goat-derived anti-mouse IgG antibody plates prepared in (1), the one with day 0 was prepared by removing the solution in each well by decantation. -The plate was washed 3 times with PBS and further washed once with PBS for activity measurement.

  First, 400 μg / mL mouse IgG (manufactured by Santa Cruz Biotechnology) was diluted with a 1% (w / w) skim milk solution to prepare 50 ng / mL, 25 ng / mL and 0 ng / mL solutions. An antigen-antibody reaction was performed by adding 100 μL to an anti-mouse IgG antibody plate and incubating at 37 ° C. for 90 minutes.

  After completion of the incubation, the solution in each well was removed by decantation, and the plate was washed 3 times with T-PBS, and further washed once with PBS. Thereto was added 100 μL of a 1.7 μg / mL HRP-labeled goat-derived anti-mouse IgG antibody solution (Cappel) diluted with PBS, and incubated at 37 ° C. for 90 minutes. After completion of the incubation, the solution in each well was removed by decantation, and the plate was washed 3 times with T-PBS, and further washed once with PBS. Thereto, 75 μL of 2 mM orthophenylenediamine dihydrochloric acid solution as a substrate for HRP was added and reacted at 25 ° C. for 40 minutes. After completion of the reaction, the degree of color development was measured at a wavelength of 490 nm (control wavelength: 600 nm).

  For each test substance, the percentage (%) of the difference in absorbance on the 11th day of storage with respect to the value obtained by subtracting the absorbance in the blank from the absorbance on the day of sample preparation (day 0) (hereinafter referred to as the absorbance difference) is obtained, and the residual activity rate is determined. did. Of these, Table 2 shows the residual activity rates when 50 ng / mL mouse IgG was added.

(3) Results In storage at 50 ° C. for 11 days, the residual activity rate of the immobilized goat-derived anti-mouse IgG antibody was 0% without addition, 73% with BSA, 76% with skim milk, and with protein hydrolyzate from wheat. 80%. On the other hand, it was 93% for the sericin hydrolyzate, indicating the highest value among the studied stabilizers. Moreover, as shown in FIG. 1, when it preserve | saved with the sericin hydrolyzate, the quantitative property with respect to mouse | mouth IgG was also maintained.
From these results, it was shown that sericin hydrolyzate has better antibody stability than immobilized protein antibody than BSA, skim milk and wheat-derived protein hydrolysates.

[Test Example 3]
Stabilization test for dry immobilized antibody-human serum albumin (hereinafter referred to as HSA) quantification system-
(1) Preparation and Storage of Dry Immobilized Goat-Derived Anti-HSA Antibody Plate The same substance as in Test Example 2 was tested as a stabilizer. Each test substance was dissolved in PBS to a concentration of 1 g / L and then filtered through a 0.22 μm filter to obtain a test substance solution.
100 μL of 8 μg / mL goat-derived anti-HSA antibody solution (Cappel) diluted with PBS was added to each well of a 96-well assay plate (flat bottom type, manufactured by Falcon) and incubated at 37 ° C. for 90 minutes. After the incubation, the solution in each well was removed by decantation. 180 μL of the test substance solution was added thereto and incubated at 37 ° C. for 90 minutes to coat the well surface of the part of the plate not coated with the goat-derived anti-HSA antibody, and the solid-phased goat-derived anti-HSA antibody Plated (day 0). Then, it dried in the 50 degreeC incubator and evaluated the storage stability. That is, the activity of the immobilized goat-derived anti-HSA antibody on the day of preparation (day 0) and on the 8th day after storage at 50 ° C. was measured according to the method of (2) to determine the residual activity rate.

(2) Method for measuring activity of immobilized goat-derived anti-HSA antibody Among the immobilized goat-derived anti-HSA antibody plates prepared in (1), the one with day 0 is obtained by removing the solution in each well by decantation, and T-PBS. The plate was washed 3 times with, and further washed once with PBS and subjected to activity measurement.

  First, HSA (manufactured by Sigma) was dissolved in 1% (w / w) skim milk solution to prepare 40 ng / mL, 20 ng / mL and 0 ng / mL solutions, and 100 μL was added to the immobilized goat-derived anti-HSA antibody plate Then, an antigen-antibody reaction was performed by incubating at 37 ° C. for 90 minutes.

  After completion of the incubation, the solution in each well was removed by decantation, and the plate was washed 3 times with T-PBS, and further washed once with PBS. Thereto, 100 μL of a 2.5 μg / mL HRP-labeled sheep-derived anti-HSA antibody solution (Binding Site) diluted with PBS was added and incubated at 37 ° C. for 90 minutes. After completion of the incubation, the solution in each well was removed by decantation, and the plate was washed 3 times with T-PBS, and further washed once with PBS. Thereto, 75 μL of 2 mM orthophenylenediamine dihydrochloric acid solution as a substrate for HRP was added and reacted at 25 ° C. for 40 minutes. After completion of the reaction, the degree of color development was measured at a wavelength of 490 nm (control wavelength: 600 nm).

  For each test substance, the percentage (%) of the difference in absorbance on the 8th day of storage with respect to the value obtained by subtracting the absorbance in the blank from the absorbance on the day of sample preparation (day 0) (hereinafter referred to as the absorbance difference) was determined, and the residual activity rate was determined. did. Of these, Table 3 shows the residual activity ratio when 40 ng / mL of HSA was added.

(3) Results When stored at 50 ° C. for 8 days, the residual activity rate of the immobilized goat-derived anti-HSA antibody was 0% without addition, 6% with BSA, 52% with skim milk, and 5 with wheat-derived protein hydrolyzate. %Met. On the other hand, in the sericin hydrolyzate, it was 66%, showing the highest value among the studied stabilizers. Moreover, as shown in FIG. 2, when it preserve | saved with the sericin hydrolyzate, the quantitative property with respect to HSA was also maintained.
From these results, it was shown that sericin hydrolyzate has better antibody stability than immobilized protein antibody than BSA, skim milk and wheat-derived protein hydrolysates.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to improve stability of an antibody and to maintain the effectiveness of the composition containing an antibody over a long term. In particular, it is excellent as a diagnostic drug used in the clinical laboratory field, and contributes greatly to the industry.

It is a graph which shows the quantitative property with respect to mouse | mouth IgG. It is a graph which shows the quantitative property with respect to HSA.

Claims (20)

A method for stabilizing an antibody, wherein the antibody can be preserved by allowing the antibody to coexist with sericin and / or a hydrolyzate thereof in a solution at 40 to 50 ° C. A method for stabilizing an antibody, comprising allowing an antibody to be stored by allowing the antibody to coexist in a solution at 50 ° C. with sericin and / or a hydrolyzate thereof. A method for stabilizing an antibody, wherein the antibody can be stored by allowing the antibody to coexist in a solution at 40 ° C with sericin and / or a hydrolyzate thereof. The method for stabilizing an antibody according to any one of claims 1 to 3, wherein the antibody is immobilized on a carrier. The method for stabilizing an antibody according to any one of claims 1 to 4 , wherein sericin and / or a hydrolyzate thereof is derived from natural sericin extracted from silkworm silk or raw silk. The method for stabilizing an antibody according to any one of claims 1 to 4 , wherein the sericin equivalent is obtained by a genetic engineering technique. The method for stabilizing an antibody according to claim 2, wherein the residual activity of the antibody after storage at a sericin concentration of 10 g / L in a solution at 50 ° C for 6 days is 84% or more. The method for stabilizing an antibody according to claim 2, wherein the residual activity of the antibody after storage at a sericin concentration of 1 g / L in a solution for 11 days at 50 ° C is 93% or more. The method for stabilizing an antibody according to claim 2, wherein the residual activity of the antibody after storage at a sericin concentration of 1 g / L in a solution at 50 ° C for 8 days is 66% or more. An antibody- stabilized storable composition, wherein the antibody coexists with sericin and / or a hydrolyzate thereof in a solution at 40 to 50 ° C. An antibody-stabilized storable composition, wherein the antibody coexists with sericin and / or a hydrolyzate thereof in a solution at 50 ° C. An antibody-stabilized storable composition, wherein the antibody coexists with sericin and / or a hydrolyzate thereof in a solution at 40 ° C. The composition according to claim 11, wherein the residual activity of the antibody after storage at a sericin concentration of 10 g / L in the solution at 50 ° C for 6 days is 84% or more. The composition according to claim 11, wherein the residual activity of the antibody after storage for 11 days at a sericin concentration of 1 g / L in a solution at 50 ° C. is 93% or more. The composition according to claim 11, wherein the residual activity of the antibody after storage at a sericin concentration of 1 g / L in a solution at 50 ° C for 8 days is 66% or more. A composition for stabilizing an antibody in a solution at 40 to 50 ° C., comprising sericin and / or a hydrolyzate thereof. A composition for stabilizing an antibody in a solution at 50 ° C., comprising sericin and / or a hydrolyzate thereof. A composition for stabilizing an antibody in a solution at 40 ° C., comprising sericin and / or a hydrolyzate thereof. A diagnostic agent comprising the composition according to any one of claims 10 to 18 . A biosensor comprising the composition according to any one of claims 10 to 18 .

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