JP2006126166A - Composition for enhancing measurement sensitivity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition for promoting immune reaction used in immunological measurements. <P>SOLUTION: The composite for promoting immune reaction contains a blocking agent of weight% concentration of higher than 0.01% and polyethylene glycol whose mean molecular weight of 2,000 to 26,000 at a weight% concentration of 1 to 10%. Any one or more of the sorts selected from among artificial synthetic polymer, normal serum. bovine serum albumin, gelatin, and casein can be used as the blocking agent, and a plurality of composites can be combined. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

    The present invention relates to a composition for promoting an immune response used in an immunoassay.

    Today, analytical methods using antibodies have become indispensable techniques in the field of research in general biology, beyond the field of immunology. Furthermore, in recent years, the analysis of genome information of various species including humans has gradually progressed, and analysis of genome information, so-called post-genomics, has attracted attention. The role of immunoassays using antibodies has been further increased due to increasing importance.

    Known immunoassay methods using antibodies include immunohistochemistry (IHC), which visualizes antigens (proteins, etc.) present in tissues using fluorescent dyes, enzymes, etc. Western blotting (WB) to measure protein expression pattern and molecular weight by electrophoresis and visualization with specific antibody, antigen or antibody is immobilized on reaction plate, and enzyme-labeled antigen or antibody is used Enzyme immunoassay (EIA) to detect minute amounts of antigens or antibodies in the sample Metric All of these technologies are widely used in laboratories of companies and research institutions. Further, as a method for specifically purifying a protein using an antibody, a purification method using an antibody column, an immunoprecipitation method, or the like is often used. Furthermore, in recent years, protein screening methods using antibodies using the phage display method and the like, pharmaceuticals using antibodies, and the like have been developed, and the use of immunological techniques is rapidly spreading with increasing demand.

    On the other hand, there are polyclonal antibodies, which are a mixture of antibodies that bind to multiple antigenic determinants in a single antigen, and monoclonal antibodies that consist of a single type of antibody that binds to a single antigenic determinant. , Each creation method is different. Polyclonal antibodies are more suitable for immunohistochemical staining, Western blotting, and the like because they are highly reactive with antigens and have little effect on changes in the three-dimensional structure of antigens. In addition, since the reactivity of the monoclonal antibody is more limited, the monoclonal antibody is more suitable for strict examination than the polyclonal antibody and has an advantage that the difference between lots is stable. Each has its strengths and weaknesses, and is selected according to the purpose of use.

    It is well known that even if antibodies are antibodies against the same antigen, performance varies greatly from production to production. Therefore, although antibodies against the same antigen are commercially available from various companies, it is important to compare the antibodies of each company before use. In addition, for polyclonal antibodies, lot-to-lot comparison is also necessary for the reasons described above. However, it takes a lot of cost and time to make comparisons between companies and lots widely, and there is a current situation that it is very burdensome to conduct preliminary studies for the intended immunological measurement. . Furthermore, it is also known that it may be very difficult to produce a high-performance antibody depending on the type of antigen, and antibodies against histidine tags and phosphorylated serine / threonine are used very frequently, but they are good. The current situation is that there is no antibody with performance.

On the other hand, an immunoassay using an antigen-antibody reaction uses an antibody that directly binds to the antigen to be detected (primary antibody) and an antibody that specifically binds to the primary antibody (secondary antibody), and further uses a secondary antibody. A method of amplifying a signal (indirect method) by using a label to which a detection label (a dye, an enzyme, etc.) has been added is frequently used. However, in this method, non-specific signals derived from the secondary antibody are also amplified compared to the method using the primary antibody added with a label directly (direct method), and therefore, a high signal-to-noise ratio. The current situation is that the ratio is difficult to obtain.

    On the other hand, it has been known for a long time that the storage stability of an antibody is deteriorated by diluting the antibody with an aqueous solution containing polyethylene glycol. Therefore, when performing an antigen-antibody reaction using an antibody diluted with an aqueous solution containing polyethylene glycol, the antibody must be diluted immediately before the start of the operation, and the same operation is divided into several times. At the time of carrying out, the current situation is that the antibody dilution operation must be performed each time.

    As described above, there has been a demand for a method for allowing an antibody to react with an antigen more efficiently than before, as the demand for immunological techniques has increased and academic and applied uses have been gradually spreading.

    The inventors of the present invention have made extensive studies in order to achieve the above-mentioned object. By adding a blocking agent and polyethylene glycol in an appropriate concentration to the reaction system, the antigen-antibody reaction efficiency is improved and the storage stability of the antibody. Has been found to improve, and the present invention has been completed.

That is, the present invention
“[Claim 1]
A composition for promoting an immune reaction, comprising a blocking agent having a weight percent concentration of 0.01% or more and a polyethylene glycol having a weight percent concentration of 1 to 10% and an average molecular weight of 2,000 to 26,000 A composition for promoting immune response, characterized by comprising:
[Section 2]
Item 2. The immune reaction promoting composition according to Item 1, wherein the blocking agent is at least one blocking agent selected from artificial synthetic polymers, normal serum, bovine serum albumin, gelatin, and casein.
[Section 3]
Item 3. The immune reaction promoting composition according to Item 1 or 2, wherein the blocking agent is casein.
[Claim 4]
Item 4. The immune reaction promoting composition according to any one of Items 1 to 3, wherein the salt concentration is 40 to 400 mM.
[Section 5]
Item 5. The immune reaction promoting composition according to Item 1 to 4, comprising a combination of two or more compositions.
[Claim 6]
Item 6. The immune reaction promoting composition according to Item 5, wherein the combination of the compositions is a combination of a primary antibody reaction promoting composition and a secondary antibody promoting composition.
[Claim 7]
Item 7. The immune reaction promoting composition according to any one of Items 4 to 6, comprising a combination of a primary antibody reaction promoting composition having a salt concentration of 40 to 100 mM and a secondary antibody promoting composition having a salt concentration of 150 to 400 mM. Composition.
[Section 8]
Item 8. The immune reaction promoting composition according to any one of Items 4 to 7, wherein the salt is sodium chloride.
[Claim 9]
Item 9. The immune reaction promoting composition according to any one of Items 1 to 8, wherein the composition contains a nonionic surfactant having a concentration by weight of 0.001 to 0.2%.
[Section 10]
Item wherein the nonionic surfactant is at least one nonionic surfactant selected from polyethylene glycol mono-p-isooctylphenyl ether and polyoxyethylene sorbitan monolaurate. The composition for promoting immune response according to 9,
[Section 11]
Item 11. The immune reaction promoting composition according to any one of Items 1 to 10, wherein the storage stability of the antibody is improved.
[Claim 12]
Item 12. A kit for use in an immunoassay comprising the immune reaction promoting composition according to any one of Items 1 to 11.
[Claim 13]
Using the immune reaction promoting composition according to any one of Items 1 to 11, immunoassays such as Western blotting, dot blotting, immunohistochemical staining, enzyme immunoassay, radioimmunoassay, immunoprecipitation, and flow cytometry are performed. Method.
[Section 14]
Item 2. The immune reaction promoting composition according to Item 1, wherein the immune reaction is an immune reaction in Western blotting or dot blotting.
[Section 15]
Item 2. The immune reaction promoting composition according to Item 1, wherein the immune reaction is an immune reaction in enzyme immunoassay (ELISA, EIA).
[Section 16]
Item 2. The immune reaction promoting composition according to Item 1, wherein the immune reaction is an immune reaction in immunostaining. "
It is.

    The present invention is a composition for promoting an immune reaction for performing an immunoassay.

    The immune reaction in the present invention refers to a reaction in which an antigen and an antibody capable of specifically binding to the antigen specifically bind, that is, an antigen-antibody reaction, and an immunoassay refers to an antigen-antibody reaction. This refers to the specific detection and purification of antigens or antibodies.

There are various analysis methods using an immune reaction, but the present invention can be applied to any method in principle. In particular, in Western blotting, dot blotting, immunostaining (immunohistological staining, immune cell staining), enzyme immunoassay (ELISA, EIA), and radioimmunoassay, no special components are particularly essential in the antigen-antibody reaction system. The composition of the invention can be suitably used because it is easy to apply.
In addition, the structure required for reactions including antigens and antibodies used in these immune reactions is not particularly limited. A person skilled in the art constructs a reaction system using a technique that can be usually used, such as selecting these appropriately according to the experimental method to be used, and further processing (for example, labeling) by appropriate means as necessary. be able to.

    As shown in Example 3, the storage stability in the present invention is obtained by diluting an antibody to an appropriate concentration using the composition of the present invention and storing the composition at 37 ° C. for 2 weeks. Judgment can be made by comparing the degree of reaction when applied to an experimental method utilizing reaction. More specifically, an antibody having the same concentration diluted according to the present invention and a buffer solution generally used for antibody dilution, more specifically, Tris-buffered saline (TBS) or phosphate-buffered saline ( PBS and the like are stored at 37 ° C. for 2 weeks, and then the enzyme immunoassay (EIA) is performed with the composition to quantitatively measure the degree of decrease in the binding power of the antibody to the antigen. be able to.

The content of polyethylene glycol used in the present invention is 1% by weight or more and 10% by weight or less, preferably 2% by weight or more and 9% by weight or less, more preferably 3% by weight or more and 8% by weight or less. When the content is too low, there is no effect on the efficiency of antigen-antibody reaction, when the content is too high, non-specific reaction increases, and when the content is too high, the antigen-antibody reaction is inhibited. Further, since the optimum polyethylene glycol concentration for maximizing the so-called S / N ratio varies depending on the combination of antigen and antibody, the ratio of specific binding to non-specific binding, so-called S / N ratio is different. It is preferred to vary the glycol concentration within the aforementioned range. The polyethylene glycol used in the present invention has an average molecular weight of 2,000 to 26,000. Preferably it is 2,500-15,000. If the average molecular weight is too low or high, there is no effect on improving the efficiency of the antigen-antibody reaction, and if the concentration is high, the antigen-antibody reaction is inhibited.
Any of these may be a commercially available product. The polyethylene glycol can be quantified by various known methods, more specifically, a refractive index measurement method, a chromatography method, or the like. The average molecular weight of polyethylene glycol is various known methods, more specifically, chromatographic methods (GPC method), viscosity methods, methods using a bundle of properties (vapor pressure method, osmotic pressure method, boiling point increase method). , Light scattering method, sedimentation velocity method (ultracentrifugation method) and the like.

The blocking agent of the present invention is intended to prevent nonspecific adsorption of antibody protein to a site where no antigen is present and to enhance a specific antigen-antibody reaction, and its concentration is preferably 0.01. % Or more, more preferably 0.01% or more and 5% or less, further preferably 0.01% or more and 1% or less, and most preferably 0.01% or more and 0.5% or less. If the content is too low, there is no effect on improving the antigen-antibody reaction efficiency or reducing the non-specific reaction, and if the content is too high, the antigen-antibody reaction is inhibited. The blocking agent here refers to the case where a specimen containing an antigen is pre-treated before the antibody reaction or the non-specific reaction of the antibody in order to prevent non-specific adsorption of the antibody to a site where no antigen is present. Is used for addition to a diluent for antibody reaction, and the form thereof is not particularly limited, and any substance may be used. -Artificial synthetic polymers such as methacryloyloxyethyl phosphorylcholine polymer, normal serum derived from animals such as rabbits and goats, bovine serum albumin, gelatin, casein, surfactants, etc. Any one or more blocking agents are preferably used, and casein is more preferable.
Any of these can be determined by adding the blocking agent antibody to an appropriate buffer and comparing the degree of non-specific reaction when the composition is applied to an experimental technique using an immune reaction. . More preferably, in enzyme immunoassay (EIA), the blocking agent is diluted with a commonly used buffer solution, more specifically, Tris buffered saline (TBS) or phosphate buffered saline (PBS). Can be quantitatively measured for the degree of non-specific adsorption of the antibody to the non-antigen by performing the antibody reaction after instilling to the specimen containing the antigen before the antibody reaction and incubating at 37 ° C. for 1 hour. it can.
Any of these may be a commercially available product.
The casein of the present invention refers to a protein that is the main component of milk protein, and its form is not particularly limited, but a protein purified from milk is particularly preferably used. Casein is known to be electrophoretically composed of three components, α, β, and γ, but the casein used in the present invention may be one of these or a mixture thereof. Further, it may be hydrolyzed enzymatically. Further, the content of casein used in the present invention is 0.01% by weight or more. Casein contained in the composition of the present invention can be easily measured by electrophoresis or the like.

The composition of the present invention can contain a nonionic surfactant. The concentration of the nonionic surfactant is preferably 0.001 to 0.2% by weight. If the concentration is too high, the antigen-antibody reaction is inhibited, and if it is too low, the nonspecific reaction increases. The form of the nonionic surfactant in the present invention is not limited, but is preferably an ether type, more specifically, polyethylene glycol mono-p-isooctylphenyl ether or the like, or an ester ether type, more specifically. One or more nonionic surfactants selected from polyoxyethylene sorbitan monolaurate and the like are used.
Any of these may be a commercially available product.

    Either one or both of the above-mentioned salt and nonionic surfactant may be contained, but it is more preferred that both are contained.

The composition of the present invention can contain a salt. Although the salt concentration of a composition is not specifically limited, Preferably it is 40-400 mM, More preferably, it is 40-350 mM, More preferably, it is 45-320 mM. If the salt concentration is too high, the antigen-antibody reaction is inhibited, and if it is too low, the nonspecific reaction increases. The form of the salt in the present invention is not limited, but sodium chloride is particularly preferably used.
Any of these may be a commercially available product. The salt concentration is measured by various known methods, more specifically, a gravimetric method using a precipitation reagent, a titration method or a colorimetric method using a chelating reagent or a colorimetric reagent, or a flame analysis using a flame color of a metal ion. For example, a method using an ion selective electrode, a method using a scattered ion sensor, and a method using a fluorescent reagent such as SPQ or MQAE.

The present invention is a composition for promoting an immune reaction for performing an immunoassay and comprises a combination of two or more compositions, for example, a combination of a composition for promoting a primary antibody reaction and a composition for promoting a secondary antibody reaction. May be. The salt concentration of each composition in that case is not particularly limited, but is preferably 40 to 400 mM.
More preferably, the salt concentration in each of the above compositions of the present invention is 40 to 100 mM for the primary antibody reaction promoting composition and 150 to 400 mM for the secondary antibody reaction promoting composition. The primary antibody here refers to an antibody that directly binds to the antigen to be detected when the antigen detection sensitivity is increased by a two-step antigen-antibody reaction, and the secondary antibody specifically refers to the primary antibody. An antibody that binds, and a secondary antibody is often added with an enzyme or a dye for detection. When the salt concentration of the composition for promoting primary antibody reaction is high, the antigen-antibody reaction is inhibited, and when the salt concentration of the composition for promoting secondary antibody is low, nonspecific reaction is increased. In addition, the salt concentration when detecting by one-step antigen-antibody reaction using an antibody directly binding to an antigen to which an enzyme, a dye or the like is added is preferably 150 to 400 mM. The form of the salt in the present invention is not limited, but sodium chloride is particularly preferably used.
Any of these may be a commercially available product. The salt concentration is measured by various known methods, more specifically, a gravimetric method using a precipitation reagent, a titration method or a colorimetric method using a chelating reagent or a colorimetric reagent, or a flame analysis using a flame color of a metal ion. For example, a method using an ion selective electrode, a method using a scattered ion sensor, and a method using a fluorescent reagent such as SPQ or MQAE.

    In addition, the composition of the present invention can further contain or select other substances that bring about a favorable effect on the present invention according to various experimental techniques to be applied. Specific examples include buffer agents (buffer solutions), anionic surfactants, cationic surfactants, enzyme stabilizers, proteolytic enzyme inhibitors, phosphatase inhibitors, and the like. Furthermore, specific examples of the buffer include glycine, phthalic acid, citric acid, β, β′-dimethylglutaric acid, succinic acid, acetic acid, histidine, maleic acid, cacodylic acid, β-glycerophosphoric acid, imidazole, Examples include phosphoric acid, arsenic acid, triethanolamine, 5,5-diethylparbituric acid, tris (hydroxymethyl) aminomethane, glycylglycine, pyrophosphoric acid, boric acid, carbonic acid, or a good buffer. More specific examples of MES, HEPPS, MOPSO, POPSO, bis-tris, ADA, PIPES, ACES, colamine chloride, BES, MOPS, TES, HEPES, acetamidoglycine, tricine, TAPS, bicine, CHES, Examples include CAPS. Of these, tris (hydroxymethyl) aminomethane, phosphoric acid, HEPES, carbonic acid and the like can be preferably used.

By using the composition for immune reaction provided by the present invention, the efficiency of the antigen-antibody reaction is improved as compared with the conventional method, and an immunoassay having high detection sensitivity and purification efficiency can be performed.

  The details of the present invention will be described in Examples. The present invention is not limited to these examples.

Example 1
Comparison with conventional composition by Western blotting As the antigen, human ERK2 protein synthesized by a cell-free protein synthesis kit PROTEIOS (manufactured by Toyobo Co., Ltd.) was used. Protein synthesis was performed at 23 ° C. for 16 hours according to PROTEIOS standard protocol (multilayer method). The synthesized protein solution was serially diluted with TBS (containing 0.1% Tween-20), and a PAG mini “Daiichi” (15-25%, 13%) polyacrylamide gel (Daiichi Chemical Co., Ltd.) was used. Used for SDS-polyacrylamide gel electrophoresis. Samples were applied as 5 types of samples of 2 times dilution, 4 times dilution, 8 times dilution, 16 times dilution and 32 times dilution, respectively, and PROTEIOS reaction composition solution without cell-free protein synthesis was used as a negative control. Applied, applied molecular weight markers, and again applied 5 samples and negative control. Electrophoresis was performed at 15 mA for 90 minutes. Thereafter, the protein was transferred from the gel subjected to electrophoresis to Immun-Blot PVDF Membrane (manufactured by Bio-Rad). The transfer was performed for 60 minutes with a membrane area of 0.8 mA. The membrane was washed with TBS (containing 0.1% Tween-20), followed by a blocking reaction. For blocking, use a blocking solution prepared by dissolving 5% by weight of skim milk (Difco) in TBS (containing 0.1% Tween-20), soak the membrane in the blocking solution and incubate at 37 ° C for 1 hour. went. Thereafter, the membrane was washed with TBS (containing 0.1% Tween-20), followed by a primary antibody reaction. The primary antibody reaction was performed as follows. First, a mixed solution was prepared by adding a hydrolyzed casein solution (ICN Biomedical Co., Ltd.) having a final concentration of 0.1 wt% and polyethylene glycol # 6,000 having a final concentration of 4% to 1 × PBS (−) (solution A). ). Further, TBS (containing 0.1% Tween-20), which is widely used as an antibody diluent in Western blotting, was used as a control (solution B). Using each mixture, anti-His-probe rabbit IgG (manufactured by Santa Cruz) diluted 2000 times was prepared as a primary antibody solution. The membrane after blocking was separated with scissors in the lane where the molecular weight marker was electrophoresed, and the left side was immersed in an antibody diluted solution with A solution and the right side was immersed in an antibody diluted solution with B solution, and incubated at 37 ° C. for 1 hour to carry out the reaction. . Each membrane was washed with TBS (containing 0.1% Tween-20), followed by a secondary antibody reaction. The secondary antibody reaction was performed using a mixed solution having the same composition as that used in the primary antibody reaction as an antibody diluent. As a secondary antibody, anti-rabbit IgG goat IgG (HRP labeled) (manufactured by Santa Cruz) was used, and the reaction was performed by diluting 20,000 times with each mixed solution. Thereafter, each membrane was washed with TBS (containing 0.1% Tween-20), followed by an enzyme substrate reaction. ECL Plus (manufactured by Amersham Biosciences) was used as a substrate solution and incubated at room temperature for 5 minutes for reaction. About the film | membrane after completion | finish of the reaction, the light emission signal was detected using the light emission image analyzer FAS-1000 (made by Toyobo Co., Ltd.).
The result is shown in FIG. It was confirmed that the measurement sensitivity was much higher when the antibody was diluted with the A liquid composition based on the present invention and compared with the measurement results when the B liquid used in the conventional method was used. It was done.

Example 2
Comparison with conventional composition by immunostaining As a sample to be stained, normal human aortic vascular endothelial cells (manufactured by Toyobo Co., Ltd.) fixed with methanol were used. Subsequently, a blocking reaction was performed. Blocking was performed by using a blocking solution prepared by dissolving skim milk (manufactured by Difco) in 1 × PBS (−) at 1 wt%, dropping the blocking solution onto the sample at room temperature for 1 hour. Thereafter, the sample was washed with 1 × PBS (−), followed by a primary antibody reaction. The primary antibody reaction was performed as follows. First, a mixed solution was prepared by adding a hydrolyzed casein solution (ICN Biomedical Co., Ltd.) having a final concentration of 0.1 wt% and polyethylene glycol # 6,000 having a final concentration of 4% to 1 × PBS (−) (solution A). ). Moreover, said blocking liquid was used as a control (B liquid). Using each mixed solution, 50-fold diluted anti-human Von Willebrand Factor mouse IgG (manufactured by Dako) was prepared as a primary antibody solution. Each antibody diluted solution was dripped at the sample after blocking, and reaction was performed for 30 minutes at room temperature. Each sample was washed with 1 × PBS (−), followed by a secondary antibody reaction. The secondary antibody reaction was performed using a mixed solution having the same composition as that used in the primary antibody reaction as an antibody diluent. As a secondary antibody, an anti-mouse IgG biotinylated antibody (manufactured by Toyobo Co., Ltd.) was used, and the reaction was performed by diluting 100 times with each mixed solution. Thereafter, each sample was washed with 1 × PBS (−), and then an avidin-biotin complex solution was dropped and reacted at room temperature for 30 minutes. Then each sample is 1
After washing with xPBS (−), an enzyme substrate reaction was performed. The substrate solution attached to HRP Immunostaining Kit (manufactured by Toyobo Co., Ltd.) was used as the substrate solution, and incubated at room temperature for 10 minutes for reaction. The sample after the reaction was washed with distilled water, sealed with glycerin, and observed with an optical microscope.
The results are shown in FIG. 2 and FIG. It was confirmed that the measurement sensitivity was much higher when the antibody was diluted with the A liquid composition based on the present invention and compared with the measurement results when the B liquid used in the conventional method was used. It was done.

Example 3
Comparison with conventional composition by ELISA (Enzyme-linked Immunosorbent Assay) MAP kinase p42 (FL) (manufactured by Santa Cruz) was used as the antigen. The antigen solution is diluted 2,000 times with carbonate buffer (15 mM sodium carbonate, 35 mM sodium bicarbonate), dispensed 100 μl each into a 96-well ELISA plate, and incubated at 37 ° C. for 1 hour to immobilize the antigen. went. Thereafter, the wells were washed with 1 × PBS (−), followed by a blocking reaction. For blocking, skim milk (Difco) dissolved in 1 × PBS (−) (containing 0.1% Tween-20) at 3% by weight was dispensed in a volume of 250 μl to each well. Incubated for hours. Thereafter, the wells were washed with 1 × PBS (−) (containing 0.1% Tween-20), followed by primary antibody reaction. The primary antibody reaction was performed as follows. First, a mixture of 1 × PBS (−) with a hydrolyzed casein solution having a final concentration of 0.001 to 1% by weight (ICN Biomedical) and polyethylene glycol # 6,000 having a final concentration of 4%. A liquid was prepared (A liquid series). Moreover, the liquid mixture which added 0.01 weight% hydrolyzed casein solution to 1xPBS (-) was prepared (B liquid). Using each mixture, anti-His-probe rabbit IgG (manufactured by Santa Cruz) diluted 2000 times was prepared as a primary antibody solution. 100 μl of each primary antibody solution was dispensed into each well and incubated at 37 ° C. for 1 hour. Thereafter, the wells were washed with 1 × PBS (−) (containing 0.1% Tween-20), followed by a secondary antibody reaction. The secondary antibody reaction was performed using a mixed solution having the same composition as that used in the primary antibody reaction as an antibody diluent. As a secondary antibody, anti-rabbit IgG goat IgG (HRP labeled) (manufactured by Santa Cruz) was used, and the reaction was performed by diluting 20,000 times with each mixed solution. Thereafter, the wells were washed with 1 × PBS (−) (containing 0.1% Tween-20), followed by enzyme substrate reaction. Using TMB (manufactured by BioFX) as a substrate solution, 100 μl was added to each well and incubated at 37 ° C. for 20 minutes. Thereafter, 100 μl of 1N sulfuric acid was added to each well to stop the reaction. About the reaction completion liquid, the light absorbency of 450 nm was measured with the plate reader.
The result is shown in FIG. It was confirmed that the measurement sensitivity was clearly different depending on the casein concentration when the antibody was diluted with the A liquid series to which polyethylene glycol was added. Moreover, it was confirmed that the measurement sensitivity of the A liquid series to which polyethylene glycol was added was remarkably higher than the measurement results of the B liquid to which polyethylene glycol was not added as shown in FIG.

Example 4
Comparison with conventional composition by ELISA (Enzyme-linked Immunosorbent Assay) MAP kinase p42 (FL) (manufactured by Santa Cruz) was used as the antigen. The antigen solution is diluted 2,000 times with carbonate buffer (15 mM sodium carbonate, 35 mM sodium bicarbonate), dispensed 100 μl each into a 96-well ELISA plate, and incubated at 37 ° C. for 1 hour to immobilize the antigen. went. Thereafter, the wells were washed with 1 × PBS (−), followed by a blocking reaction. For blocking, skim milk (Difco) dissolved in 1 × PBS (−) (containing 0.1% Tween-20) at 3% by weight was dispensed in a volume of 250 μl to each well. Incubated for hours. The wells were then washed with 1 × PBS (−) (containing 0.1% Tween-20)
Followed by a primary antibody reaction. The primary antibody reaction was performed as follows. First, based on a solution in which the sodium chloride concentration was reduced to 50 mM from 1 × PBS (−), a hydrolyzed casein solution (ICN Biomedical) having a final concentration of 0.01% by weight and polyethylene glycol # 6 having a final concentration of 4% were prepared. 000 was added (liquid C). First, based on a solution obtained by increasing the sodium chloride concentration from 1 × PBS (−) to 300 mM, a hydrolyzed casein solution (ICN Biomedical) having a final concentration of 0.01% by weight and polyethylene glycol # 6 having a final concentration of 4% , 000 was added (Liquid D). Using each mixture, anti-His-probe rabbit IgG (manufactured by Santa Cruz) diluted 2000 times was prepared as a primary antibody solution. 100 μl of each primary antibody solution was dispensed into each well and incubated at 37 ° C. for 1 hour. Thereafter, the wells were washed with 1 × PBS (−) (containing 0.1% Tween-20), followed by a secondary antibody reaction. The secondary antibody reaction was performed using a mixed solution having the same composition as that used in the primary antibody reaction as an antibody diluent. As a secondary antibody, anti-rabbit IgG goat IgG (HRP labeled) (manufactured by Santa Cruz) was used, and the reaction was performed by diluting 20,000 times with each mixed solution. The primary antibody reaction and the secondary antibody reaction are a combination of the primary antibody reaction using the C solution or D solution, and the secondary antibody reaction using the C solution or D solution. went. Thereafter, the wells were washed with 1 × PBS (−) (containing 0.1% Tween-20), followed by enzyme substrate reaction. Using TMB (manufactured by BioFX) as a substrate solution, 100 μl was added to each well and incubated at 37 ° C. for 20 minutes. Thereafter, 100 μl of 1N sulfuric acid was added to each well to stop the reaction. About the reaction completion liquid, the light absorbency of 450 nm was measured with the plate reader.
As a result, for each of the combinations of primary antibody reaction: secondary antibody reaction C: C, C: D, D: C, and D: D, the value of the signal-to-noise ratio (S / N ratio) is C: D> D: D> C: C> D: C. From this, it was confirmed that the highest S / N ratio was obtained by increasing the salt concentration of the secondary antibody reaction solution compared to the primary antibody reaction solution.

Example 5
Comparison of storage stability by dilution of antibody MAP kinase p42 (FL) (manufactured by Santa Cruz) was used as the antigen. The antigen solution is diluted 2,000 times with carbonate buffer (15 mM sodium carbonate, 35 mM sodium bicarbonate), dispensed 100 μl each into a 96-well ELISA plate, and incubated at 37 ° C. for 1 hour to immobilize the antigen. went. Thereafter, the wells were washed with 1 × PBS (−), followed by a blocking reaction. For blocking, skim milk (Difco) dissolved in 1 × PBS (−) (containing 0.1% Tween-20) at 3% by weight was dispensed in a volume of 250 μl to each well. Incubated for hours. Thereafter, the wells were washed with 1 × PBS (−) (containing 0.1% Tween-20), followed by primary antibody reaction. The primary antibody reaction was performed as follows. First, a mixed solution was prepared by adding a hydrolyzed casein solution having a final concentration of 0.01% by weight (ICN Biomedical) and polyethylene glycol # 6,000 having a final concentration of 4% to 1 × PBS (−) (solution E). ). Moreover, the liquid mixture which added polyethyleneglycol # 6,000 of final concentration 4% to 1xPBS (-) was prepared (F liquid). Moreover, the liquid mixture which added 0.01 weight% hydrolyzed casein solution to 1xPBS (-) was prepared (G liquid). Moreover, the solution comprised only with 1xPBS (-) was prepared (H liquid). Using each mixture, anti-His-probe rabbit IgG (manufactured by Santa Cruz) diluted 2000 times was prepared as a primary antibody solution. Each primary antibody solution was stored for 2 weeks at 37 ° C. and then used for the study. 100 μl of each primary antibody solution was dispensed into each well and incubated at 37 ° C. for 1 hour. Thereafter, the wells were washed with 1 × PBS (−) (containing 0.1% Tween-20), followed by a secondary antibody reaction. For the secondary antibody reaction, a mixed solution having the same composition as that used in the primary antibody reaction was used as an antibody diluent, and the same was stored at 37 ° C. for 2 weeks, and then used for the examination. As a secondary antibody, anti-rabbit IgG goat IgG (HRP labeled) (manufactured by Santa Cruz) was used, and the reaction was performed by diluting 20,000 times with each mixed solution. afterwards,
The wells were washed with 1 × PBS (−) (containing 0.1% Tween-20), followed by enzyme substrate reaction. Using TMB (manufactured by BioFX) as a substrate solution, 100 μl was added to each well and incubated at 37 ° C. for 20 minutes. Thereafter, 100 μl of 1N sulfuric acid was added to each well to stop the reaction. About the reaction completion liquid, the light absorbency of 450 nm was measured with the plate reader.
As a result, each of the signal intensities of E solution added with polyethylene glycol and hydrolyzed casein, F solution added with only polyethylene glycol, G solution added with only hydrolyzed casein, and H solution not added with both were E>G>H> F, and the decrease in storage stability of the antibody due to polyethylene glycol is eliminated by the addition of hydrolyzed casein, and the storage stability is rather improved compared to the case where polyethylene glycol is not added. confirmed.

    By using the composition for promoting immune reaction of the present invention, immunoassays such as Western blotting, dot blotting, immunohistochemical staining, enzyme immunoassay, radioimmunoassay, immunoprecipitation, flow cytometry can be efficiently performed. It is great to contribute to the world.

The figure which shows the result of having performed the comparison by Western blotting. The figure which shows the result of having performed immunostaining using A liquid. The figure which shows the result of having performed immunostaining using B liquid. The figure which shows the result of having performed ELISA using the A liquid series which added polyethyleneglycol. The figure which shows the result of having performed ELISA using B liquid which did not add polyethyleneglycol.

Claims (16)

  A composition for promoting an immune reaction, comprising a blocking agent having a weight percent concentration of 0.01% or more and a polyethylene glycol having a weight percent concentration of 1 to 10% and an average molecular weight of 2,000 to 26,000 A composition for promoting immune response, characterized by comprising:   The composition for promoting immune reaction according to claim 1, wherein the blocking agent is at least one blocking agent selected from artificial synthetic polymers, normal serum, bovine serum albumin, gelatin, and casein.   The composition for promoting immune reaction according to claim 1 or 2, wherein the blocking agent is casein.   The composition for promoting immune reaction according to any one of claims 1 to 3, wherein the salt concentration is 40 to 400 mM.   The composition for promoting immune response according to claim 1, comprising a combination of two or more compositions.   The composition for promoting immune responses according to claim 5, wherein the combination of the compositions is a combination of a composition for promoting a primary antibody reaction and a composition for promoting a secondary antibody.   The immune reaction promotion according to any one of claims 4 to 6, comprising a combination of a composition for promoting a primary antibody reaction having a salt concentration of 40 to 100 mM and a composition for promoting a secondary antibody having a salt concentration of 150 to 400 mM. Composition.   The composition for promoting immune reaction according to any one of claims 4 to 7, wherein the salt is sodium chloride.   The composition for promoting immune reaction according to any one of claims 1 to 8, wherein the composition contains a nonionic surfactant having a concentration by weight of 0.001 to 0.2%.   The nonionic surfactant is one or more nonionic surfactants selected from polyethylene glycol mono-p-isooctylphenyl ether and polyoxyethylene sorbitan monolaurate. Item 10. The immune reaction promoting composition according to Item 9.   The composition for promoting immune reaction according to any one of claims 1 to 10, wherein the storage stability of the antibody is improved.   A kit for use in an immunoassay comprising the immune response promoting composition according to any one of claims 1 to 11.   Using the immune reaction promoting composition according to any one of claims 1 to 11, immunoassay such as Western blotting, dot blotting, immunohistochemical staining, enzyme immunoassay, radioimmunoassay, immunoprecipitation, flow cytometry, etc. How to do.   2. The immune reaction promoting composition according to claim 1, wherein the immune reaction is an immune reaction in Western blotting or dot blotting.   The composition for promoting immune reaction according to claim 1, wherein the immune reaction is an immune reaction in enzyme immunoassay (ELISA, EIA).   2. The immune reaction promoting composition according to claim 1, wherein the immune reaction is an immune reaction in immunostaining.