JP2009162535A - Reagent for producing solid phase, and solid phase produced by using reagent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for suppressing or avoiding a nonspecific reaction. <P>SOLUTION: A reagent for capturing specifically a measuring object, and a reagent for producing a solid phase by a label binding assay method, containing a vegetable-originated polypeptide as an effective component, are used. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a reagent for preparing a solid phase for a label binding assay and a solid phase for a label binding assay prepared using the reagent.

Technical background

Depending on the combination of antigen and antibody, nucleic acid strand and complementary strand, aptamer and specific molecule for aptamer, ligand and receptor, etc., one of the combinations can be used directly or indirectly. Various label-binding assays have been devised and put into practical use for measuring the analyte in a sample from the degree of accumulation or inhibition of accumulation resulting from specific binding pair formation.
Among these, immunological measurement methods that utilize antigen-antibody reactions for reactions that form specific binding pairs depending on the combination can be applied to many measurement objects, and are excellent in operability and speed. Is used for point-of-care testing (POC testing), and is used in many clinical settings.
As a POC test based on an immunological measurement method, a test using a membrane immunoassay method has become widespread. The membrane immunoassay method includes, for example, mixing a labeled antibody with an antigen in a sample (measurement target in this case) to form a specific binding pair, and then using the membrane to which the antibody is immobilized. It is developed by developing on the (solid phase), forming a ternary specific binding pair of labeled antibody-antigen-immobilized antibody on the membrane (solid phase), and measuring the amount of accumulated label. .

  The subject of the immunological measurement method, which is a label binding assay, is the suppression and avoidance of so-called non-specific reactions in which label accumulation occurs regardless of the presence or absence of the measurement target. In the method of measuring the amount of accumulated label using a solid phase on which an antibody is immobilized, such as the membrane immunoassay method described above, there are two major non-specific reactions, one of which is the analyte ( In the above example, a non-specific reaction related to the background in which accumulation of the label is observed at the site where the antibody on the solid phase is immobilized or not, regardless of the presence or absence of the antigen) The other is a so-called false positive in which the label accumulates at the antibody immobilization site as if the antigen is present, even though the antigen is not present in the sample.

  As the cause of the former (non-specific reaction related to the background), the label itself and / or the accumulation of the labeled antibody at a site where the antibody is not immobilized can be considered, and as the cause of the latter (false positive), It is conceivable that the label itself and / or the accumulation of labeled antibody on the immobilized antibody is considered. Furthermore, there may be a case where components other than the sample-derived measurement target (for example, denatured protein or adhesive substance) are involved in the accumulation of the non-specific label.

  Two methods are employed as methods for suppressing and avoiding these non-specific reactions. One is a method by blocking, and the other is a method in which a sample and a labeled antibody are contained in a solution containing specific components when developed on a solid phase.

  Examples of the blocking method include animal-derived proteins such as bovine serum albumin (BSA), casein, and ovalbumin (Non-Patent Document 1, Patent Documents 1 and 2, etc.) and plant-derived proteins such as soybean peptide (Patent Document 3). ) And the like as a blocking agent, and a method for blocking has been reported. Recently, in addition to the superiority and inferiority of the blocking effect, from the viewpoint of prevention of zoonotic infection such as BSE, polymer substances such as polyethylene glycol (patent document 4), sericin derived from sputum (patent document 5), etc. A method used as a blocking agent, a blocking agent containing a plant-derived polypeptide as an active ingredient (Patent Document 6), and the like have been reported.

  Specific examples of the method of incorporating a sample or labeled antibody in a solution containing a specific component when developing on a solid phase include components that can suppress or avoid nonspecific reactions in so-called sample diluent or labeled antibody solution A method of containing a component (such as BSA) used as a blocking agent, a specific amino acid, or a specific surfactant (Patent Documents 7, 8, etc.) has been reported.

However, all of the above methods using a blocking agent involve immobilizing an antibody on an insoluble carrier to form a solid phase, and then covering the entire solid phase or the antibody immobilization site with the blocking agent, thereby improving the blocking effect. When the concentration of the blocking agent is increased for the purpose, the blocking agent itself becomes insoluble and precipitates, or the antigen recognition site of the antibody is masked.
The method of devising the composition of the sample diluent or labeled antibody solution described above is that the detection sensitivity of the membrane immunoassay method is such that the contact time between the sample or labeled antibody and the solid phase on which the antibody is immobilized (the flow rate of the sample or labeled antibody) ) Could not be adopted.
JP 06-130062 JP 06-160385 A JP 07-270413 A JP 2006-226882 A JP2007-248373 JP 2006-047255 A JP-A-2005-291780 JP-A-2005-291784 New Chemistry Laboratory 12, Molecular Immunology III (Tokyo Chemical Doujin)

  While there are problems with the conventional methods for suppressing and avoiding nonspecific reactions that devise the composition of blocking or sample diluent or labeled antibody solution as described above, at the time of preparing a solid phase by immobilizing the antibody on an insoluble carrier No method has been reported for immobilizing antibodies in the presence of antibodies and substances other than antibodies. One reason for this is that there is a general understanding that an antibody immobilized on an insoluble carrier preferably has a high specific activity. Components other than antibodies (buffers, salts, preservatives, preservatives, etc.) It is speculated that it is considered disadvantageous to reduce the specific activity of the antibody by coexisting with the other). The term specific activity includes concepts such as titer or purity, and so on.

  The present invention provides a method for suppressing / avoiding non-specific reactions other than the conventional method for suppressing / avoiding non-specific reactions in which the composition of blocking, sample diluent or labeled antibody solution is devised under the above circumstances. It is aimed.

  It has been considered that an antibody (solid phase preparation reagent) that has been immobilized on an insoluble carrier so far to produce a solid phase preferably has a high specific activity. In general, components other than antibodies (excluding buffers, salts, stabilizers, preservatives, preservatives, etc.) do not coexist and are used as reagents for solid phase preparation with high antibody specific activity. . In contrast, the present inventors use a solution in which an antibody and a plant-derived polypeptide are mixed as a reagent for preparing a solid phase, immobilize the antibody on an insoluble carrier, and use this solid phase for a label binding assay. As a result, it was found that false positives that occur in negative samples can be suppressed and avoided without affecting the measurement of positive samples.

Therefore, the present invention includes a reagent for preparing a solid phase in a label binding assay, which contains an antibody and a plant-derived polypeptide as active ingredients, a solid phase for a label binding assay prepared using the reagent, and the solid phase. Provided is a labeled binding assay characterized by using a phase.
That is, the present invention has the following configuration.
(1) A reagent for producing a solid phase in a label binding assay method, which contains a reagent for specifically capturing a measurement target and a plant-derived polypeptide as active ingredients.
(2) The reagent for solid phase preparation according to (1) above, wherein the plant-derived polypeptide is a polypeptide that can be obtained by degrading a plant-derived protein.
(3) In the above (2), the plant is a plant selected from the group consisting of wheat, barley, corn, rice, soybeans, red beans, kidney beans, broad beans, almonds, peanuts, potatoes, sweet potatoes, taro and taro. The reagent for solid phase preparation as described.
(4) Solid phase preparation according to (3) above, wherein the plant-derived protein is a protein selected from the group consisting of gliadin, zein (zein), glutenin, gluten, hordein, oryzenin, glycinin, patatin and conglycinin. Reagent.
(5) The reagent for specifically capturing the measurement target includes any one selected from the group consisting of an antibody, an antigen, a nucleic acid chain, an aptamer, a specific substance for the aptamer, a ligand, and a receptor. The reagent for solid phase preparation according to any one of (4) to (4).
(6) A solid phase for a label binding assay prepared by applying the solid phase preparation reagent according to any one of (1) to (5) above to an insoluble carrier.
(7) The solid phase according to (6) above, wherein the insoluble carrier comprises a membrane.
(8) A labeled binding assay method using the solid phase described in (6) or (7) above.
(9) The labeled binding assay method according to (8), which is a flow-through type, lateral flow type, or dipstick type membrane assay method.
(10) The label binding assay method according to (8) or (9) above, wherein the label is metal colloid particles or colored latex particles.

  The reagent for preparing a solid phase provided by the present invention can produce a solid phase for a label binding assay method in which non-specific reactions are suppressed / avoided.

  The “reagent for preparing a solid phase in a labeled binding assay method” in the present invention contains “a reagent for specifically capturing a measurement object” and “a plant-derived polypeptide” as active ingredients.

  In the present invention, “label binding assay” refers to a reaction that forms a specific binding pair depending on the combination of an antigen and an antibody, a nucleic acid chain and its complementary strand, an aptamer and a specific molecule for an aptamer, a ligand and a receptor, etc. , And one of the combinations is directly or indirectly labeled, and an assay method in which a measurement object in a sample is measured from the accumulation of the label or the degree of inhibition of the accumulation.

  In the present invention, “reagent for specifically capturing a measurement object” refers to an object to be immobilized on an insoluble carrier, and the measurement object is formed by forming a specific binding pair with the measurement object. The substance to be captured. Specifically, it refers to one of substances that form a specific binding pair depending on the combination thereof, such as an antigen and an antibody, a nucleic acid chain and its complementary strand, an aptamer and a specific molecule for an aptamer, and a ligand and a receptor. Any of these can be used as “a reagent for specifically capturing a measurement object” in the present invention.

  Hereinafter, the present invention will be described in detail with reference to an example in which an antibody is used as a “reagent for specifically capturing a measurement target” and immobilized on an insoluble carrier.

  The antibody that can be used as the “reagent for specifically capturing the measurement target” may be any of a polyclonal antibody, a monoclonal antibody, and a fragment containing an antigen recognition site. Methods known to those skilled in the art can be used for the kind of animal for collecting the antibody, the immunization method, the establishment of antibody-producing cells (preparation of hybridoma), the purification method of the antibody, and the like. The concentration of the antibody in the “reagent for preparing a solid phase in the labeled binding assay” is preferably 1 μg / mL to 10 mg / mL, more preferably 0.1 mg / mL to 5 mg / mL.

  Plants for obtaining a “plant-derived polypeptide” that can be contained in the “reagent for preparing a solid phase in a label binding assay method” of the present invention include wheat, barley, oats, corn, japonica rice, and indica rice. , Jabanca rice, African rice, glutinous rice, soybeans, red beans, kidney beans, broad beans, almonds, peanuts, potatoes, sweet potatoes, taros, taros and the like. Of these, soybean, wheat, corn, potato and rice are preferred, and soybean and corn are more preferred.

  The “plant-derived polypeptide” is preferably a polypeptide obtained by decomposing a protein derived from the aforementioned plant. Examples of the “plant-derived protein” include a protein extracted from the plant or a fraction containing the protein. Specific examples of the protein include gliadin, zein (zein), glutenin, gluten, hordein, oryzinin, glycinin, and patatin. And storage proteins such as conglycinin, functional proteins such as lectins, amylases, enzymes involved in respiration and photosynthesis, and structural proteins derived from roots, stems, leaves, flowers, fruits, seeds and the like. Of these, storage proteins are preferred.

The “plant-derived polypeptide” can be obtained by degrading the protein by treatment with an enzyme, acid, alkali, etc., but is not limited thereto. Further, it may be chemically synthesized or produced by genetic recombination.
As the decomposition method, enzymatic hydrolysis is preferred. Enzymes that can be used for hydrolysis are those belonging to proteases, as long as they can decompose proteins into polypeptides, such as proteinases derived from animals such as pepsin, trypsin, chymotrypsin, plant-derived proteases such as papain, ficin, bromelain, Other examples include proteases derived from microorganisms such as bacteria, molds, and radioactive bacteria. Examples of acid decomposition include acids such as hydrochloric acid, sulfuric acid, phosphoric acid, and nitric acid. Examples of alkali decomposition include sodium hydroxide, potassium hydroxide, and sodium carbonate.

  The “polypeptide” after being decomposed preferably has a weight average molecular weight of 200 Da to 300,000 Da, and more preferably a weight average molecular weight of 300 Da to 200,000 Da. Further, free amino acids generated during protein hydrolysis may be mixed as long as the performance of the “reagent for preparing a solid phase in a labeled binding assay method” of the present invention is not impaired. Moreover, you may use together the polypeptide derived from two or more types of plants, or two or more types of protein of the same plant.

  Preferred “plant-derived polypeptides” include hydrolysates of soy protein, wheat protein, potato protein, corn protein, rice bran protein, etc. Among them, corn protein hydrolyzate is preferred.

  As the hydrolyzate of corn protein, for example, a peptide having a molecular weight of 200 Da to 4000 Da and having a free amino acid content of 1% or less based on the total amino acids is preferable. Among them, the amino acid composition (% by weight) is aspartic acid 2.5 to 12.5, threonine 2.5 to 6.0, serine 4.0 to 6.0, glutamic acid 15.0 to 50.0, Glycine 2.0-5.5, Alanine 2.0-13.5, Valine 4.0-8.0, Cysteine 0.0-1.5, Methionine 1.0-2.0, Isoleucine 3.0- 6.0, leucine 6.0-15.0, tyrosine 1.0-4.0, phenylalanine 2.0-5.5, lysine 0.5-7.0, histidine 0.5-3.0, arginine What is 1.0-8.0 and proline 5.0-13.0 is more preferable.

  As a commercially available product that can be used as the “plant-derived polypeptide” of the present invention, ApplyDuo (registered trademark, Seikagaku Biobusiness Co., Ltd., Code # 200140) can be mentioned.

  The concentration of the “plant-derived polypeptide” in the “solid phase preparation reagent in the label binding assay” is 0.005 to 30% (w / v), preferably 0.01 to 20% (w / v), More preferably, it is 0.03 to 5%.

The “reagent for preparing a solid phase in a labeled binding assay method” of the present invention is in a solution state when used. Hereinafter, various conditions in the solution state will be described. The “reagent for preparing a solid phase in a labeled binding assay method” of the present invention is preferably used after being dissolved in a buffer solution. Examples of the buffer solution include commonly used buffers such as phosphate buffer, Tris buffer, and Good's buffer. The pH of the buffer is preferably in the range of 6.0 to 9.5, more preferably 6.5 to 8.5, and even more preferably 7.0 to 8.0.
The buffer may further contain salts such as NaCl, stabilizers such as sucrose, preservatives, preservatives such as procrine, and the like. The salts include those that are present in the step of adjusting the pH of the buffer solution, such as sodium hydroxide, in addition to those that are included for adjusting the ionic strength, such as NaCl.
These components other than the active ingredients of the “reagent for preparing a solid phase in a label binding assay” of the present invention may reduce the effect of suppressing or avoiding the nonspecific reaction of the present invention, As long as the performance (for example, the amount immobilized on an insoluble carrier and the ability to form a specific binding pair) is not impaired, it can be appropriately formulated.

  The “reagent for preparing a solid phase in a labeled binding assay method” of the present invention is mixed with “plant-derived polypeptide” immediately before immobilizing “reagent for specifically capturing a measurement object” on an insoluble carrier. It may be prepared, or a premixed product may be stored frozen and thawed at the time of use, or a product that has been freeze-dried stored may be dissolved in distilled water or the like.

In the case of a flow-through type solution, the “reagent for preparing a solid phase in a labeled binding assay” solution prepared as described above has a lateral flow type (typically used for immunochromatography as a symbol or a symbol such as +). In the case of a test stick), the “solid phase for a labeled binding assay method” of the present invention can be produced by coating in a line on a membrane insoluble carrier.
In the present invention, the term “flow-through membrane assay” means that the measurement sample or the like passes vertically through the “solid phase for labeled binding assay” of the present invention (in this case, an antibody-immobilized membrane). The term “lateral flow membrane assay” refers to a method in which the measurement sample is developed so as to move horizontally on the antibody-immobilized membrane. In the case of a “dipstick membrane assay”, a lateral flow type antibody-immobilized membrane is inverted in a measurement sample solution, and one end of the membrane is immersed, so that the measurement sample can be sucked up by capillary action. It refers to the method that is deployed.

The “solid phase for label-binding assay” of the present invention is obtained by immobilizing a “reagent for specifically capturing a measurement object” on an insoluble carrier using the reagent for preparing a solid phase of the present invention. The above-described normally used blocking agent can be coated in the form of a solution or vapor to perform blocking.
The form of the solid phase (insoluble carrier) when producing the “solid phase of the labeled binding assay method” of the present invention using the “reagent for preparing the solid phase in the labeled binding assay method” of the present invention is well-like, particle Any of a shape, a plate shape, and a film shape may be used. As the material of the solid phase, plastic, latex, cellulose and the like can be used. When the “solid phase for label binding assay” is for POC testing, it is preferable to use a porous membrane made of nitrocellulose or the like.

  The labeling substance in the “label binding assay method” of the present invention may be any substance that is used in so-called immunoassays and hybridization assays, such as enzymes, dyes, fluorescent substances, metal colloid particles such as gold colloids, and colored latex particles. But you can use it. In the case of a POC inspection that requires simplicity, metal colloid particles and colored latex particles are preferable in terms of visualization of label accumulation.

  As used herein, the term “measurement” is to be interpreted in the broadest sense unless otherwise specified, and is commonly used as “test”, “detection”, “analysis”, “identification”, It includes concepts such as “determination”, and includes cases where the result of measurement is “quantitative”, “semi-quantitative”, and “qualitative”.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, the scope of the present invention is not limited to the following Example.

[Example 1]
Measurement of antigen using solid phase prepared by the reagent for preparing solid phase of the present invention / when label is metal colloidal particles (1) Preparation of solid phase (antibody-immobilized membrane) for label binding assay method 20 mmol / L Tris 0.75 mg / mL of mouse anti-influenza A virus monoclonal antibody against buffer solution (pH 8.0), Applied Duo (registered trademark, Seikagaku Biobusiness Co., Ltd., Code # 200140), containing plant-derived polypeptide of the present invention ) 0% (no addition) to 5.0% (v / v) (the concentration of the plant-derived polypeptide of the present invention is 0% to 0.3% (w / v) as the protein concentration in terms of BSA), Sucrose was added to 2.5% (w / v) to obtain a reagent for preparing a solid phase for influenza A (antibody for type A).
Similarly, mouse anti-influenza B virus monoclonal antibody is 1.0 mg / mL, and Appli Duo is 0% (no addition) to 2.5% (v / v) in 10 mmol / L phosphate buffer (pH 7.2) ( The concentration of the plant-derived polypeptide of the present invention is such that the protein concentration in terms of BSA is 0% to 0.15% (w / v)), and sucrose is added to 2.5% (w / v). A reagent for preparing a solid phase for influenza for type (antibody for type B) was used.
In addition, to the 10 mmol / L phosphate buffer (pH 7.2), goat anti-mouse IgG antibody was added at 0.75 mg / mL and sucrose at 2.5% (w / v), and the control line (CTRL) was added. A solid phase preparation reagent.
The above three kinds of solid phase preparation reagents were applied to a nitrocellulose membrane in the order of an antibody for type A, an antibody for type B, and an antibody for CTRL at intervals in a line, and dried with a dryer. This was used as an antibody-immobilized membrane. In addition, the main molecular weight of the plant-derived polypeptide in ApplyDuo is 6000 Da or less as estimated by SDS-PAGE.

(2) Preparation of labeled antibody pad Colloidal gold-labeled anti-influenza A virus mouse monoclonal antibody and gold in 20 mmol / L Tris buffer (pH 7.5) containing 1.3% (w / v) casein and 4% sucrose Colloidally labeled anti-influenza B virus mouse monoclonal antibody was added and mixed to obtain a labeled antibody solution. The labeled antibody solution was applied to a glass fiber sheet and dried with a dryer to obtain a labeled antibody pad.

(3) Preparation of test stick using solid phase for labeled binding assay method of the present invention The antibody-immobilized membrane prepared in (1) above is pasted on a plastic adhesive sheet, and a labeled antibody pad and sample as shown in FIG. A pad and a water absorption pad were arranged. That is, the labeled antibody pad prepared in the above (2) is arranged near the end on the side coated with the anti-A type antibody (upstream in the development of the measurement sample on the solid phase), and the labeled antibody pad is placed on the labeled antibody pad. Sample pads were placed so as to overlap. On the other hand, a water-absorbing pad was placed at the end of the side to which the CTRL antibody was applied (downstream in the development of the measurement sample on the solid phase). Further, it was covered with a transparent plastic seal. The laminated sheet was cut to a width of 4 mm to obtain a test stick. The sheet length at this time was 98 mm.

(4) Measurement (dipstick membrane assay)
For the nasal swab samples (one case) that were negative from type A and type B influenza viruses collected from healthy adults, 0.3% Tween20 and 0.25% (w / v) BSA were extracted after viral antigen extraction treatment. The sample was diluted with a 20 mmol / L phosphate buffer solution (pH 7.6). A test stick was immersed in a tube containing the measurement sample, and the presence or absence of a red line appearing at each antibody application site of the test stick was measured 10 minutes later. The measurement under each application condition of AppliedDuo was performed twice (n = 2) from the virus antigen extraction process. The results are shown in Table 1. In the table, + means that a red line was observed, and the larger the value, the darker the coloring. N represents that no red line was observed.

  When a solid phase prepared using a reagent for preparing a solid phase without addition of Appli Duo was used, a red line was observed at the site of application of the antibody for type A in both measurements, regardless of the type A influenza virus negative sample. When using a solid phase prepared with a solid phase preparation reagent with 1.0% Applied Duo added, the color of the red line decreases from 2.5+ with no addition to 1.5+, 2.5% and 5%. When a solid phase prepared with a solid phase preparation reagent added with 0.0% Appli Duo was used, no red line was observed. From the above, it was confirmed that non-specific reaction (false positive) can be suppressed / avoided by preparing a solid phase using a reagent for solid phase preparation to which Appli Duo was added.

[Example 2]
Measurement of antigen using solid phase prepared by the reagent for preparing solid phase of the present invention / when labeling is colored latex (1) Preparation of antibody-immobilized membrane Mouse against 10 mmol / L Tris buffer (pH 7.2) 0.75 mg / mL of anti-influenza A virus monoclonal antibody, 0% (no addition) of Appli Duo, 1% (v / v) (the concentration of the plant-derived polypeptide of the present invention is 0. 06% (w / v)) and sucrose were added to 2.5% (w / v) to obtain a solid phase preparation reagent for type A.
Similarly, 10 mg / mL of mouse anti-influenza B virus monoclonal antibody and 0%, 1% (v / v) of Applied Duo (from the plant of the present invention) in 10 mmol / L phosphate buffer (pH 7.2) The polypeptide concentration was 0.06% (w / v) as the protein concentration in terms of BSA, and sucrose was added to 2.5% (w / v). did.
Further, to 10 mmol / L phosphate buffer (pH 7.2), anti-HRP (horseradish peroxidase) antibody was added to 1.8 mg / mL, and sucrose to 2.5% (w / v), A reagent for preparing a solid phase for CTRL was used.
The above three kinds of solid phase preparation reagents were applied to a nitrocellulose membrane in the order of an antibody for type A, an antibody for type B, and an antibody for CTRL at intervals in a line, and dried with a dryer. This was used as an antibody-immobilized membrane. In addition, the main molecular weight of the plant-derived polypeptide in ApplyDuo is 6000 Da or less as estimated by SDS-PAGE.

(2) Preparation of labeled antibody pad Colored latex-labeled anti-influenza A virus mouse monoclonal antibody, colored latex label in 50 mmol / L Tris buffer (pH 8.5) containing 2% (w / v) casein and 10% sucrose Anti-influenza B virus mouse monoclonal antibody and colored latex-labeled HRP were added and mixed to obtain a labeled antibody solution. The labeled antibody solution was applied to a glass fiber sheet and dried with a dryer to obtain a labeled antibody pad.

(3) Preparation of test stick using solid phase for labeled binding assay method of the present invention The antibody-immobilized membrane prepared in (1) above is pasted on a plastic adhesive sheet, and a labeled antibody pad and sample pad as shown in FIG. A water absorbing pad was placed. That is, the labeled antibody pad prepared in the above (2) is arranged near the end on the side coated with the anti-A type antibody (upstream in the development of the measurement sample on the solid phase), and the labeled antibody pad is placed on the labeled antibody pad. Sample pads were placed so as to overlap. On the other hand, a water-absorbing pad was placed at the end on the side where the CTRL (anti-HRP) antibody was applied (downstream in the development of the measurement sample on the solid phase). Further, it was covered with a transparent plastic seal. The laminated sheet was cut to a width of 4 mm to obtain a test stick. The sheet length at this time was 98 mm.

(4) Measurement (dipstick membrane assay)
After nasal swab samples (10 cases) negative for influenza A virus and influenza B virus collected from healthy adults (10 cases), 0.3 (w / v)% Tween 20 and 0.25% ( w / v) Diluted with 20 mmol / L phosphate buffer (pH 7.5) containing BSA to prepare a measurement sample. The test stick was immersed in the tube containing the measurement sample, and the determination was made based on the presence or absence of colored lines (A type and B type) appearing at each antibody application site of the test stick 10 minutes later. The measurement under each addition condition was performed twice (n = 2) from the virus antigen extraction process. The results are shown in Table 2. In the table, + means that a colored line was observed, and the larger the value, the darker the color. N represents that coloring was not observed.

In the case of using a solid phase prepared with a reagent for solid phase preparation to which no Appli Duo was added, coloring was performed at the site where the A-type antibody was applied in three samples Nos. 7, 8, and 9 despite the samples that were negative for influenza A virus A line was observed. On the other hand, when using a solid phase prepared with a reagent for preparing a solid phase with 1.0% Applied Duo added, the colored line observed when no Applied Duo was added was not observed.
From the above, it was confirmed that non-specific reaction (false positive) can be suppressed / avoided by preparing a solid phase using a reagent for solid phase preparation to which Appli Duo was added.
In combination with the results of Example 1, when a solid phase was prepared using the reagent for preparing a solid phase of the present invention, a nonspecific reaction (false positive) was caused regardless of whether the label was a metal colloid particle or a colored latex particle. It was confirmed that it can be suppressed and avoided.

  Using a mixed solution of a reagent for specific capture of a measurement object and a plant-derived polypeptide as a solid phase preparation reagent, the solid phase formed by immobilizing the reagent on an insoluble carrier is labeled binding assay If it is used, non-specific reactions can be suppressed and avoided, and accurate measurement can be realized.

It is a figure which shows an example of the test stick of this invention.

Claims (10)

  A reagent for producing a solid phase in a label binding assay method, which contains a reagent for specifically capturing a measurement target and a plant-derived polypeptide as active ingredients.   The reagent for solid phase preparation according to claim 1, wherein the plant-derived polypeptide is a polypeptide that can be obtained by degrading a plant-derived protein.   The solid phase according to claim 2, wherein the plant is a plant selected from the group consisting of wheat, barley, corn, rice, soybeans, red beans, kidney beans, broad beans, almonds, peanuts, potatoes, sweet potatoes, taro and taro. Reagent for preparation.   The reagent for solid phase preparation according to claim 3, wherein the plant-derived protein is a protein selected from the group consisting of gliadin, zein (zein), glutenin, gluten, hordein, oryzenin, glycinin, patatin and conglycinin.   The reagent for specifically capturing the measurement object comprises any one selected from the group consisting of an antibody, an antigen, a nucleic acid chain, an aptamer, a specific molecule for the aptamer, a ligand, and a receptor. The reagent for solid-phase preparation as described in any one.   A solid phase for a label binding assay, which is prepared by applying the solid phase preparation reagent according to any one of claims 1 to 5 to an insoluble carrier.   The solid phase according to claim 6, wherein the insoluble carrier comprises a membrane.   A labeled binding assay method, wherein the solid phase according to claim 6 or 7 is used.   The labeled binding assay method according to claim 8, which is a flow-through type, lateral flow type or dipstick type membrane assay method.   The label-binding assay method according to claim 8 or 9, wherein the label is a metal colloid particle or a colored latex particle.