JP4571999B1 - Method for suppressing false positives derived from specimens - Google Patents

Abstract

An antigen detection method capable of obtaining a more reliable result is provided.
A first antibody specific to an antigen to be detected and labeled with a detectable substance, and a second antibody specific to the antigen to be detected and immobilized on a substrate And a sample capable of containing the antigen in the presence of an N-substituted maleimide compound, and determining the presence or absence of the antigen by detecting a signal derived from a label contained in the obtained immune complex. A method for detecting an antigen.
[Selection figure] None

Description

  The present invention relates to an antigen detection method. Specifically, the present invention relates to a method for suppressing a false positive reaction in an antigen detection method using an antigen-antibody reaction.

  In recent years, consumers' awareness of food safety and safety has increased greatly, and food allergy is one example. Under the Food Sanitation Law, it is recommended to indicate that ingredients that are likely to cause food allergies are included from the viewpoint of preventing health hazards caused by food allergies to consumers. In particular, seven items of “specific raw materials” of shrimp, wheat, buckwheat, egg, milk and peanuts, which are highly necessary to display in consideration of the number and severity of food allergies, include these specific raw materials. Manufacturers etc. are obliged to display this.

  In addition, by displaying "Contains specified raw materials" or "Does not include specific raw materials", it is possible for consumers to accept their products with a sense of security. Is also beneficial to manufacturers.

  In processed foods produced by mixing various raw materials, it is complicated to trace the history of individual raw materials, and it is desirable that the obtained products can be directly inspected. In addition, even though it is not used as a raw material, it may be mixed (contaminated) in the production line. Therefore, from the viewpoint of product maintenance and prevention of sudden accidents, it is necessary to quickly and easily measure whether or not a specific raw material is contained in food even at the production site. Furthermore, in consideration of the safety of protecting consumers, the detection requires high accuracy.

  In order to improve detection accuracy, for example, in Patent Document 1, when allergen is extracted from food, sodium dodecyl sulfate (hereinafter referred to as “SDS”) as a surfactant and 2-mercaptoethanol as a reducing agent are used. And a method for efficiently extracting an allergen contained in a specific raw material.

  Patent Document 2 discloses a method of suppressing a false positive reaction that can occur between 2-mercaptoethanol and gold colloid used in the method of Patent Document 1. In this method, sulfite is used instead of 2-mercaptoethanol.

  As mentioned above, some methods are known to those skilled in the art as prior art, but at present, consumers can ingest food more safely, and producers can grasp the state of products more accurately. Therefore, it is desired to develop a further technique that can obtain more accurate results.

JP 2008-298664 A JP 2009-133712 A

  In view of the above situation, an object of the present invention is to provide an antigen detection method capable of obtaining a more accurate result.

The above object is achieved by the present invention as follows:
A first antibody specific to the antigen to be detected and labeled with a detectable substance; a second antibody specific to the antigen to be detected and immobilized on a substrate; and the antigen An antigen detection comprising: reacting a sample that may contain an N-substituted maleimide compound in the presence of the N-substituted maleimide compound; and detecting a signal derived from a label contained in the obtained immune complex to determine the presence or absence of the antigen. Method.

  The present invention provides an antigen detection method capable of obtaining more accurate results.

It is a schematic diagram which shows an example of the immunochromatography apparatus according to this invention. It is a schematic diagram which shows an example of the immunochromatography apparatus according to this invention. It is a schematic diagram which shows an example of the immunochromatography apparatus according to this invention.

1. Definitions “Subject” may be a substance that may contain an antigen to be detected or a substance suspected of containing an antigen to be detected, such as meat, seafood, eggs, milk, cereals, legumes. , Moss, vegetables, wild plants, seaweed, seeds, fruits, herbs and their processed foods, cosmetics, pharmaceuticals, for example, substances present in the environment such as tap water, lake water, seawater and sludge, living organisms such as blood and biopsies It may be a derived substance. “Sample” generally refers to at least a portion collected from a subject. It is possible to prepare a sample to be actually brought into the test from such a specimen.

  A “sample” is a substance derived from a subject and brought directly into a reaction system for performing the method according to the present invention. Steps such as pulverization, homogenization, separation, extraction and dilution may be performed in advance so as to be suitable for application to the method. Here, the “reaction system” refers to a place where a target reaction is performed, and may be, for example, a reaction vessel, a test tube, a flow path, and a substrate surface.

  “Antigen” refers to any component capable of immunologically specific binding to an antibody, and is a substance to be detected by this method. Preferred antigens include, for example, proteins, polypeptides and substances containing them.

  The “antibody” may be an antibody capable of recognizing and binding a specific substance as an antigen, and may be IgG, IgM, IgA, IgD and IgE and fragments thereof, and is a monoclonal antibody. Or a polyclonal antibody. In the method, the first antibody and the second antibody used may be the same or different.

  As used herein, “specific” indicates a property that allows an antibody to bind immunologically and selectively to an antigen to be detected.

  The “detectable substance” may be any substance that can confirm the presence of the substance by generating any known detectable signal such as color development, luminescence, and fluorescence. . Also, a detectable substance may produce a detectable signal by itself, or may produce a detectable signal by encountering an auxiliary means that assists in producing the signal. Such auxiliary means may be, for example, substances such as enzyme substrates and light such as excitation light and laser light used in treatments such as enzyme treatment and / or excitation light irradiation. A detectable substance may be understood as a “labeling substance”.

  Examples of detectable substances may be colored microparticles and specific enzymes. The “colored microparticle” generally only needs to be a detectable substance for labeling to detect an antibody. For example, a metal colloid such as a gold colloid, a silver colloid, a platinum colloid, and a composite colloid thereof, for example, Any latex particles such as colored latex may be used. An example of a specific enzyme may be horseradish peroxidase.

2. Antigen detection method One aspect of the present invention is specific to an antigen to be detected and labeled with colored microparticles, and is specific to the antigen to be detected and immobilized on a substrate. A second antibody and a sample that can contain the antigen are reacted in the presence of an N-substituted maleimide compound, and a signal derived from a label contained in an immune complex of the antigen, the first antibody, and the second antibody is detected. To detect the presence or absence of the antigen.

  A feature of the present invention is that a sample is reacted with a first antibody and a second antibody labeled with a detectable substance in the presence of an N-substituted maleimide compound. Thereby, non-specific binding between the detectable substance and the thiol group (that is, SH group) contained in the sample is suppressed. Since the SH group has a property of being chemically adsorbed to a detectable substance, particularly a gold surface, a false positive reaction occurs due to the SH group contained in the sample. However, the presence of an N-substituted maleimide having an SH group modifying action can accurately reflect only the antigen-antibody reaction to be specifically bound by the first antibody and the second antibody.

The N-substituted maleimide compound used is a compound of formula I

Here, n is an integer of 1 to 30, m is an integer of 2 to 30, and C _n H _m is one or more hetero groups such as N, S and May form one or more cyclic structures optionally containing O and / or the like, or may be a straight chain or branched chain, and one or more than one H is O, S, Br, Cl, it may be substituted by like NH _2, OH and / or NO _2. The cyclic structure may be a 3-6 membered ring.

  Examples of N-substituted maleimide compounds are N-methylmaleimide, N-ethylmaleimide, N-phenylmaleimide, 1,2-bis (maleimide) ethane, 1,6-bismaleimide hexane, 3-maleimidopropionic acid, 4,4 '-Bismaleimide diphenylmethane, 6,7-methylenedioxy-4-methyl-3-maleimidocoumarin, bis (3-ethyl-5-methyl-4-maleimidophenyl) methane, N, N'-1,3-pheny Rangemaleimide, N, N′-1,4-phenylenedimaleimide, N, N′-1,4-phenylenedimaleimide, N- (1-pyrenyl) maleimide, N- (2,4,6-trichlorophenyl) Maleimide, N- (4-aminophenyl) maleimide, N- (4-nitrophenyl) maleimide, N-bromomethyl-2,3- Dichloromaleimide, N-cyclohexylmaleimide, 3-maleimidobenzoic acid N-succinimidyl, 3-maleimidopropionate N-succinimidyl, 4-maleimidobutyrate N-succinimidyl, 6-maleimidohexanoic acid N-succinimidyl, N- [4- (2 -Benzimidazolyl) phenyl] maleimide, N- (9-acridinyl) maleimide and the like, but are not limited thereto. Also preferred are N-methylmaleimide and N-ethylmaleimide. The N-substituted maleimide compound can be obtained from, for example, Tokyo Chemical Industry Co., Ltd. and Wako Pure Chemical.

  When an N-substituted maleimide compound is brought into an antigen-antibody reaction system, when a sample is prepared from a specimen, for example, it is added to a solution for performing the extraction and dilution, and the sample and the first antibody are added. It may be performed by adding at the time of mixing and / or by adding at the time of mixing the sample, the first antibody, and the second antibody. Since the N-substituted maleimide compound does not need to be removed thereafter, it may be present in a system in which the sample reacts with the first antibody and the second antibody. When the N-substituted maleimide compound is added prior to the antigen-antibody reaction, for example, when the sample is prepared, the SH group as a contaminant that does not contribute to the antigen-antibody reaction contained in the sample is added during the antigen-antibody reaction. It may exist in the form of a complex with a substance having it, for example, a protein or polypeptide.

  The sample may be prepared from the specimen as follows, for example. First, an extract is added to a sample collected from a subject and homogenized. Next, this is stirred, heated, and then subjected to rough heat. Thereafter, the mixture is centrifuged and filtered, and the filtrate is collected as a sample extract. The obtained sample extract is diluted with a diluent so that the antigen contained therein has a concentration suitable for the reaction.

  For example, in order to prepare a sample using food as a specimen, the following operation may be performed. Grind and homogenize food. Add the extract to the homogenized specimen. Stir with a vortex mixer. Centrifuge. Filter and collect the filtrate. The collected filtrate is diluted with a diluent as necessary. What is necessary is just to use the obtained solution as a sample.

The extract used here may contain a reducing agent such as Na ₂ SO ₃ , a surfactant such as SDS, Tween 20, NP-40 and Triton X-100, and is known per se depending on the antigen to be detected. Any of these extracts may be used. When the N-substituted maleimide compound is present in the extraction step, the N-substituted maleimide compound may be added to the extract. When an N-substituted maleimide compound is added to the extract, the concentration is about 0.01% to about 10%, preferably about 0.1% to about 5%, or more preferably about 0.4% to about 2%. Should be included.

  The diluent used herein may include a buffer such as Tris, a protein such as bovine serum albumin (BSA), a surfactant such as Tween 20, a chelating agent such as EDTA, and a preservative such as Procrine 300. The pH value may be 6-9. When the N-substituted maleimide compound is present in the dilution step, the N-substituted maleimide compound may be added to the diluted solution. Further, an N-substituted maleimide compound may be contained in both the extract and the diluent. When an N-substituted maleimide compound is added to the diluent, the concentration is about 0.01% to about 10%, preferably about 0.1% to about 5%, more preferably about 0.5% to about 2%. Should be included.

  Addition of an N-substituted maleimide compound may be performed during the reaction. In that case, it may be added to the sample, added at the time of mixing the sample and the first antibody, or added at the time of mixing the sample, the first antibody and the second antibody. However, it is preferable that the N-substituted maleimide compound is present in the sample preparation step in order to sufficiently bind the SH group of the contaminant.

  The reaction of the sample, the first antibody, and the second antibody may be performed simultaneously. In this case, the sample, the first antibody and the second antibody are simultaneously present in one reaction system, the antigen, the first antibody and the second antibody contained in the sample are brought into contact with each other, and their immune complexes What is necessary is just to form a body. Or you may react in steps. That is, after the sample and the first antibody are contacted to obtain the antigen and the first immune complex contained in the sample, the first immune complex and the second antibody are contacted to obtain the second What is necessary is just to obtain an immune complex. Contact in this case may be performed by mixing in the liquid phase in the container, or may be performed by movement of the liquid phase by development in a reaction system in which development is obtained by capillary action.

  The method of the present invention may be classified into antigen detection methods such as immunochromatography and ELISA (enzyme-linked immunosorbent assay).

  When used in an immunochromatography method, it may be used in combination with an immunochromatography apparatus as described later.

  When used in an ELISA method, a first antibody that is specific to the antigen to be detected and labeled with a detectable substance, and a second antibody immobilized on a substrate such as a 96-well plate, for example The presence or absence of the antigen may be determined by reacting a sample that can contain the antigen in the presence of an N-substituted maleimide compound and detecting a signal derived from a label contained in the obtained immune complex. In the ELISA method, the detectable substance may be, for example, horseradish peroxidase.

  According to the method of the present invention, it is possible to suppress the occurrence of a false positive reaction in an antigen detection method using an antigen antibody.

3. Immunochromatography kit One aspect of the present invention is a substrate, a developing unit for developing a reaction component on the substrate, an upstream of the developing unit, specific to the antigen to be detected, and colored fine particles An immunochromatography apparatus comprising: a first antibody labeled with a second antibody specific to the antigen immobilized in an observable region downstream of the development portion;
An N-substituted maleimide compound as a false positive reaction inhibitor;
An extract and / or a diluent,
Is an immunochromatography kit.

  The N-substituted maleimide compound is dissolved in an appropriate solvent, such as water, Tris buffer, hydrochloric acid buffer, borate buffer, acetate buffer, citrate buffer, tartrate buffer, and ammonia buffer, in an extract. It may be provided in a dissolved form and / or dissolved form in a diluent. The extract and diluent may be the composition described above.

  The immunochromatography kit may further include a container for preparing a sample, and may include a positive control and / or a negative control.

(1) Immunochromatography apparatus (a) Housing type FIGS. 1 and 2 show an example of an immunochromatography apparatus. FIG. 1 is a cross-sectional view of the immunochromatography apparatus of FIG. 2 along line kk ′. FIG. 2 is a plan view of the immunochromatography apparatus.

  The immunochromatographic apparatus 1 includes a base 2, a developing part 3 for developing a reaction component on the base 2, an outer wall 4 constituting the developing part 3, an adding part 5 opened upstream of the developing part 3, and an addition Use the first antibody 6 conjugated downstream from the position of the part 5, the second antibody 7 immobilized further downstream, and the region where the second antibody 7 is immobilized. A window 8 is provided for a person to observe. Here, the upstream in the deployment unit 3 refers to the deployment start point side, and the downstream refers to the deployment end point side. The development is started by adding the sample to the adding section 5. Here, “attachment” refers to a state in which the first antibody 6 is attached to a desired location, and the attachment state is, for example, an external influence such as contact with moisture or a change in the environment. This is an attachment that can be eliminated by the first antibody 6 into the environment.

  The expansion | deployment part 3 should just be comprised by the expansion | deployment member 9 and / or the flow path 10 which can obtain a capillary phenomenon. The material constituting the deployment member 9 may be any material that can obtain a known capillary phenomenon, such as paper, resin, and porous material.

  The sample is added to the developing unit 3 from the adding unit 5. The sample added from the addition part 5 is developed downstream by capillary action. In order for the added sample to move at an appropriate speed in the downstream direction of the developing unit 3, the absorber 12 may be disposed at a location corresponding to the adding unit 5.

  The added sample is first contacted with the conjugated first antibody 6. The conjugated first antibody 6 is released from the solid phase to the liquid phase by contact with the liquid. The attachment of the first antibody 6 is known per se to any surface constituting the development portion 3, that is, the inside or the surface of the development member 9, the inner wall of the flow path 7, or the inner surface of the base body 2. This is achieved by releasably attaching the liquid by any means.

  When the sample contains an antigen, the antigen specifically binds to the first antibody 6 to form a first immune complex. The first immune complex is developed and specifically binds to the second antibody 7 to form a second immune complex. Here, the second antibody 7 is any known per se with respect to any surface constituting the developing portion 3, that is, the inside or the surface of the developing member 9, the inner wall of the flow path 10, or the inner surface of the base 2. It is fixed by the means. The region where the second antibody 7 is immobilized is a region that can be observed through the window 8. By observing the signal from the detectable substance contained in the second immune complex from the window 8, the presence or absence of the antigen in the sample is determined. Therefore, the window 8 functions as a determination unit. In view of ease of observation, the second antibody 7 is preferably immobilized inside or on the surface of the developing member 9. The signal from the detectable substance contained in the second immune complex may be performed, for example, by detecting color development, luminescence, and fluorescence, depending on the nature of the substance. FIG. 2 shows a state in which the signal 21 from the detectable substance is observed from the window 8.

  As the immunochromatography apparatus, an immunochromatography apparatus known per se may be used.

  With the immunochromatography kit of the present invention, it is possible to detect antigens quickly and easily with high accuracy.

(B) Stick type for immersion One example of a further immunochromatographic apparatus is shown in FIG. This example is an example of an immersion stick type device. Please refer to FIG.

  The immunochromatographic apparatus 31 includes a developing member 32 that can obtain a capillary phenomenon, an immersion part 33 provided on the development start point side of the developing member 32, and a first antibody 6 provided downstream of the immersion part 33. And the immobilization region 35 of the second antibody 7 provided downstream thereof, and the region 36 provided further downstream thereof.

  In the inspection, the immersion part 33 is directly immersed in the sample. Thereafter, the sample develops by capillary action. When the sample contains an antigen, the antigen specifically binds to the first antibody 6 in the region 34 to form a first immune complex. The first immune complex is developed and specifically binds to the second antibody 7 to form a second immune complex. By observing a signal from a detectable substance contained in the second immune complex, the presence or absence of an antigen in the sample is determined. An absorption region for absorbing the liquid that has moved due to the expansion may be provided in the region 36 and / or downstream thereof. Although not shown, a confirmation unit that can confirm that the inspection has been performed correctly may be provided in the region 36. Furthermore, an area for the tester to hold the immunochromatography apparatus 31 (that is, a holding area) may be provided outside the area 36. The holding region is preferably provided outside the development end point. Thereby, the possibility of artificially affecting the inspection can be suppressed.

(2) Immunochromatography kit for detecting a specific raw material The immunochromatography kit may be used for confirming whether a specific raw material is used in a food or whether a specific raw material is included.

The immunochromatography kit in that case is
A base, a development part for developing a reaction component on the base by capillary action, a first part that is combined with the upstream of the development part, is specific to a specific food-derived raw material, and is labeled with a colloidal gold An immunochromatography apparatus comprising: the first antibody; and a second antibody specific to the food-derived specific raw material immobilized in an observable region downstream of the development part;
A diluent containing an N-substituted maleimide compound as a false positive reaction inhibitor and a surfactant, reducing agent, buffer, chelating agent and / or preservative;
An immunochromatography kit comprising

  Preferred food-derived specific raw materials are, for example, eggs, milk, wheat, buckwheat and peanuts. These specific raw materials are detected as antigens. The antigen to be detected may be, for example, ovalbumin in eggs, casein in milk, gliadin in wheat, buckwheat soluble protein in buckwheat, and peanut soluble protein in peanut. Examples of the first antibodies against these are colloidal gold-labeled anti-hen egg albumin polyclonal antibody for eggs, colloidal gold-labeled anti-casein polyclonal antibody for milk, colloidal gold-labeled anti-gliadin polyclonal antibody for wheat, and colloidal gold-labeled anti-buckwheat protein for buckwheat. In the case of a polyclonal antibody, peanut is a colloidal gold labeled anti-peanut protein polyclonal antibody. As a method for producing these antibodies, any method known per se may be used.

  Examples of preferred second antibodies are, like the above-mentioned first antibody, anti-chicken ovalbumin polyclonal antibody in eggs, anti-casein polyclonal antibody in milk, anti-gliadin polyclonal antibody in wheat, anti-buckwheat protein polyclonal antibody in buckwheat, In peanuts, it is an anti-peanut protein polyclonal antibody, but is not limited thereto. The first antibody and the second antibody included in one immunochromatography apparatus may be the same antibody or different.

  The antibody according to the present invention may be a polyclonal antibody or a monoclonal antibody. Monoclonal antibodies are selected from hybridomas obtained by fusing spleen cells of immunized mice with parental cells for cell fusion such as myeloma cell lines using techniques known to those skilled in the art. Then, the fused cells are cultured in vitro or in vivo, and a monoclonal antibody having higher specificity than this culture mixture can be collected.

  Further, as the antibody of the present invention, in addition to the polyclonal antibody and the monoclonal antibody as described above, a reactive fragment of the antibody which can be obtained by enzymatic digestion of those antibodies can also be used. Examples of such antibody fragments include Fab fragments, Fab ′ fragments, F (ab ′) 2 fragments, F (v) fragments, H chain monomers or dimers, L chain monomers or dimers, one H chain and one The dimer which consists of the L chain of this is contained. The fragment can be obtained, for example, by digesting a complete antibody with a protease such as pepsin or papain, or by treating with a reducing agent as necessary after digestion. The H chain and L chain monomers can also be obtained by separating the purified chain after treating the complete antibody with a reducing agent such as dithiothreitol. Each of the first antibody and the second antibody may be any of the polyclonal antibody, the monoclonal antibody or a fragment thereof, or two or more from the group consisting of the polyclonal antibody, the monoclonal antibody and a fragment thereof. Any combination selected in (1) may be used. The combination of the first antibody and the second antibody may be any combination selected from the group consisting of the polyclonal antibody, the monoclonal antibody, and fragments thereof.

  With the immunochromatography kit of the present invention, it is possible to reliably and quickly detect an antigen.

4). False positive reaction inhibitor A further aspect of the present invention is a false positive reaction inhibitor containing an N-substituted maleimide compound as an active ingredient. The false positive reaction inhibitor is used in the antigen detection method as described above. Particularly preferably, it is used in a method of detecting an antigen by specific binding to an antibody labeled with colored fine particles such as colloidal gold labeled antibody and latex particles in an immunochromatography apparatus.

  The N-substituted maleimide compound may be provided dissolved in the diluent and extractant described above, or other solvents such as water, Tris buffer, hydrochloric acid buffer, borate buffer, acetate buffer. Alternatively, it may be provided in a state dissolved in a citrate buffer, a tartrate buffer, an ammonia buffer, or the like.

[Example]
Hereinafter, the false positive reaction inhibitory effect of the N-substituted maleimide compound in the antigen detection method will be described with an example.

Example 1 Suppression of false positive reaction ・ Preparation of standard products According to the method described in the 2004 “Food Testing and Expense Expense Report”, “Development of Standards for Food Inspection Methods Containing Allergic Substances”, By replacing% SDS with 0.6% SDS and 0.5% 2-ME with 0.1M Na ₂ SO ₃ , eggs, milk, wheat, buckwheat and peanut standards were prepared.

Preparation of extract 0.6% SDS, 0.1M Na ₂ SO ₃ , 0.15M Tris-HCl (pH 7.4), 0.1% BSA, 0.04% Tween, 2 mM EDTA and 0.0025% An extract containing proclin 300 was prepared.

Extraction 19 mL of the extract was added to 1 g of homogenized rice and stirred for 30 seconds with a maximum output vortex mixer. After heating in a boiling water bath for 10 minutes, after removing the rough heat, it was centrifuged at 3000 × g for 20 minutes at 25 ° C. (BEKKMANCOULTER, Avanti J-HC). The supernatant was collected and filtered through 5A filter paper (ADVANTEC) to obtain a sample extract. Moreover, the same operation was performed on soybeans, barbs, wah, acne, lycrisp (rye), barley malt, and wheat.

-Preparation of diluent As a control diluent, a diluent containing 0.15 M Tris-HCl (pH 7.4), 0.1% BSA, 0.04% Tween, 2 mM EDTA and 0.0025% proclin300 was prepared.

  In addition, as N-ethylmaleimide (hereinafter referred to as NEM) addition dilutions, 1% NEM, 0.15 M Tris-HCl (pH 7.4), 0.1% BSA, 0.04% Tween, 2 mM EDTA and 0 A dilution containing 0025% proclin 300 was prepared. Similarly, NEM addition dilutions containing 0.3% and 0.5% NEM were prepared.

-Dilution 250 ng / mL of each standard product or each sample extract was added to the control diluent and NEM-added diluent at a ratio of 900 μL to obtain a sample for use in the reaction with the antibody.

・ Determination 200 μL of the obtained sample was a Morinaga specific raw material immunochromatography kit, Nanotrap (registered trademark) egg (ovalbumin), Nanotrap (registered trademark) milk (casein), Nanotrap (registered trademark) wheat (gliadin) The reaction was started by dripping onto a dropping part of a test stick provided in Nanotrap (registered trademark) buckwheat (buckwheat soluble protein) or Nanotrap (registered trademark) peanut (peanut soluble protein). The determination was made after 15 minutes by confirming the presence or absence of the determination line. Regarding the judgment line, “−” was negative, “±” was weakly positive, and “+” was positive.

·result

  From the above results, it was found that the false positive reaction of rice and other cereals was suppressed by adding NEM. The false positive of soybean was also reduced to about 30% by adding NEM. The false positive in the antigen detection method using gold colloid is considered to be caused by a substance having an SH group present in each sample extract chemically bound to the gold colloid and aggregated. By adding NEM, it was thought that the SH group that may bind to the gold surface also binds to the maleimide group, so that the aggregation of colloidal gold causing false positives could be suppressed.

Example 2 Effect and effective concentration of N-substituted maleimide compound In the same manner as in Example 1, preparation of a standard product, preparation of an extract and extraction were performed.

-Preparation of diluent As a control diluent, a diluent containing 0.15 M Tris-HCl (pH 7.4), 0.1% BSA, 0.04% Tween, 2 mM EDTA and 0.0025% proclin300 was prepared.

  Also, as NEM-added diluent, prepare a diluent containing 2% NEM, 0.15M Tris-HCl (pH 7.4), 0.1% BSA, 0.04% Tween, 2 mM EDTA and 0.0025% proclin 300 did. Similarly, 0.0001%, 0.001%, 0.01%, 0.1%, 0.3%, 0.5%, 1% NEM or N-methylmaleimide (hereinafter referred to as NMM) A NEM additive diluent or NMM additive diluent was prepared.

・ Dilution 250 ng / mL of each standard product or each sample extract, 100 μL of the control diluent, NEM-added diluent or NMM-added diluted solution was added at a ratio of 900 μL to prepare a sample for reaction with the antibody. Obtained.

・ Determination 200 μL of the obtained sample was a Morinaga specific raw material immunochromatography kit, Nanotrap (registered trademark) egg (ovalbumin), Nanotrap (registered trademark) milk (casein), Nanotrap (registered trademark) wheat (gliadin) The reaction was started by dripping onto a dropping part of a test stick provided in Nanotrap (registered trademark) buckwheat (buckwheat soluble protein) or Nanotrap (registered trademark) peanut (peanut soluble protein). The determination was made after 15 minutes by confirming the presence or absence of the determination line. Regarding the judgment line, “−” was negative, “±” was weakly positive, and “+” was positive.

·result

  From the above results, it was confirmed that NEM significantly suppresses false positive reaction from 0.01%. Furthermore, although a very thin line was observed even at a low concentration of 0.001%, a false positive reaction could be suppressed. Similarly, it was confirmed that NMM having an N-substituted maleimide group also suppresses false positive reaction.

  From the above examples, the N-substituted maleimide compound was effective in suppressing false positives in the antigen detection method. N-substituted maleimide compounds can suppress false positive reactions from a concentration as low as 0.001%, and in order to obtain the effect, N-substituted maleimide compounds can be used up to a limit concentration at which N-substituted maleimide compounds can be dissolved. .

  The present invention is useful for determining the presence or absence of specific raw materials in foods. Furthermore, it is widely useful in fields where it is necessary to detect an antigen by antigen-antibody reaction, for example, fields such as medicine, medicine, cosmetics, agriculture and fishery, and industries related to basic research thereof.

1. 1. Immunochromatography apparatus Substrate 3. Development part 4. Outer wall 5. Addition part 6. First antibody 7. Second antibody 8. Windows 9. Developing part 10. Flow path
11. Confirmation section 12. Absorber 13. Absorber 21. Signal 31. Immunochromatography device 32. Deployment member 33. Immersion part 34. Assembly region 35. Immobilization region 36. region

Claims (12)

Mixing a specimen and an extract containing sulfite to extract at least one component selected from the group consisting of proteins, polypeptides and substances containing them; and
A sample obtained by extraction, a first antibody specific to the target antigen and labeled with colored microparticles, and a second antibody specific to the antigen and immobilized on the substrate And reacting in the presence of N-methylmaleimide or N-ethylmaleimide, a method for suppressing false positives derived from a specimen . In the reaction, the first immune complex is formed by the reaction between the first antibody and the sample, and the second immune complex is formed by the reaction between the first immune complex and the second antibody. The method of claim 1, comprising forming a body. The method according to claim 1 or 2, wherein N-methylmaleimide or N-ethylmaleimide is brought into the reaction system by being added when preparing a sample to be subjected to an antigen-antibody reaction. The method according to any one of claims 1 to 3 , wherein the colored fine particles are metal colloids or latex particles. The method according to claim 4, wherein the metal colloid is a gold colloid. The method according to any one of claims 1 to 5 , wherein all the steps are performed in an immunochromatography apparatus in which components are developed by capillary action. An antigen detection method using the method according to any one of claims 1 to 6,
  Mixing a specimen and an extract containing sulfite to extract at least one component selected from the group consisting of proteins, polypeptides and substances containing them,
  A sample obtained by extraction, a first antibody specific to the target antigen and labeled with colored microparticles, and a second antibody specific to the antigen and immobilized on the substrate And in the presence of N-methylmaleimide or N-ethylmaleimide, and
  Determining the presence or absence of the antibody by detecting a signal derived from a label contained in the immune complex obtained by the reaction
An antigen detection method comprising: A base, a development part for developing a reaction component on the base, a first antibody attached upstream of the development part, specific to the antigen to be detected and labeled with colored microparticles ; An immunochromatography apparatus comprising: a second antibody specific to the antigen immobilized in an observable region downstream of the development part;
An extract containing sulfite;
N-methylmaleimide or N-ethylmaleimide as a false positive reaction inhibitor derived from the specimen ,
An immunochromatography kit using the method according to any one of claims 1 to 7 . The immunochromatography kit according to claim 8 , wherein the colored fine particles are metal colloids or latex particles . A base, a development part for developing a reaction component on the base by capillary action, a first part that is combined upstream of the development part, specific to a specific raw material in food, and labeled with a colloidal gold An immunochromatography apparatus comprising: one antibody; and a second antibody that is immobilized in an observable region downstream of the development portion and specific to the specific raw material;
A diluent containing N-methylmaleimide or N-ethylmaleimide as a false positive reaction inhibitor derived from a specimen, and a surfactant, reducing agent, buffer, chelating agent and / or preservative;
An extract containing sulfite;
An immunochromatography kit using the method according to any one of claims 1 to 7 . The immunochromatography kit according to claim 10 , wherein the specific raw material is derived from at least one selected from the group consisting of eggs, milk, wheat, buckwheat and peanuts. In an immunochromatography apparatus, for use in a method of detecting an antigen by specific binding between a target antigen contained in a sample extracted from a specimen with a sulfite-containing extract and an antibody labeled with colored microparticles A false positive reaction inhibitor derived from a specimen containing N-methylmaleimide or N-ethylmaleimide as an active ingredient.

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