WO2007132928A1

WO2007132928A1 - Composition for dilution of antibody

Info

Publication number: WO2007132928A1
Application number: PCT/JP2007/060176
Authority: WO
Inventors: Wataru Matsuyama
Original assignee: Kagoshima University
Priority date: 2006-05-15
Filing date: 2007-05-11
Publication date: 2007-11-22
Also published as: JP2007333723A

Abstract

Disclosed is a composition for diluting an antibody, which can improve the accuracy of an immunological analysis. The composition comprises agar, carrageenan or a mixture thereof.

Description

Description: Antibody dilution composition.

The present invention relates to an antibody dilution composition capable of improving the accuracy of immunological analysis, a method for producing and using the same, and an immunological analysis kit including the antibody dilution composition. Background art

Immunostaining is a method for investigating whether a target protein or target antibody is present in a sample (eg, blood or cell lysate). Research in a wide range of fields such as immunology and molecular biology It is used in. In addition, the amount of the target protein and the presence or absence of activation can be examined by the immunostaining.

In immunostaining, the experimental materials such as antibodies and serum used are diluted with buffer. In immunostaining, the most important problem in detecting the target protein is the phenomenon that non-specific bands other than the Pand corresponding to the target protein appear. This phenomenon is also called “background goes up”.

Therefore, conventionally, in order to prevent the appearance of such non-specific panda, for example, a blocking solution (for example, Plocace (registered trademark) Catalog number: UK-B25 (manufactured by Sumitomo Dainippon Pharma Co., Ltd.)) Or a method of diluting an antibody using a commercially available buffer (for example, DAKO Antibody Diluent with Background Reducing Components Catalog number: S3022 (Dako Cytomation)).

However, non-specific pandas often appear in immunostaining even when these conventional methods are used.

Therefore, methods and reagents that suppress the appearance of non-specific bands in immunostaining are desired. ·

On the other hand, Patent Document 1 discloses a composition for promoting an immune reaction used for immunological measurement. Patent Document 2 discloses a buffer solution for stabilizing an antigen. Furthermore, Patent Document 3 discloses a stabilizing solution for proteins and peptides.

However, to date, for the purpose of improving the accuracy of immunoassay and / or suppressing non-specific detection in immunoassay of an antibody dilution composition containing agar or carrageenan. Use was not known.

Patent Document 1 International Publication No. 2005/121790 Pamphlet

Patent Document 2 Special Table 2001-517799

Patent Document 3 Japanese Patent Publication No. Hei 10-500704

An object of the present invention is to provide an antibody dilution composition for improving the accuracy of immunological analysis and / or suppressing non-specific detection in immunological analysis in view of the above-described circumstances. And

As a result of intensive studies to solve the above problems, it was found that the accuracy of immunological analysis can be improved by using an antibody dilution composition containing agar or carrageenan. It came to complete.

The present invention includes the following.

(1) An antibody dilution composition for improving the accuracy of immunoassay and / or suppressing nonspecific detection in immunoassay, comprising agar, carrageenan or a mixture thereof object.

(2) The antibody dilution composition according to (1), wherein the carrageenan is λ carrageenan.

(3) The composition for diluting an antibody according to (1), further comprising sugar and gelatin.

(4) The antibody dilution composition according to (1), comprising one or more components selected from the group consisting of a salt, a chelating agent, a buffer and a nonionic surfactant.

(5) The antibody dilution composition according to (1), wherein the concentration of the agar is 0.1 to 5% by weight. 6

(6) The antibody dilution composition according to (1), wherein the concentration of the agar is 0.2 to 3% by weight.

(7) The antibody dilution composition according to (1), wherein the concentration of the force radinan is 0.01 to 5% by weight.

(8) The antibody dilution composition according to (1), wherein the concentration of the carrageenanan is 0.03 to 2% by weight.

(9) The antibody dilution composition according to (3), wherein the sugar is sucrose.

(1 0) concentration of the sugar is from 0.0017 to 0., Characterized in that ⁸ is 3 molar, (3) an antibody dilution composition.

(1 1) The antibody dilution composition according to (3), wherein the gelatin concentration is 0.01 to 2% by weight.

(1 2) The composition for diluting an antibody according to (4), wherein the salt is sodium chloride.

(1 3) The antibody dilution composition according to (4), wherein the concentration of the salt is 10 mM to 2 M.

(14) The antibody dilution composition according to (4), wherein the chelating agent is ethylenediamine tetraacetic acid.

(15) The antibody dilution composition according to (4), wherein the chelating agent has a concentration of lmM to 100 mM.

(16) The antibody dilution composition according to (4), wherein the buffer is Tris.

(17) The antibody dilution composition according to (4), wherein the concentration of the buffer solution is 10 mM to 1 M.

(18) The antibody dilution composition according to (4), wherein the nonionic surfactant is Tween 20 or Triton X-100.

(19) The antibody dilution composition according to (4), wherein the concentration of the nonionic surfactant is 0.01 to 1% by volume.

(20) An immunological analysis method comprising using the antibody dilution composition according to any one of (1) to (19). (21) The method according to (20), wherein the immunological analysis is Western blotting or immunostaining.

(22) An immunological analysis kit comprising the antibody dilution composition according to any one of (1) to (19) and an antibody.

(23) The immunological analysis kit according to (22), wherein the immunological analysis is Western plotting or immunostaining.

(24) The following steps:

(i) A step of stirring a mixed solution in water containing one or more components selected from the group consisting of a salt, a chelating agent, a buffer solution and a nonionic surfactant:

(ii) incubating the mixture produced in step (i) under heating;

(iii) cooling the mixed solution; and

(iv) adding carrageenan and stirring;

A method for producing an antibody dilution composition.

(25) The method according to (24), wherein the mixed solution in water of the step (i) further contains gelatin.

(26) The method according to (24), wherein in the step (ii), the incubation temperature is 35 ° C. to 40 ° C.

(27) The method according to (24), wherein in the step (iii), the mixed solution is cooled to 4 ° C to 30 ° C.

(28) The method according to (24), wherein in step (iv), sugar is added together with carrageenan and stirred.

This specification includes the contents described in the specification and / or drawings of Japanese Patent Application Nos. 2007-24437 and 2006-135032 which are the basis of the priority of the present application. Brief Description of Drawings

FIG. 1 shows the results of Western plotting using the antibody dilution composition containing agar according to the present invention (left figure: conventional method, right figure: agar-containing antibody dilution composition according to the present invention). use).

FIG. 2 shows the use of the antibody dilution composition containing carrageenan according to the present invention. Estan blotting results are shown. The left and right diagrams of A show the results of using the conventional method and the results of using the composition for diluting antibody containing carrageenan. The left and right diagrams of B show the results of using the conventional method and the results of using the carragionan-containing antibody dilution composition, respectively. C shows the result of using the antibody dilution composition of the present invention that does not contain carrageenan (left figure) and the result of using the antibody dilution composition of the present invention that contains carrageenan (right figure). Indicates. The left and right diagrams of D show the results using the conventional method and the results using the composition for diluting antibody containing carrageenan.

FIG. 3 shows the results of immunohistochemical staining of lung tissue using an antibody dilution composition containing carrageenan according to the present invention (A: conventional method, B .: composition for antibody dilution containing carrageenan). C: negative control using rabbit IgG as the primary antibody (use composition for dilution of antibody containing carrageenanane), D: hematoxylin eosin staining).

FIG. 4 shows the results of immunohistochemical staining of kidney tissue using an antibody dilution composition containing carrageenan according to the present invention (A: conventional method, B: composition for antibody dilution containing carrageenan). C: negative control using goat IgG as the primary antibody (using carrageenan-containing antibody dilution composition), D: hematoxylin eosin staining). BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

The antibody dilution composition according to the present invention is a composition containing agar, carrageenan or a mixture thereof. The antibody dilution composition according to the present invention is used for diluting an antibody used for immunological analysis. If the antibody dilution composition according to the present invention is used, the accuracy of immunological analysis can be improved.

Specifically, non-specific detection of proteins other than the target protein (antigen) can be suppressed in immunological analysis. For example, by using the antibody dilution composition according to the present invention in Western blotting or immunostaining, the appearance of non-specific panda can be suppressed.

In the composition of the present invention, in addition to agar or carrageenan, one or more components selected from the group consisting of salts, chelating agents, buffers and nonionic surfactants are particularly preferred. Or contains all these components. Others, for example, sodium azide, glyce

'Roll, ushi serum albumin (BSA), 2-mercaptoethanol, dithiothreitol, gelatin, sugar, etc. may be contained in the mixture. It is particularly preferable to contain gelatin and sugar. Examples of the sugar that can be used in the present invention include sucrose and cellulose, and particularly preferably includes sucrose. The addition of sugar is effective in increasing the solubility of carrageenan. Addition of gelatin is effective for improving the versatility of the composition. At this time, the sugar concentration in the mixed solution is adjusted to 0.0001 to 0.83 molar concentration, preferably 0.005 to 0.33 molar concentration. The concentration of gelatin in the mixed solution is adjusted to be 0 · 01 to 2% by weight, preferably 0.05 to 1% by weight. Further, as the water used for mixing the components of the composition of the present invention, for example, double-distilled water (D2, distilled water, sterilized water, etc.) can be used.

Examples of the agar used include special grade powder agar, powder agar, powder agar, thread agar, square agar, etc. Special grade powder agar is particularly preferable. The concentration of agar in the mixture is adjusted to 0.1 to 5% by weight, preferably 0.2 to 3% by weight.

Examples of the carrageenan used include κ carrageenan, t carrageenan, and λ carrageenan, and it is particularly preferable to use carrageenan. The concentration of color Jinan in the mixed solution is from 0.01 to 5 weight ^_0/0, preferably from 0.03 to 2 wt%.

On the other hand, the form of the salt is not limited, and examples thereof include sodium chloride (NaCl), potassium chloride, magnesium chloride, calcium chloride and the like, and NaCl is particularly preferable. The concentration of the salt in the mixed solution is adjusted to 10 mM to 2 M, preferably 100 mM to 1.5 M.

Examples of chelating agents include: ethylene: diamintetraacetic acid (EDTA), hydroxyl acetylenic diamine triacetic acid (HEDTA), dihydroxyxetyl diethylenediamine diacetic acid (DHEDDA), diethylenetriaminepentaacetic acid (DTPA) EDTA metal salts and the like, and EDTA is particularly preferable. When using EDTA, it is preferable to use, for example, ρΗ7.0 · 8.5 to 8.5, especially ρΗ8. The concentration of the chelating agent in the mixture is lmM to 100mM, preferably 3mM to 50mM.

Examples of buffers include tris (tris (hydroxymethyl) aminomethan; hereinafter referred to as “Ttis”), glycine, phthalic acid, citrate, succinate, acetic acid, Examples thereof include phosphoric acid, boric acid, carbonic acid, and a good buffer (MES, PIPES, MOPS, HEPES, Bistris, etc.), and Tris is particularly preferable. When using Tris, it is preferable to use, for example, ρΗ7 · 0 to 8.0, especially ρΗ7.4. The concentration of the buffer in the mixed solution is adjusted to 10 mM to 1 M, preferably 30 mM to 600 mM.

In addition, as nonionic surfactants, for example, Tween 20 (Polyoxyethylene Sorbitan Monolaurate), Triton X-100 (Octylphenolpoly (ethylene glycolether)) Chileen cornoreetenore))), Tween 80, Brij 35, Triton X-114, Nonidet P-40 (NP-40), Octyl b-glucoside, etc., especially Tween 20 or Triton X-100 are preferred. . The concentration of the nonionic surfactant in the mixture is 0.01 to 1% by volume, and preferably 0.03 to 0.6% by volume.

In another aspect of the present invention, a method for producing the antibody dilution solution of the present invention is provided. In the method for producing an antibody dilution composition of the present invention containing agar, first, the mixture containing agar is stirred. In the stirring, the temperature is, for example, 10 to 30 ° C, preferably 15 to 20 ° C. The stirring speed is, for example, 200 to 1500 rpm, preferably 600 to 800 rpm. The stirring time is, for example, 1 to 6 hours, preferably 1 to 2 hours. Next, the mixed solution after stirring is incubated. In the incubation, the temperature is, for example, 35-40 ° C, preferably 37 ° C. The incubation time is, for example, 5-24 hours, preferably 8-12 hours. By the method described above, the antibody dilution composition containing agar according to the present invention can be produced.

The method for producing the antibody dilution composition of the present invention containing carrageenan comprises (i) one or more components selected from the group consisting of a salt, a chelating agent, a buffer solution and a nonionic surfactant. Stirring the mixed solution in water containing: (ii) incubating the mixed solution produced in step (i) under heating; (ii i) cooling the mixed solution; and (iv) Adding carrageenan and stirring.

In the above step (i), in water containing all components (eg, gelatin, salt, chelating agent, buffer, nonionic surfactant, etc.) except for force radinan and sugar (if included) Stir the mixture. The temperature during stirring is, for example, 10-30 ° C, preferably 15-20 ° C. The stirring speed is, for example, 200 to 1500 rpm, preferably 600 to 800 rpm. Ma The stirring time is, for example, 1 to 6 hours, preferably 1 to 2 hours. Then, the above process

Incubate the mixture after stirring in (ii). In the incubation, the temperature is, for example, 35 to 40 ° C, preferably 37 ° C. The incubation time is, for example, 5 to 24 hours, preferably 8 to 12 hours. After incubation, process

In (iii), the mixture is cooled to 4 ° C to 30 ° C, preferably 4 ° C to 20 ° C, most preferably 4 ° C to 12 ° C, and then the carrageenan is added in step (iv). In addition, stir. The stirring speed in the step (iv) is, for example, 200 to 1500 rpm, preferably 600 to 800 rpm, and the stirring time is, for example, 1 to 6 hours, preferably 1 to 2 hours. By the method described above, the composition for antibody dilution containing a cardinan according to the present invention can be produced. In a preferred embodiment of the present invention, the mixed solution in the step (i) contains gelatin. At that time, the concentration of gelatin contained in the mixed solution is 0.01-2% by weight, preferably 0.05-! weight. / Adjust to ₀ . This improves the versatility of the composition liquid.

In another preferred embodiment of the present invention, in step (iv), sugar is added together with carrageenan and stirred. The amount of sugar added is from 0.0019 to 0.83 molar, preferably from 0.005 to 0.33 molar. As a result, the dissolution rate of the present invention is increased, and sufficient dissolution of the force radinan in the mixed solution can be achieved.

The antibody dilution composition of the present invention containing a mixture of agar and carrageenan is produced in the same manner as above except that agar is added and stirred with carrageenan in the step (iv). Can do. Incidentally, quantities of ingredients can be added at this time is as follows: Karaji one Nan, 0.001 to 5 wt%, preferably from 0.005 to 1 weight ^_0/0; agar, from 0.001 to 3 wt%, preferably 0.015 to 1 weight ^_0/0; gelatin, 0.005 to 2 wt%, preferably from 0.01 to 1% by weight;. sugar, 0.00017 to 0 83 molar, preferably 0.00083 to 0.165 molarity.

When the antibody dilution composition according to the present invention is used in immunological analysis, for example, D2W, distilled water, sterilized water, or the like that is appropriately diluted may be used. . The concentration of agar or carrageenan in the antibody dilution composition at the time of use is, for example, 0.1 to 0.5% by weight, preferably 0.2 to 0.3% by weight, particularly preferably 0.28% by weight. The

The salt concentration in the antibody dilution composition at the time of use is, for example, 50 mM to 200 mM, Preferably it is 100 mM-150 mM, Most preferably, it is 120 mM.

Furthermore, the concentration of the chelating agent in the antibody dilution composition at the time of use is, for example, ImM to 8 mM, preferably 3 raM to 5 mM, particularly preferably 4 mM.

The concentration of the buffer in the antibody dilution composition at the time of use is, for example, 10 mM to 80 mM, preferably 30 mM to 60 mM, and particularly preferably 50 mM.

The concentration of the nonionic surfactant in the antibody dilution composition at the time of use is, for example, 0.01 to 1% by volume, preferably 0.03 to 0.06% by volume, particularly preferably 0.05% by volume. %.

The composition for antibody dilution according to the present invention is used for dilution of an antibody used for immunological analysis. The antibody to be diluted with the antibody dilution composition according to the present invention is not particularly limited. For example, monoclonal antibody, polyclonal antibody, antibody fragment (for example, Fab, F (ab ′) ₂ ), antiserum and ascites (Including monoclonal antibodies). Further, the antibody dilution composition according to the present invention can be used for diluting a labeled antibody (for example, fluorescently labeled antibody, enzyme-labeled antibody). Examples of the antibody dilution rate include 2 times, 5 times, 10 times, 50 times, 100 times, 200 times, 500 times, 1000 times, 5000 times, and 10,000 times.

By using the antibody diluted in this way, the accuracy of immunological analysis can be improved. Examples of immunological analysis include immunostaining, immune cell staining, immunohistochemical staining, enzyme immunoassay (for example, ELISA, EIA), radioimmunoassay, fluorescent antibody method, and flow cytometry. For example, the primary antibody and / or the secondary antibody diluted with the composition for antibody dilution of the present invention against the membrane after completion of Western plotting or in the immunological analysis of a biological tissue or cell. Non-specific detection can be suppressed by using color development or immunostaining. In addition, when immunologically coloring or staining using an antibody diluted with the antibody dilution composition according to the present invention, it is not necessary to use a conventionally used blocking solution. Furthermore, the antibody dilution composition according to the present invention can also be used for antibody storage. Furthermore, the antibody dilution composition according to the present invention can be provided as an immunological analysis kit together with the antibody. In the kit, the antibody dilution composition according to the present invention and the antibody can be provided in the same container or in separate containers. Also In this case, the antibody includes the antibodies exemplified above.

EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.

Example 1: Western blotting using an antibody dilution composition containing agar according to the present invention

[Materials and Methods]

1. Antigens and antibodies

As antigens, 1 × 10 ⁸ C0S-1 cell lysate (hereinafter referred to as “protein A”) and IX 10 ⁸ MCF-7 cell lysate (hereinafter referred to as “protein B”) were used.

As the antibody, a mouse monoclonal anti-Phospho-Estrogen Receptor a (Serl8) antibody (# 2511) manufactured by Cell Signaling was used.

2. Preparation of agar-containing antibody dilution composition according to the present invention

An antibody dilution composition containing agar according to the present invention was prepared as follows.

First, 240 ml of 5M NaC1, 80 ml of 0.5M EDTA (pH8), 500 ml of 1M Tris (pH7.4), 5 ml of Tween 20 and 28 g of special grade powder agar (Wako Pure Chemical Industries, Ltd.) are mixed and combined with D2W 1L It was.

The mixture was then stirred at 17 ° C for 1 hour at 700 rpm. The mixed solution after stirring was incubated at 37 ° C for 10 hours to prepare a 10-fold concentration antibody dilution composition at the time of use. Furthermore, the same composition was diluted 1-fold with D2W to prepare an antibody dilution composition.

3. Electrophoresis, Western blotting and color development

Electrophoresis, Western plotting and color development were performed as follows. 3-1. Electrophoresis and Western blotting

Protein A 10 i l and Protein B 10 i l were mixed with 10 / l sample buffer (20% glycerol, 6% SDS, 10% 2-mercaptoethanol), respectively, and incubated at 95 ° C for 10 minutes.

After cooling, 20 μ1 of these two samples were applied to 10% Tris-Glycine polyacrylamide gel (Invitrogen), and electrophoresis was performed at 15 mA for 90 minutes. After electrophoresis, the protein was transferred from the electrophoresed gel to Hybond-ECL Membrane (Amersham). The transfer was performed for 60 minutes at a membrane area of X lmA.

3-2. Western plotting using conventional methods

The membrane obtained after Western blotting in Section 3-1 was washed with TBS (containing 0.1% Tween-20), and then subjected to color development.

In the case of using the conventional method, the washed membrane was immersed in a blocking buffer (Block Ace: manufactured by Dainippon Sumitomo Pharma Co., Ltd.) and incubated at 37 ° C for 1 hour (blocking). '

After blocking, the membrane was washed with TBS (containing 0.1% Tween-20), followed by primary antibody reaction. As a primary antibody, a mouse monoclonal anti-Phospho-Estrogen Receptor a (Serll8) antibody diluted 1000 times with Dako Cytomation DAKO Antibody Diluent with Background Reducing Components (Catalog number: S3022) was used. The primary antibody reaction was performed by incubation at 4 ° C for 10 hours.

After the primary antibody reaction, the membrane was washed with TBS (containing 0.1% Tween-20), and a secondary antibody reaction was performed. As a secondary antibody, an HRP-labeled anti-mouse IgG antibody (Amersham) diluted 1000-fold with Dako Cytomation's Secret Antibody Diluent with Background Reducing Components was used. The secondary antibody reaction was performed by incubation at 20 ° C for 15 minutes.

After the secondary antibody reaction, the membrane was washed with TBS (containing 0.1% Tween-20). The membrane was developed with an Amersham ECL-plus kit. After color development, the membrane was exposed to Hyperfilm ECL manufactured by Amersham and analyzed.

3-3. Western blotting using the antibody dilution composition of the present invention

When using the antibody dilution composition prepared in Section 2, wash the membrane obtained after Western blotting in Section 3-1 with TBS (containing 0.1% Tween-20). The primary antibody reaction was performed directly without blocking. As the primary antibody, mouse monoclonal antibody diluted 1000-fold with the antibody dilution composition prepared in Section 2

Phospho-Estrogen Receptor CK (Serll ⁸ ) antibody was used. Primary antibody reaction at 4 ° C

This was done by incubation for 10 hours. After the primary antibody reaction, the membrane was washed with TBS (containing 0.1% T _Ween -20) and subjected to a secondary antibody reaction. As a secondary antibody, an HRP-labeled anti-mouse IgG antibody (Amersham) diluted 1000 times with the antibody dilution composition prepared in Section 2 was used. The secondary antibody reaction was performed by incubation at 20 ° C for 15 minutes.

After the secondary antibody reaction, the membrane was washed with TBS (containing 0.1% Tween-20), and the membrane was developed using an Amersham ECL-plus kit. After color development, the membrane was exposed to Hyperfilm ECL manufactured by Amersham and analyzed.

[Result] '

As shown above, the results of Western plotting (Section 3-2) performed by the conventional method and the results of Western blotting (Section 3-3) using the antibody dilution composition according to the present invention are shown in FIG. Shown in In FIG. 1, M represents a molecular weight marker (kDa). The band indicated by the arrow is the band of the target protein (Phospho-Estrogen Receptor (Serl8)).

As shown in FIG. 1, in the Western plotting using the antibody dilution composition according to the present invention, the appearance of non-specific panda was much suppressed as compared with the conventional method. Example 2: Wet stamping using an antibody dilution composition containing carrageenan according to the present invention

[Materials and methods]

1. Antigens and antibodies

As the antigen, the above proteins A and 8, and 1 × 10 ⁸ THP-1 cell lysate (hereinafter referred to as “proteins”) were used.

For proteins A and B, the mouse monoclonal antibody anti-Phospho-Estrogen Receptor (Serl l8) antibody (# 2511) manufactured by Cell Signaling is used, and for protein C, a rabbit polyclonal anti-DDR1 antibody is used. (Santa-Cruz sc-532) was used.

2. Preparation of composition for dilution of antibody containing carrageenan according to the present invention '

In the following manner, an antibody dilution composition containing carrageenan according to the present invention was prepared.

First, 200raM NaCl 11.7g, 40mM EDTA (pH8.0) 80ml, 500mM Tris (pH7.4) 500ml, 0.05% Tween 20 0.5ml and 0.1% gelatin lg were mixed and made up to 1L with D2W. The mixture was stirred at 800 rpm for 2 hours at 16 ° C and then incubated at 37 ° C for 10 hours. The mixture was then cooled to room temperature and 0.3 weight. / ₀ sucrose followed by addition of 0.1% by weight Ecarazinin (Wako Pure Chemical Industries, Ltd. 035-09693) and stirring at 800 rpm for 2 hours, antibody dilution composition containing carrageenan The thing was completed.

3. Electrophoresis, Western blotting and immunostaining

FIG. 2 shows the results of Western blotting using the carrageenanan-containing antibody dilution composition produced as described above.

The Western plotting shown in FIGS. 2A-B was performed according to the method described in Example 1. For Western plotting for protein C (FIG. 2B), a rabbit polyclonal anti-DDR1 antibody diluted 150-fold was used as the primary antibody.

FIG. 2C shows a comparison between Western blotting results using the antibody dilution composition of the present invention containing carrageenanan and Western blotting results using the antibody dilution composition of the present invention not containing carrageenan. Indicates.

In the experiment shown in FIG. 2C, electrophoresis to transfer were performed by the method described in Example 1. In the method using the carrageenan-free antibody dilution composition, the mouse monoclonal anti-actin antibody (Santa-Cruz sc-8432) was diluted 200-fold with the carrageenan-free antibody dilution composition after transfer, and 4 ° Washed after 10 hours incubation with C, secondary antibody reaction was performed with HRP-labeled anti-mouse IgG antibody diluted 1000-fold with the composition for dilution of antibody free of carrageenanan, and the method described in Example 1 was used. Color was developed. The method using the composition for diluting a carrageenan-containing antibody was performed in the same manner as described above except that the composition for diluting an antibody containing a carrageenan was used instead of the composition for diluting an antibody containing no carrageenan. .

In the experiment shown in Figure 2D, the sample buffer 20 / i 1 described in Example 1 was used.

Human brain lysate (IMGENEX Corporation. # 40141) and Human Spinal Cord lysate (GENETX, Inc. # GTX15 ³⁷² ) were mixed in an amount of 10 μg, and electrophoresis to transfer were carried out by the method described in Example 1. After transfer, use conventional methods After washing with the method described in Example 1, blocking is performed, and serum obtained from a patient with Satoyoshi disease (antibodies to the brain and spinal cord are produced in the serum in this disease)

HRP diluted 2x with Dako's Antibody Diluent with Background Deducing Components (# S3022), incubated for 10 hours at 4 ° C, then washed with Dako's Antibody Diluent with Background Deducing Components 1000 (¾ ■) A secondary antibody reaction was performed with the labeled anti-human IgG antibody, and the color was developed and analyzed by the method described in Example 1. On the other hand, in the method using the composition for diluting an antibody containing carrageenanan, after the above transfer, the patient serum was diluted 2-fold with the composition for diluting the antibody containing carrageenan without performing the procking and transferred at 4 ° C. Incubate for 10 hours, wash, perform secondary antibody reaction with HRP-labeled anti-human IgG antibody diluted 1000-fold with carrageenan-containing antibody dilution composition, and develop color as described in Example 1 And analyzed.

[Result]

As shown in FIGS. 2A, 2B, and 2D, Western plotting using an antibody dilution composition containing carrageenan according to the present invention shows a much more non-specific band than the conventional method. I was holding it down. Further, FIG. 2C shows that the above-described effect of the antibody dilution composition according to the present invention is significantly related to the presence of carrageenan. Example 3: Immunohistochemical staining of lung tissue using an antibody dilution composition containing carrageenan according to the present invention

Organized pneumonia lung tissue was dehydrated after formalin fixation according to standard protocols, then embedded in paraffin, sliced into a preparation with a thickness of 4 and deparaffinized using xylene. went. The preparation of 2% 0 ₂ and Inkyubeto methyl ether 10 minutes was de Peruokishidaze reaction. Thereafter, it was washed with PBS.

In the conventional method, the prepared slide is washed with PBS containing 4% BSA and 1% horse serum.

Perform blocking reaction for 10 minutes, then Dako Antibody Diluent with

Rabbit-Positive Nano Diluted 150-fold with Background Deducing Components

Primary antibody reaction was performed by incubating with DDR1 antibody (Santa-Cruz S C-532) at 4 ° C for 10 hours. After washing with PBS, Antibody Diluent with Dako

Biotin labeled anti-rabbit diluted 1000 times with Background Deducing Components Perform secondary antibody reaction with IgG antibody (VECTOR BA-1000), wash with PBS, perform tertiary reaction with VECTOR VECTASTAIN ABC Kit Standard (PK6100), and develop color with 3,3'-diaminobenzidine tetrahydrochloride I let you.

In the method of using the composition for diluting an antibody containing carrageenan according to the present invention, the rabbit polyclonal anti-DDR1 diluted 150-fold with the composition for diluting the antibody containing carrageenan without performing the procking after washing. The primary antibody reaction was performed by incubating with the antibody (Santa-Cruz sc-532) at 4 ° C for 10 hours. After washing with PBS, a secondary antibody reaction was performed with a biotin-labeled anti-rabbit IgG antibody (VECTOR BA-1000) diluted 1000-fold with a composition for diluting antibody containing radinan. After washing with PBS, VECTOR Tertiary reaction was performed with VECTASTAIN ABC Kit Standard (PK6100), and color was developed with 3,3-diaminobenzidine tetrahydrochloride.

[Result]

As described above, the results of immunohistochemical staining performed by the conventional method and the results of immunohistological staining using the composition for diluting antibody containing carrageenan according to the present invention are shown in FIG. 3 (A: conventional method, B : Carrageenannan-containing antibody dilution composition, C: Negative control using rabbit IgG as the primary antibody (Carraginanin-containing antibody dilution composition), D: Hematoxylin eosin staining). In Fig. 3, the browned area is positive. As can be seen from FIG. 3, when the normal diluent is used, the whole is stained brown and difficult to determine, whereas when the antibody dilution composition of the present invention is used, the positive part is very clear. Recognize.

Example 4: Immunohistochemical staining of renal tissue using the antibody dilution composition containing carrageenan according to the present invention

For kidney tissue of acute progressive glomerulonephritis, using goat polyclonal anti-TRAIL receptor 3 antibody (R & DSystem, AF630) as primary antibody and biotin-labeled anti-goat IgG antibody (VECTOR BA-5000) as secondary antibody Immunohistochemical staining was performed in the same manner as in Example 3.

[Result] '

Fig. 4 shows the results of immunohistochemical staining performed by the conventional method and the results of immunohistochemical staining using the carrageenan-containing antibody dilution composition according to the present invention (A: conventional method, B: one Using the Nan-containing antibody dilution composition, C: (using Karaji one nan-containing antibody dilution composition) negatives Control Using catcher formic I _g G primary antibody, D: the Ma Toki cylinder E O Jin staining). In Fig. 4, the browned area is positive. As can be seen from FIG. 4, the positive portion is clearly cleared by the method using the antibody dilution composition of the present invention, compared to the method using a normal diluent. Industrial applicability

According to the antibody dilution composition of the present invention, non-specific detection can be suppressed, thereby improving the accuracy of immunological analysis.

All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

The scope of the claims

1. a composition for antibody dilution for improving the accuracy of immunoassay and / or suppressing non-specific detection in immunoassay, comprising agar, carrageenan, or a mixture thereof .

2. The antibody dilution composition according to claim 1, wherein the carrageenan is a carrageenan.

3. The antibody dilution composition according to claim 1, further comprising sugar and gelatin.

4. The antibody dilution composition according to claim 1, comprising at least one component selected from the group consisting of a salt, a chelating agent, a buffer and a nonionic surfactant. .

5. The antibody dilution composition according to claim 1, wherein the concentration of the agar is 0.1 to 5% by weight.

6. The antibody dilution composition according to claim 1, wherein the concentration of the agar is 0.2 to 3% by weight.

7. The antibody dilution composition according to claim 1 ^, wherein the concentration of carrageenan is 0.01 to 5% by weight.

8. The antibody dilution composition according to claim 1, wherein the concentration of the carrageenan is 0.03 to 2% by weight.

9. The antibody dilution composition according to claim 3, wherein the sugar is sucrose.

10. The antibody dilution composition according to claim 3, wherein the sugar concentration is 0.0017 to 0.83 molar.

1 1. The antibody dilution composition according to claim 3, wherein the gelatin concentration is 0.01 to 2% by weight.

1 2. The antibody dilution composition according to claim 4, wherein the salt is sodium chloride.

1 3. The resistance according to claim 4, wherein the concentration of the salt is 10 mM to 2 M. Composition for body dilution.

14. The antibody dilution composition according to claim 4, wherein the chelating agent is ethylene diamine tetraacetic acid.

15. The antibody dilution composition according to claim 4, wherein the chelating agent has a concentration of lmM to 100raM.

16. The antibody dilution composition according to claim 4 ', wherein the buffer is Tris.

17. The antibody dilution composition according to claim 4, wherein the buffer solution has a concentration of 10 mM to 1 M.

18. The antibody dilution composition according to claim 4, wherein the nonionic surfactant is Tween 20 or Triton X-100.

19. The antibody dilution composition according to claim 4, wherein the concentration of the nonionic surfactant is 0.01 to 1% by volume.

20. An immunological analysis or method comprising using the antibody dilution composition according to any one of claims 1 to 19.

21. The method according to claim 20, characterized in that the immunological analysis is Western plotting or immunostaining.

2 2. An immunological analysis kit comprising the antibody dilution composition according to any one of claims 1 to 19 and an antibody.

23. The immunological analysis kit according to claim 22, wherein the immunological analysis is Western blotting or immunostaining.

2 4. The following steps:

(i) a step of stirring a mixed solution in water containing one or more components selected from the group consisting of a salt, a chelating agent, a buffer, and a nonionic surfactant;

(i i) incubating the mixture produced in step (i) under heating;

(ii i) cooling the mixture; and

(iv) adding carrageenan and stirring;

A method for producing an antibody dilution composition.

2 5. The mixed solution in water of the above step (i) further contains gelatin 25. The method of claim 24.

26. The method according to claim 24, wherein in the step (ii), the incubation temperature is 35 ° C to 40 ° C.

In 2 7 above step (iii), mixture 4 ° (:., Characterized in that cooling to ~ 30 ° C, according to claim ² 4 The method according &.

28. The method according to claim 24, wherein in step (iv), sugar is added together with carrageenan and stirred.