KR20180039654A - Reagent compositions for immunoassay and uses thereof - Google Patents

Abstract

[PROBLEMS] To provide a means for suppressing the progress of a nonspecific reaction and hence the occurrence of false positives in an immunological measurement method using feces as a sample while suppressing a decrease in reactivity with a detection target to a minimum.
[MEANS FOR SOLVING PROBLEMS] In immunoassay using a fecal sample, a reagent composition containing an oxidizing agent is used.

Description

Reagent compositions for immunoassay and uses thereof

To a reagent composition for immunoassay and a use thereof.

Recently, in clinics and small hospitals, there has been an increasing demand for "knowing the test result while consulting a patient", and the conventional outpatient outpatient examination to the point-of-care testing (POCT) has been carried out . Test strips based on the immunochromatographic method according to the spread of POCT have been used as in vitro diagnostic drugs. The immunochromatography method does not require the preparation of reagents at the time of examination but allows a test sample such as blood, urine, or feces suspension (hereinafter, simply referred to as a "sample") to be placed on the test strip It is possible to detect an object in the sample only by a simple operation such as dropping it directly after passing through the object to be detected.

Test strips (hereinafter, simply referred to as "test strips") based on immunochromatography are generally porous membranes having a sample supply section, a labeling reagent holding section, a chromatographic medium, and a detection section. A labeling reagent such as a labeling antibody is allowed to elute and develop at the development start site of the chromatographic medium so that the labeling reagent can reach the detection portion after passing through the chromatographic medium after contact with the sample, And the like are fixed to constitute a detection unit. Here, when the sample is dropped onto the sample feeding portion and reaches the labeling reagent holding portion, the detection target in the sample specifically binds to the labeling antibody to form a complex. The complex is developed in the downstream direction of the chromatographic medium and binds to the immobilized antibody. Therefore, it is possible to qualitatively or quantitatively analyze the detection target by detecting the sandwich-type complex by the detection antibody, the detection target and the immobilized antibody in the detection part. An example of a marker constituting the labeling reagent is gold colloid particles, and it is possible to qualitatively detect it by a red line formed by gold colloid particles. It is also possible to quantitatively detect the object to be detected in the sample on the basis of the degree of the red line.

However, for the purpose of early detection of the infectious disease and the identification of the cause thereof, tests for examining the presence of viruses or bacteria such as norovirus and rotavirus in the test sample are known. Detection of a pathogen from a fecal sample is very useful since fungi or viruses that cause infection are often released in the feces. Among the pathogenic agents of infectious diseases, bacteria are detected by methods such as culture or genetic testing, and viruses are detected by genetic testing or electron microscopy. In these methods, however, techniques and time , There is a disadvantage that a special device is required, and furthermore, it takes a long time for detection, and a large sample can not be processed. Norovirus and rotavirus may become serious in children and elderly people. In virus detection, in order to prevent the spread of infection and to be able to determine treatment policy and isolation measures early, it is necessary to measure in a short time . On the other hand, the detection of pathogens from the fecal samples by using an immunological measurement method such as an immunochromatography method has an advantage that it is possible to carry out the test with a simple, short time and high sensitivity.

However, in the immunological measurement method such as immunochromatography, in the analysis of the sample collected from the patient, the analyte may not be present in the sample but may be determined to be positive by non-specific reaction (false positive (false positive). Various techniques for suppressing the progress of non-specific reactions have been proposed for the purpose of preventing the occurrence of such false positives in immunoassay. For example, a method in which a specific component is contained in a solution when a sample or a labeled antibody is developed on a solid phase (for example, Patent Document 1), a method for producing a solid phase in immobilizing an antibody (for example, 2), a method for suppressing nonspecific reaction by heterologous antibody (for example, Patent Document 3), and the like have been proposed.

In particular, as a method for suppressing non-specific reactions when a fecal sample is used, there is a method of filtering a sample with a filter including a strong rigid filter because the solid impurity is a nonspecific substance (see, for example, Patent Document 4 ), a method of treating a sample with a buffer having a pH adjusted (for example, Patent Document 5), and as a technique for suppressing non-specific reactions caused by a filtration operation using a filter material, (For example, Patent Document 6), and the like.

JP (Special Feature) 2009-517632 A JP 2009-162535 A JP 2011-197014 A JP 2008-122372 A JP 2004-301684 A JPH09-203735E

According to the studies of the present inventors, even if various techniques proposed in the prior art have been used by Patent Documents 4 to 6, it is still possible to sufficiently suppress the progress of the non-specific reaction and the occurrence of false positives in the case of using the fecal samples It turns out that there is a case that can not be done.

It is therefore an object of the present invention to provide a means for suppressing the progress of a nonspecific reaction and hence the occurrence of false positives in an immunological measurement method using feces as a sample while suppressing a decrease in reactivity with a detection target to a minimum. The purpose.

The inventors of the present invention have conducted extensive studies to solve the above problems. As a result, in the immunological measurement method using feces as a sample, the inventors found that the above problems can be solved by bringing the fecal sample into contact with the oxidizing agent before reaching the detection portion, thereby completing the present invention.

That is, according to one aspect of the present invention, there is provided a reagent composition for use in an immunoassay using a fecal sample, which comprises an oxidizing agent.

According to another aspect of the present invention, there is provided a kit for analyzing a sample, comprising: a sample supply unit; a chromatographic medium disposed downstream of the sample supply unit; and an anti-detection target antibody labeled with a metal colloid or the like A test reagent holding section in which a reagent is retained in a releasable manner and a detecting section which is located on the downstream side of the mark reagent holding section as a part of the chromatography medium, ≪ / RTI > and a reagent composition according to the present invention.

According to still another aspect of the present invention, there is provided a kit for analyzing an analyte, comprising: a sample supply unit; a chromatographic medium disposed downstream of the sample supply unit; and an anti-detection target antibody labeled with a metal colloid or the like A labeling reagent holding section in which the labeling reagent is kept in a releasable state and a detecting section which is located on the downstream side of the labeling reagent holding section as a part of the chromatography medium, A method for detecting or quantifying an object to be detected in a fecal sample by using a reagent composition according to the above-described form is also provided.

According to still another aspect of the present invention, there is also provided a method for inhibiting a nonspecific reaction in an immunoassay, which comprises contacting a reagent composition according to the above-described aspect with a fecal sample.

INDUSTRIAL APPLICABILITY According to the present invention, in immunological measurement using feces as a sample, it is possible to suppress the progress of nonspecific reaction and the occurrence of false positives, while minimizing the deterioration of reactivity with the detection target.

1 is a schematic cross-sectional view of a kit for immunochromatography for norovirus detection according to an embodiment of the present invention.

[Reagent composition]

One aspect of the present invention is a reagent composition for use in an immunoassay using a fecal sample, which comprises an oxidizing agent.

The reagent composition according to this embodiment is used for the immunoassay of a fecal sample. The reagent composition may be contained in a solution such as an extraction solution or a developing solution for extracting a fecal sample, or may be contained in a dry state on a member to be contacted with the sample, such as a pad or a filter.

The "immunological measurement method" is a method of detecting and quantifying an object to be detected by using a substance (for example, an antibody or an antigen) that specifically binds to a detection target object to be measured. In the present invention, an immunoassay method (preferably a sandwich immunochromatography method described later) is typically used as the immunoassay, but any other ELISA method, latex agglutination method, immunoabsorbing method and the like can be used .

Here, when the object to be detected is a substance having antigenicity, an immunological measurement method can be carried out using an antibody that specifically recognizes the object to be detected. In this case, the specific form of the antibody is not particularly limited, and examples thereof include antisera prepared from the serum of an animal immunized with the detection subject, polyclonal antibodies obtained from the immunoglobulin fraction purified from the antiserum, (F (ab ') ₂ , Fab, Fab', or Fv] obtained by cell fusion using spleen cells of an animal immunized with an antibody or a fragment thereof (for example, F The preparation of these antibodies can be carried out by a known method. In particular, sandwich immunoassays are preferred which form monoclonal antibody-antigen-monoclonal antibody complexes. In addition, when the object to be detected is an antibody (that is, an immunoglobulin molecule for a specific antigen), an immunoassay can be carried out using an antibody against a substance (antigen) or immunoglobulin molecule that the antibody specifically recognizes have. When the subject to be detected is the same, an immunological measurement method can be carried out by using lecithin protein or the like in addition to the antibody against the sugar.

As for the "fecal sample", the specific form thereof is not particularly limited as far as it is a sample derived from the feces, and the fecal source from which the fecal sample is derived may be any of a light meal, a normal stool, a Yanbian, a diarrhea, and a water stain. Incidentally, the fecal sample usually contains about 60% by mass of water, about 40% by mass of food waste, intestinal membrane cells, intestinal bacteria, etc., but the fecal sample contains many components that are not absorbed in the body but excreted in the body And it has a peculiar characteristic that the component, shape, pH, etc. vary in accordance with the discharged state, and that there are many solid impurities. For this reason, there are various situations and causes of false positives, and means for suppressing the progress of the nonspecific reaction and the occurrence of false positives in the fecal samples are very useful. INDUSTRIAL APPLICABILITY According to the present invention, it is possible to suppress the progress of nonspecific reaction in fecal samples and the occurrence of false positives.

In the immunological measurement method such as immunochromatography, determination (detection) of presence or absence of an object to be detected or measurement (quantification) of the presence of an object to be detected is performed. In the present invention, the "object to be detected" is not particularly limited and may be any substance that can be contained in the fecal sample. Examples of the object to be detected include viruses (eg, Norovirus, Sapovirus, Rotavirus, Adenovirus, etc.), bacteria (eg, dysentery bacteria, Salmonella spp., Enteric haemorrhagic Escherichia coli, Campylobacter, etc.) Or vascular occult blood to be subjected to colorectal cancer screening. Particularly, by using viruses or bacteria as detection targets, the superiority of the present invention, which enables quick determination while suppressing the occurrence of false positives, becomes prominent. The substance (antigen) specifically recognized by the anti-detection target antibody is not particularly limited as long as it is a substance specifically present in each of the above-mentioned detection targets. In addition to proteins, peptides, antigens and antibodies, A sugar (particularly a sugar portion of a glycoprotein, a saccharide portion of a glycolipid, etc.), a complex saccharide and the like.

(Oxidizing agent)

With regard to the specific form of the oxidizing agent contained in the reagent composition according to this embodiment, any oxidizing agent capable of exerting the effects of the present invention is not particularly limited and conventionally known oxidizing agents are used. The oxidizing agent contained in the reagent composition according to this embodiment is included in the fecal sample and oxidizes any component that causes non-specific reactions. Examples of the oxidizing agent include, for example, one or more kinds selected from the group consisting of halogen oxoates, salts of transition metal complexes, peroxides, organic mercury compounds, permanganates, chromates, oxydases, nitrites and superoxide .

As the halogen oxoacid salt, there may be mentioned, for example, salts of organic acids such as hypochlorous acid, chlorous acid, chloric acid, perchloric acid, hypochlorous acid, babochromic acid, ascorbic acid, bromic acid, perbromic acid, hypochlorous acid, Alkali metal salts, alkaline earth metal salts and ammonium salts, and in particular, alkali metal salts of iodic acid and periodic acid (for example, potassium iodate, sodium iodate, potassium periodate), alkaline earth metal salts , Calcium iodate) and ammonium salts (for example, ammonium iodate).

Examples of salts of transition metal complexes include alkali metal salts, alkaline earth metal salts and ferricyanides of ammonium salts, and potassium ferricyanide is particularly preferred.

As the peroxide, there may be mentioned, for example, organic peroxides, in particular peroxide element and hydrogen peroxide being preferred.

As the organic mercury compound, thimerosal is preferable.

As the permanganate, for example, a permanganate of an alkali metal or an alkaline earth metal may be mentioned, and potassium permanganate is particularly preferable.

Examples of the oxidase include glucose oxidase, cholesterol oxidase, choline oxidase, glucose oxidase, lactide oxidase, pyruvate oxidase, sarcosine oxidase, xanthine oxidase and uricase. And particularly, glucose oxidase is preferable. The oxidase functions as an oxidoreductase for the substrate present in the feces, and the generated hydrogen peroxide is used as the oxidizing agent. Among them, from the viewpoint that the effect of suppressing the non-specific reaction and thus the occurrence of false positives is particularly excellent, it is preferable to use one or more kinds selected from the group consisting of potassium iodate, potassium periodate and potassium perisocyanate And potassium iodate is most preferable from the viewpoint of the visibility of the measurement result and the effect of suppressing the nonspecific reaction to the concentration.

It is preferable that the oxidizing agent contained in the reagent composition according to the present embodiment has an oxidation-reduction potential of 100 mV or more when it is defined from the viewpoint of oxidation-reduction potential. By having such an oxidation-reduction potential, the effect of suppressing the non-specific reaction and hence the occurrence of false positives can be reliably demonstrated. The oxidation-reduction potential of the oxidizing agent is measured by the following method. The value of the redox potential is more preferably 200 mV or more, still more preferably 300 mV or more, and particularly preferably 400 mV or more. On the other hand, the upper limit of the oxidation-reduction potential of the oxidizing agent is not particularly limited, but is usually 900 mV or less, preferably 500 mV or less.

Measuring method of oxidation-reduction potential: The oxidizing agent is dissolved in purified water and 10 mL of a 10 mM solution is prepared. For the obtained solution, the redox potential is measured using a 3.33 ㏖ / L KCl-Ag / AgCl electrode.

[Immunological measurement kit (immunochromatography kit)]

In the reagent composition according to the above-described form, the oxidizing agent may be contained in various forms. Hereinafter, details of the kit for immunological measurement in which the reagent composition according to the above-described form is used will be described in detail prior to the description of the form of containing the oxidizing agent, with reference to the drawings with reference to the case where the immunological measurement method is immunochromatography do.

1 is a schematic cross-sectional view of an immunochromatography kit for the detection of norovirus according to an embodiment of the present invention.

The immunochromatography kit 10 shown in Fig. 1 has a test strip 100 as a body of the kit. The test strip 100 is provided with a plastic adhesive sheet 110 as the lowest layer and an antibody immobilization membrane 120 as a chromatographic medium (insoluble carrier; stationary phase) is laminated on the adhesive sheet 110. Hereinafter, description will be made assuming that the upstream side of the sample supply portion side of the antibody-immobilized membrane 120 and the downstream direction of the sample (absorptive portion side) are downstream.

On the upstream side of the antibody-immobilized membrane 120, a labeling reagent holding pad 130 (labeling reagent holding section) is further laminated. A sample pad 140 (sample supply part) is further laminated on the labeling reagent holding pad 130. An anti-norovirus antibody (for example, an antagonist GI mouse monoclonal antibody and an anti-norovirus GII mouse monoclonal antibody), which is an anti-detection target antibody, is immobilized on the surface of the gold colloid particle, And the labeling antibody (labeling reagent) made from the labeled antibody is eluted. In the antibody-immobilized membrane 120, on the downstream side of the labeling reagent holding pad 130, an anti-norovirus antibody (for example, an anti-norovirus GI mouse monoclonal antibody and an antagonist GII mouse monoclonal antibody A control line 124 (control unit) on which an anti-immunoglobulin antibody is immobilized is arranged on the downstream side of the test line 122. The control line 124 (control unit) Further, in the antibody-immobilized membrane 120, an absorbent pad 150 is laminated on the downstream side of the control line 124.

The kit for immunochromatography 10 includes a filter 200 for filtering a sample before being supplied to the sample pad 140 (sample supply part) in addition to the test strip 100 which is the body of the kit, And a developing solution (300). The kit for immunochromatography 10 may further include other components (for example, a user's manual, a sample collection instrument, a sample extraction container, etc.) as necessary. Further, it is preferable that the test strip 100 is stored and mounted in a dedicated housing made of plastic (including a sample supply window portion and a detection window portion) not shown and is in the form of an immunochromatographic test device.

When the kit for immunochromatography 10 is used, for example, in the case of using the extracting solution 300, the feces sample is mixed with a solution for extraction in a container such as a feces container or a microtube, (200) to obtain a sample in the form of a filtered sample. At this time, the filtration filter 200 may be used as a member different from the container, or may be used as a member integrated with the container lid or the like. By constituting the filtration filter 200 as a member integrated with the lid of the container, the suspension is discharged from the container and the filtration treatment is performed to obtain a sample in the form of a filtrate, so that the filtration operation after the extraction can be performed easily.

Subsequently, when a sample in the form of the filtered product obtained above is dropped on the sample pad 140, the sample in the form of a filtered product functions as a developing solution. After passing through the sample pad 140, the sample reagent holding pad 130 ). When a sample to be detected (for example, Norovirus) is present in the sample, when the sample moves through the labeling reagent holding pad 130 by capillary phenomenon, the antilarovirus antibody constituting the labeling reagent, The complex (gold colloid particle-antagonist antibody-norovirus (antigen) complex) is formed by the antigen-antibody reaction of the virus (antigen). The complex moves with the sample further down through the antibody immobilization membrane 120 by capillary action. When the complex reaches the test line 122 (detection portion) installed in the middle of the antibody immobilization membrane 120, the anti-norovirus antibody which is the anti-detection target antibody immobilized on the test line 122 is immobilized on the complex immobilized membrane 120 (Antigen) is inserted between two anti-norovirus antibodies (an antibody immobilized on gold colloid particles) and an antibody immobilized on a test line) constituting the labeling reagent) by causing an antigen-antibody reaction with the antigen So that the sandwich complex thus formed is formed. Here, since one of the antibodies constituting the sandwich complex is immobilized on the test line 122, the sandwich complex stays on the test line 122 to show a red line by the integration of the gold colloid particles. With this line, it becomes possible to visually confirm the presence of the object to be detected (norovirus) in the sample. In the absence of the object to be detected (norovirus) in the sample, the antigen-antibody complex formed by the antigen and the antibody constituting the labeling reagent is not formed and the sandwich complex in the test line is not formed. There is no red line. That is, if the test line 122 (detecting portion) is colored with the color of the gold colloid (for example, red to reddish brown), it is determined that the object to be detected (norovirus) is present in the sample. On the other hand, it is judged that the object to be detected (norovirus) does not exist in the sample if the test line has no change in color tone and remains in the film member.

In addition, the anti-norovirus antibody-binding gold colloid (labeling reagent) not involved in the reaction is captured by the anti-immunoglobulin antibody immobilized on the control line 124 (control section). As a result, since the control line 124 indicates a red line, it is confirmed that the reaction on the test strip 100 proceeds normally.

As described above, the use form of the kit has been described in detail in the case of using the extraction solution 300 in the use of the kit. However, in the case of using the developing solution 300 instead of the extraction solution, for example, A filtrate similar to that described above is supplied to the sample pad 140 by dropping the developing solution 300 on the sample while the feces sample is placed on the filtration filter 200 overlying the filter 140. The subsequent behavior is the same as described above, and therefore, a detailed description thereof will be omitted.

(Containing form of the oxidizing agent (reagent composition)

The reagent composition containing the oxidizing agent according to the above-mentioned form can be contained in various forms as follows in an immunoassay kit (immunochromatography kit).

(1) a form to be added to the extraction solution or the developing solution 300;

(2) a form added to the filtration filter 200;

(3) a form to be added to the sample pad 140;

(4) a form added to the labeling reagent holding pad 130;

(5) A form added upstream of the test line 122 of the antibody-immobilized membrane.

Here, in the above-described forms (3) to (5), the test strip 100 contains an oxidizing agent, but any of them can be used as an antibody immobilizing membrane 120 (chromatographic medium) The oxidizing agent is contained in any portion up to the upstream of the test line 122 (detection portion). Therefore, in all the forms of (1) to (5), the sample or its filtrate and the mixture of the extraction solution or the developing solution come into contact with the oxidizing agent upstream of the test line 122 (detection portion) . Thus, the non-specific reaction due to any component contained in the fecal sample is suppressed by the presence of the oxidizing agent. As a result, occurrence of false positives can be suppressed. Of the above-mentioned forms (1) to (5), the forms (1) to (4) wherein the contact between the mixture and the oxidizing agent are carried out upstream of the antibody immobilization membrane 120 are preferable, (1) to (3) in which contact is performed upstream of the labeling reagent holding pad 130 is more preferable. From the viewpoint of suppressing the occurrence of false positives at a lower oxidant concentration, the above-mentioned form (1) or (4) is more preferable, and from the viewpoint that the reaction between the oxidant and the sample can be uniformly conducted, 1) is most preferable.

Hereinafter, each component of the immunoassay kit (immunochromatography kit) will be described in detail.

(Extraction solution / developing solution)

The solution for extraction and the developing solution differ in their use forms as described above, but a solution of the same composition can serve as both the solution for extraction and the developing solution. The extraction solution and the developing solution include a buffer solution consisting of a buffer and a solvent (usually, water). As the buffer, a buffer which can be adjusted to a pH suitable for an intended immune reaction is used. Generally, the pH of the buffer solution is preferably 5 to 10, and in this case, a commonly used buffer solution such as a phosphate buffer solution, a Tris buffer solution or a good buffer solution may be used. As a buffer to give a pH of 6 to 8 suitable for the immune response, Good buffer such as HEPES or PIPES is preferable. In addition to the buffer, sodium chloride and the like may be added to the extraction solution and the developing solution, and known additives may be further added. Examples of the additive include a protein (for example, bovine serum albumin, casein, gelatin, etc.) for the purpose of promoting an antigen-antibody reaction or inhibiting a nonspecific reaction, a polymer compound (for example, polyethylene glycol (For example, Tween (registered trademark) 20, Triton (registered trademark) X-100 and the like), ionic surfactants (such as dextran, methylcellulose, polyvinylpyrrolidone and the like) Surfactants or polyanions (e.g., dextran sulfate, heparin, polystyrene sulfonic acid, hyaluronic acid, chondroitin sulfate, etc.) or salts thereof; Sodium azide as an antimicrobial agent, long-chain alkyl quaternary ammonium salt, and the like.

Here, when an oxidizing agent is contained in the extraction solution or the developing solution, the lower limit of the concentration of the oxidizing agent in these solutions is preferably 0.05 mM or more, more preferably 0.1 mM or more, more preferably 1 mM or more, Preferably at least 2.5 mM. However, when a peroxide group is used as the oxidizing agent, the lower limit of the oxidizing agent concentration is preferably 1 mM or more, and in the case of using glucose oxidase that produces hydrogen peroxide as the oxidizing agent, the lower limit of the oxidizing agent concentration is preferably 5 units / mL or more More preferably 10 units / mL or more, and still more preferably 100 units / mL or more. On the other hand, the upper limit of the oxidizing agent concentration is preferably 100 mM or less, more preferably 10 mM or less, still more preferably 4 mM or less, and most preferably 3 mM or less, from the viewpoints of storage stability of the solution and inhibition of the immune response to be. Further, the upper limit of the glucose oxidase is preferably 2000 units / mL or less, more preferably 1000 units / mL or less. However, when thimerosal or potassium permanganate is used as the oxidizing agent, the upper limit of the concentration of the oxidizing agent is preferably 10 mM or less, more preferably 1 mM or less, from the viewpoint of inhibition of the immune response.

(Antibody Immobilized Membrane)

The main phase of the antibody-immobilized membrane 120 is an insoluble carrier made of a porous material that functions as a chromatographic medium (immobilized phase) of immunochromatography. Examples of the porous body which is a constituent material of the antibody immobilization membrane 120 include a nitrocellulose membrane, a cellulose membrane, an acetyl cellulose membrane, a polysulfone membrane, a polyether sulfone membrane, a nylon membrane, a glass fiber, , And a nitrocellulose membrane is particularly preferably used. The moving speed of the dropped sample on the membrane 120 can be changed in accordance with the material and size of the membrane, and the speed suitable for the measurement of the object to be detected can be set.

(Sample pad)

The sample pad 140 functions as a sample supply unit, but may also serve to filter the insoluble particles in the sample as well as to receive the sample dropped after passing through the filter 200. Examples of the constituent material of the sample pad 140 include materials having uniform properties such as cellulose filter paper, glass fiber, polyurethane, polyacetate, cellulose acetate, nylon, cotton and the like.

(Labeling reagent holding pad)

The labeling reagent holding pad 130 functions as a labeling reagent holding section for holding the labeling reagent as described above. As a constituent material of the labeling reagent holding pad 130, for example, cellulose filter paper, glass fiber and nonwoven fabric, and the like, preferably glass fiber is used.

The labeling reagent held by the labeling reagent holding pad 130 is a complex of a substance that specifically binds to the detection target and a suitable labeling substance. As described above, when the object to be detected is an antibody, an antibody against a substance (antigen) or immunoglobulin molecule specifically recognized by the antibody is used as the " substance specifically binding to the detection subject " When the object is an antigenic substance, an anti-detection target antibody (preferably a monoclonal antibody) that specifically recognizes the detection target is used. On the other hand, the "label" is preferably colored particles, and examples thereof include metal particles or metal colloids such as gold, silver, platinum or copper; Metal oxide particles such as iron oxide or metal oxide colloids; Non-metallic particles such as selenium, tellurium, and sulfur; Colored latex particles including those colored with a dye or the like, and gold colloid is particularly preferable.

(Test line)

The test line 122 is located on the downstream side of the labeling reagent holding pad 130 in the antibody immobilizing membrane 120 and a substance specifically binding to the detection target is arranged as the immobilizing reagent, Function. 1, the detection unit is formed in a line shape as the test line 122, but the shape of the detection unit is not limited to this, and the shape of the detection unit may be changed depending on the shape in which the immobilization reagent is locally disposed, And may be any shape. However, the shape of the detecting portion is preferably a line shape, more preferably a line shape having a width of 0.5 to 1.5 mm. Methods for immobilizing the labeling reagent on the labeling reagent holding pad 130 and for immobilizing the reagent (such as an antibody) immobilized on the test line 122 are not particularly limited and conventionally known methods can be used. As these immobilization methods, there is a method in which a reagent to be immobilized is directly immobilized on a chromatographic medium by physical means or chemical means. The direct immobilization method includes physical adsorption or covalent bonding. In general, when the chromatographic medium is a nitrocellulose membrane or a cellulose ester membrane composed of mixed niches, physical adsorption can be carried out. In order to prevent the accuracy of the analysis from deteriorating due to nonspecific adsorption after immobilization, a blocking treatment may be carried out by a known method as necessary.

(Control line)

The control line 124 is located on the downstream side of the test line 122 in the antibody-immobilized membrane 120, and a substance that specifically binds to the labeling reagent is disposed as an immobilizing reagent. For example, when a gold colloid-labeled mouse monoclonal antibody is used as a labeling reagent, for example, if an anti-mouse IgG antibody is immobilized, it functions as a control part. When the labeling reagent moves to the control line 124, the labeling reagent is accumulated by reacting with the immobilized reagent disposed on the control line 124. As a result, it is confirmed that the reaction on the test strip 100 proceeds normally because the control line 124 exhibits color development due to the integration of the labeling reagent. 1, the control unit is also formed in a line shape as the control line 124. However, the shape of the control unit is not limited to this, and the shape of the control unit may be changed depending on the shape in which the immobilizing reagent is locally arranged, And may be any shape. However, the shape of the control portion is preferably a line shape, more preferably a line shape having a width of 0.5 to 1.5 mm.

(Absorbing pad)

The absorbing pad 150 is a member having a function of absorbing and removing unreacted labeling substances or the like that are not insolubilized in the detecting portion while the sample or the developing solution is absorbed by moving the chromatographic medium. The constituent material of the absorbent pad 150 is not particularly limited, and for example, an absorbent material such as cellulose filter paper, nonwoven fabric, cloth, or cellulose acetate is used.

These members may be a single porous member sheet even if the sample can be moved by the capillary phenomenon and the sample pad, the marking reagent holding pad, the test line, the control line, and the absorbent pad may be attached to the substrate.

(Filtration filter)

The filtration filter 200 is installed in a container such as a fountain container or a microtube, and is used to filter an extract containing a sample and to remove microfined solids (impurities). By using the filtration filter, the solid matter (impurities) which interfere with the inspection can be removed by filtration. The constituent material of the filtration filter is not particularly limited as long as it is inactive with respect to the object to be detected. Examples of the constituent material include porous materials such as a malt filter (filter), a filter paper and a resin sintered filter, fibers such as glass fiber, Or the like, and it is preferable to use a malt filter or a resin sintered filter having a predetermined pore diameter.

The method of holding the oxidizing agent in the respective parts such as the filtration filter, the sample feeding part, and the labeling reagent holding part in contact with the sample may be any known method. For example, a filtration filter, a sample feeding part, A method in which an oxidizing agent is added to a solution, a buffer solution or a cleaning solution used in the production of a holding part and the like, and the solution is applied, dropped, impregnated or sprayed on each member and dried and kept by freeze drying or wind drying have.

When each member retains the oxidizing agent, the lower limit of the amount of the oxidizing agent (impregnated amount) to which the sample comes into contact per test is preferably 0.1 n mol / test or more, and more preferably 0.5 n mol / test or more. In particular, when the sample pad or the filtration filter maintains the oxidizing agent, the lower limit of the impregnation amount is preferably 0.5 nmol / test or more, more preferably 1.0 nmol / test or more, particularly preferably 2.0 nmol / Or more. By impregnating the oxidant with the pads, the amount of the oxidant in the pads can be controlled so as to be able to react with the sample in an amount required for the measurement, and the amount of the oxidizer can be effectively suppressed.

Example

Hereinafter, preferred embodiments of the present invention will be described with reference to Examples, but the technical scope of the present invention is not limited to the following Examples.

[Example 1: inhibition of non-specific reaction by addition of various oxidizing agents]

(Manufactured by EIKEN CHEMICAL CO., LTD.) Which is an immunochromatographic kit for detecting norovirus having the structure shown in Fig. 1 , And the effect of the present invention (inhibition of nonspecific reaction) by the use of an oxidizing agent was confirmed. The test strip of ImmunoCatch (TM) -noR has a constitution in which the labeling reagent holding pad 130 is disposed on the antibody immobilizing membrane 120 made of a nitrocellulose membrane, (130) is impregnated with an anti-norovirus GI mouse monoclonal antibody-binding gold colloid solution and an antaroor virus GII mouse monoclonal antibody-binding gold colloid solution. In addition, the test line 122, which is a detection unit, is immobilized with an anti-norovirus GI mouse monoclonal antibody and an anti-norovirus GII mouse monoclonal antibody as immobilized antibodies. An anti-mouse IgG rabbit antibody is immobilized on the control line 124, which is a control unit. In addition, the solution for extraction with ImmunoCatch (R) -Ro is composed of HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer (pH 7.0), sodium azide ≪ / RTI > As a result, the minimum detection sensitivity to Immunocake (R) -no was 2.5 ng / mL for Norovirus GI and 0.25 ng / mL for Norovirus GII.

In this embodiment, first, oxidizing agents of the kind and concentration shown in Table 1 below are added (Example) or not (Comparative Example) to the extraction solution attached to Immunocake (R) , A solution for extraction was prepared.

To each of the extraction solutions (1 mL) prepared above, fecal samples (20 to 50 mg) collected from humans were added, stirred, and suspended. Here, as the fecal samples, there were used three kinds of positive specimens which confirmed the presence of norovirus as an object to be detected by the RT-PCR method, false positive specimens which confirmed the absence of norovirus, and negative specimens.

Next, the suspended sample was filtered with a filtration filter attached to an ImmunoCatch (R) - Noro, and then dropped three times (90 to 135 μL) into the sample well (sample feeding section). After dropwise addition, the mixture was allowed to stand at room temperature for 15 minutes, and then the line of the detection portion was confirmed. Specifically, the intensity of the reflected light of the test line was measured using an immunochromatograph reader C10066-10 (manufactured by Hamamatsu Photonics K.K.). The results are shown in Table 1 below. On the other hand, the numerical value [%] shown in Table 1 indicates the relative value when the reflected light intensity of the sample (false positive sample or positive sample) showing the color development rate (calculated by the following formula) to be. In addition, "-" in Table 1 indicates negative (test line is not colored). In addition, the reflected light intensity of the false positive sample to which the oxidizing agent was not added was 48.0 mAbs, and the reflected light intensity of the positive sample was 335.7 mAbs. In addition, the same measurement was carried out for the extraction solution to which the oxidizing agent was added (or not added), but no reflected light was detected for any extraction solution.

(%) = (Reflected light intensity (mAbs) in the detection portion of each sample / reflected light intensity (mAbs) in the detection portion in the absence of the oxidizing agent) x 100

Values of the oxidation-reduction potentials of some of the oxidizing agents used in this example are shown in Table 1 below. Here, the oxidation-reduction potential of the oxidizing agent was measured by the following method.

Measuring method of oxidation-reduction potential: The oxidizing agent is dissolved in purified water and 10 mL of a 10 mM solution is prepared. The redox potential of the obtained solution was measured using a 3.33 mol / L KCl-Ag / AgCl electrode.

It can be seen from the results shown in Table 1 that the occurrence of false positives can be significantly suppressed by adding various oxidizing agents to the extraction solution and bringing the fecal sample into contact with the oxidizing agent in the upstream of the detection part. This is thought to be due to suppression of non-specific reactions due to any components contained in the fecal samples. On the other hand, since the color development rate of the positive specimen was not significantly lowered by the addition of the oxidizing agent, it was confirmed that the advantage of the immunoassay that it would be useful for rapid examination and diagnosis was not undermined.

It has also been found that the effect of suppressing the progress of nonspecific reaction and generation of false positives by the addition of an oxidizing agent is expressed depending on the concentration of the oxidizing agent. It is possible to exhibit the above-described effect even with an extremely low concentration of about 0.1 mM in many oxidants. On the other hand, depending on the type of the oxidizing agent (for example, thimerosal, potassium permanganate), the coloring rate of the positive specimen decreases with the increase in the concentration of the oxidizing agent, and so- Is limited to a few mM at most, it has been found that it is possible to prevent both false positives and false negatives.

[Example 2: Suppression of non-specific reactions by addition of various iodates as oxidizing agents]

The effect of suppressing the occurrence of false positives by addition of an oxidizing agent was confirmed by the same method as in Example 1, except that various iodic acid salts were used as oxidizing agents. The results are shown in Table 2 below. The values of the oxidation-reduction potentials are shown in Table 2 below for some of the oxidizing agents used in this example (the method of measuring the oxidation-reduction potential is the same as described above).

As shown in Table 2, the same results as those shown in Table 1 were also obtained when various iodates were added as an oxidizing agent. From these results, it was confirmed that the effect of the present invention in suppressing the occurrence of non-specific reactions and the generation of false positives according to the same was demonstrated even when various iodates were used as oxidizing agents.

[Example 3: Suppression of nonspecific reaction by addition of oxidizing agent to each part of the test strip or the filter filter]

In the first and second embodiments described above, the oxidizing agent is added to the solution for extracting the sample. In this embodiment, the oxidizing agent (potassium iodate) is not added to the solution for extraction but a part of the constituent elements of the test strip or the filtering filter , It was confirmed whether or not the same effect (inhibition of non-specific reaction) as described above was exhibited.

(Confirmation of the addition effect of oxidizing agent to the labeling reagent holding pad)

Potassium iodate was added to the anti-NV-GI mouse antibody binding gold colloid solution and the anti-NV-GII mouse antibody binding gold colloid solution, and the resulting solution was impregnated with a labeling reagent holding pad (glass fiber filter paper) The labeling reagent holding pad of this example containing the oxidizing agent of each impregnated amount shown in Table 3 was obtained. The supported concentration of the labeling reagent in the obtained labeling reagent holding pad was the same as that in the Immunocat (registered trademark) - Noro. Using the labeling reagent holding pad thus obtained, the intensity of reflected light in each specimen was measured by the same method as in Examples 1 and 2, except that no oxidizing agent was added to the extraction solution, And the color development rate was calculated. The results are shown in Table 3 below.

(Confirmation of the effect of addition of oxidizing agent to the sample pad)

Potassium iodate was added to the purified water, and the obtained solution was impregnated with a sample pad (cellulose membrane) and dried to obtain a sample pad of this embodiment containing oxidizers of the respective impregnated amounts shown in Table 3 below. Using the sample pads thus obtained, the reflected light intensity in each sample was measured by the same method as in the above-described Example 1 and Example 2, except that the oxidizing agent was not added to the extraction solution, and the color development rate Respectively. The results are shown in Table 3 below.

(Confirmation of effect of addition of oxidizing agent to filtration filter)

Potassium iodate was added to the purified water, the obtained solution was impregnated into the cellulose membrane, and dried to obtain an oxidizing agent-containing cellulose membrane containing the oxidizing agent of each impregnated amount shown in Table 3 below. Except that the obtained oxidizing agent-containing cellulose membrane was overlaid on a filtration filter attached to a specimen extraction container with an ImmunoCatch (TM) -no to perform filtration of the extract solution and no oxidizing agent was added to the extraction solution. The intensity of reflected light in each specimen was measured by the same method as in Example 2, and the color development rate was calculated. The results are shown in Table 3 below. Incidentally, the impregnated amount shown in Table 3 below is the amount of potassium iodate contained in each member, which is an amount that reacts with the sample per one test.

As shown in Table 3, the suppression of the non-specific reaction by the addition of the oxidizing agent according to the present invention and the suppression effect of the occurrence of the false positive according to the present invention are not limited to the solution for extraction, It was confirmed that even if it was included, it was demonstrated to be likewise exerted.

The present application is based on Japanese Patent Application No. 2015-169742 filed on August 28, 2015, the disclosure of which is incorporated by reference in its entirety.

10: kit for immunochromatography 100: test strip
110: Plastic adhesive sheet
120: Antibody-immobilized membrane (chromatographic medium)
122: test line (detection unit) 124: control line (control unit)
130: Label reagent holding pad (label reagent holding section)
140: sample pad (sample feeding part) 150: absorbing pad
200: Filtration filter 300: Extraction solution or developing solution

Claims (18)

A reagent composition for use in an immunoassay using a fecal sample, the reagent composition comprising an oxidizing agent. The method according to claim 1, wherein the oxidizing agent is one or two selected from the group consisting of halogen oxoates, salts of transition metal complexes, peroxides, organic mercury compounds, permanganates, chromates, oxydases, nitrites, Lt; / RTI > The method according to claim 1 or 2, wherein the oxidizing agent is one or more selected from the group consisting of iodate, periodate, ferricyanide, peroxide, hydrogen peroxide, thimerosal, permanganate and glucose oxidase At least two reagent compositions. 4. The reagent composition according to any one of claims 1 to 3, wherein the oxidizing agent is at least one selected from the group consisting of potassium iodate, potassium periodate and potassium ferricyanide. 5. The reagent composition according to any one of claims 1 to 4, wherein the immunoassay is an immunochromatographic method. A solution or solution for extraction of a fecal sample, comprising the reagent composition according to any one of claims 1 to 5. The extracting solution or solution according to claim 6, wherein the concentration of the oxidizing agent is 0.05 to 100 mM. A sample supply unit,
A chromatographic medium located on the downstream side of the sample supply unit,
A labeling reagent holding part which is located in a part of the chromatographic medium and in which the labeling reagent is eluted,
A detection reagent containing a immobilized antibody, which is located on the downstream side of the label reagent holding portion as a part of the chromatographic medium,
A test strip,
A test strip comprising the reagent composition of any one of claims 1 to 5. 9. The test strip according to claim 8, wherein the reagent composition is contained in any portion from the sample supply portion to the upstream of the detection portion in the chromatographic medium. A filter filter for filtering a fecal sample, comprising the reagent composition according to any one of claims 1 to 5. A method for detecting an object to be detected in a feces sample, comprising at least one of the extraction solution or the developing solution according to claim 6 or 7, the test strip according to claim 8 or 9, and the filtration filter according to claim 10 Or immunoassay for quantification. A sample supply unit,
A chromatographic medium located on the downstream side of the sample supply unit,
A labeling reagent holding part which is located in a part of the chromatographic medium and in which the labeling reagent is eluted,
A detection reagent containing a immobilized antibody, which is located on the downstream side of the label reagent holding portion as a part of the chromatographic medium,
A method for detecting or quantifying an object to be detected in a fecal sample by immunoassay using a test strip containing a test strip,
Use of the reagent composition according to any one of claims 1 to 5. 13. The method of claim 12,
A step of supplying a sample or a filtrate thereof and a mixture of an extraction solution or a developing solution to the sample supply section;
And detecting the complex of the detection target and the labeling reagent in the detection section,
Further comprising the step of contacting the mixture with the oxidant upstream of the detecting portion. 14. The method according to claim 13, wherein the step of bringing the mixture into contact with the oxidizing agent is performed upstream of the labeling reagent holding unit. The method according to claim 13 or 14, wherein the extracting solution or the developing solution contains the oxidizing agent, and the step of bringing the mixture into contact with the oxidizing agent is carried out by mixing the sample with the extracting solution or the developing solution Done. 16. The method according to claim 15, wherein the concentration of the oxidizing agent in the extraction solution or the developing solution is 0.05 to 100 mM. 17. The method according to any one of claims 12 to 16, wherein the object to be detected is a virus, a bacterium or an occlusive blood. A method for inhibiting a nonspecific reaction in an immunoassay, which comprises contacting the reagent composition according to any one of claims 1 to 5 with a fecal sample.

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