Method for detecting substance to be detected in sample, and method for suppressing false negative in immunochromatography for food inspection

Abstract

Provided is a method for suppressing false negative in immunochromatography or the like. A method for detecting a substance to be detected in a sample, which comprises treating the sample or an extract of the sample with a compound having a functional group having an acid dissociation constant (pKa) of 3 or less in water at 25 DEG C, or a salt thereof; and a method for suppressing false negative in immunochromatography or nucleic acid chromatography for detecting a substance to be detected in a sample, which comprises treating the sample or an extract of the sample with the compound or a salt thereof.

Description

Technical Field

[0001] This invention relates to the examination of substances in food ingredients, and particularly to methods for detecting substances in food ingredients. The invention also relates to methods for suppressing false negatives in immunochromatography or nucleic acid chromatography. More specifically, it relates to techniques for suppressing false negatives in immunochromatography or nucleic acid chromatography for detecting substances in edible meat or shellfish. Background Technology

[0002] Immunological assays such as immunochromatography and latex assays are important for rapidly and reliably identifying the presence of infections or immune disorders. However, false negatives—the so-called "false negatives"—are problematic in the analysis of samples actually collected from patients, where the tested substance is present but the sample is still marked negative. False negatives provide incorrect information about the disease, thus delaying the determination of the cause and potentially leading to serious consequences such as exacerbating the condition due to inappropriate interventions. Therefore, from the perspective of the primary purpose of these simplified testing methods, suppressing false negatives is an extremely important issue.

[0003] For example, Patent Document 1 reports a method in which, when detecting influenza virus from samples derived from biological mucosa such as nasopharyngeal mucosa and saliva, the sample is treated with piperazine-1,4-bis(2-ethanesulfonic acid (PIPES)) or sodium dextran sulfate in order to suppress false negatives caused by biological defense components contained in the sample.

[0004] However, the method in Patent Document 1 is characterized by blocking biological defense components such as IgA using PIPES, etc., and it does not describe the applicability of this method to samples other than those derived from biological mucosa.

[0005] Patent document 2 reports a method for inhibiting the sedimentation of colloidal gold particles bound to antibodies or antigens using dextran sulfate. This method addresses the problem that, due to the tendency of colloidal gold particles to sediment, it is necessary to homogenize their concentration during immunoassays, which can easily lead to measurement errors.

[0006] However, Patent Document 2 makes no mention of using dextran sulfate to suppress false negatives. Furthermore, biological samples such as blood, urine, feces, and marrow were used as subjects.

[0007] Patent Document 3 relates to a method for adding agglomeration promoters such as dextran sulfate and chondroitin sulfate to a assay based on immunoagglomeration. A key feature of Patent Document 3 is background correction, which involves subtracting the obtained background value from the agglomeration reaction of interfering substances and tiny particles contained in the sample.

[0008] However, Patent Document 3 makes no mention of using sulfated dextran or similar substances to suppress false negatives.

[0009] Furthermore, "polyanions" possess two or more anionic groups within a single molecule, resulting in a negative charge as a whole. As mentioned above, polyanions are widely used in immunoassays for purposes such as preventing nonspecific adsorption and inhibiting the sedimentation of colloidal gold particles; however, there are no examples of their application in the analysis of analytes in food ingredients. Additionally, food ingredients contain various proteins, including those that may adversely affect antigen-antibody reactions; however, no relationship has been reported between polyanions and techniques for suppressing false negatives that may arise due to proteins in food ingredients.

[0010] Existing technical documents

[0011] Patent documents

[0012] Patent Document 1: Japanese Patent No. 6116268

[0013] Patent Document 2: Japanese Patent No. 5442179

[0014] Patent Document 3: Japanese Patent No. 5323328 Summary of the Invention

[0015] The problem that the invention aims to solve

[0016] Specific microorganisms in food, especially meat, fish, and shellfish, can cause food poisoning. Therefore, accurate and rapid techniques for detecting the presence and hygiene of microorganisms are crucial. However, the presence of proteins in food can lead to false negatives. This is particularly true in tests using immunochromatography and other methods for detecting substances in meat, fish, and shellfish, where proteins can clog the sample pad, resulting in false negatives.

[0017] Therefore, the subject of this invention is to develop a method for detecting a substance in food that can suppress false negatives, and is accurate and rapid. In particular, the subject of this invention is to suppress false negatives when using immunochromatography or similar methods to examine the substance in edible meat, fish, shellfish, etc.

[0018] Methods for solving problems

[0019] In order to solve the above-mentioned problems, the inventors conducted in-depth research and found that when using immunochromatography and other methods to test for the substances to be detected in food, by using polyanion pretreatment of the sample, false negatives caused by the cationic proteins or their aggregates inherent in the food can be suppressed, and the substances to be detected in the sample can be detected quickly and accurately.

[0020] In particular, the inventors discovered that when testing food samples using immunochromatography or similar methods, pre-treating the samples with polyanions can eliminate sample pad blockage and suppress false negatives. It is speculated that sample pad blockage is caused by the binding of inherent cationic proteins or their aggregates present in samples such as edible meat, fish, and shellfish to labeled antibodies such as negatively charged colloidal gold particles, and this blockage is considered the main cause of false negatives. Furthermore, it is hypothesized that if the positive charge of these cationic protein aggregates can be neutralized beforehand, the binding of these aggregates to colloidal gold particles may be inhibited. Based on this hypothesis, pre-treating samples with polyanions capable of neutralizing positive charges successfully suppressed false negatives, thus completing this invention.

[0021] That is, the present invention provides the following solution.

[0022] [1] A method for detecting a substance in a sample, wherein the method comprises: treating the sample or an extract thereof with a compound having a functional group having an acid dissociation constant (pKa) of less than 3 in water at 25°C.

[0023] [2] A method for suppressing false negatives in an immunochromatographic or nucleic acid chromatography method for detecting a substance in a sample, wherein the method comprises: treating the sample or an extract of the sample with a compound or a salt thereof having a functional group having an acid dissociation constant (pKa) of less than 3 in water at 25°C.

[0024] [3] According to the method of [2], the false negative is caused by aggregates of proteins from the sample source.

[0025] [4] According to the method of [2], the false negative is caused by blockage of protein aggregates from the sample source, which is a blockage that occurs upstream of the detection section when the sample is added to an immunochromatographic test strip (10) containing at least a sample pad (2), a spreader (3) and a detection section (4) in sequence from the upstream side to the downstream side.

[0026] [5] The method according to any one of [1] to [4], wherein the sample is a food sample containing protein or an environmental sample containing food residue containing protein.

[0027] [6] The method according to any one of [1] to [5], wherein the sample is edible meat or fish and shellfish.

[0028] [7] According to the method described in [6], wherein the edible meat is selected from the group consisting of chicken, pork, beef, horse meat, mutton, venison and whale meat, and the fish and shellfish are selected from the group consisting of tuna, salmon, sea bass, shrimp, squid, octopus and shellfish.

[0029] [8] The method according to any one of [1] to [7], wherein the compound has three or more phosphate groups, sulfite groups and / or sulfate groups.

[0030] [9] The method according to any one of [1] to [8], wherein the molecular weight of the compound is in the range of 5,000 to 500,000.

[0031]

[10] The method according to any one of [1] to [9], wherein the compound or its salt is polyphosphate, heparin or dextran sulfate, or its salt.

[0032]

[11] The method according to any one of [1] to

[10] , wherein the compound or its salt is polyphosphoric acid or polyphosphate (degree of polymerization of phosphoric acid 60 to 100), and the concentration of polyphosphoric acid or polyphosphate in the sample treatment solution after the sample or the extract of the sample is treated with the compound or its salt is in the range of 0.25 to 2.5% (w / v).

[0033]

[12] The method according to any one of [1] to

[10] , wherein the compound or its salt is heparin or heparin salt, and the concentration of heparin or heparin salt in the sample treatment solution after the sample or the extract of the sample is treated with the compound or its salt is in the range of 0.0005 to 0.5% (w / v).

[0034]

[13] The method according to any one of [1] to

[10] , wherein the compound or its salt is dextran sulfate or dextran sulfate salt, and the concentration of dextran sulfate or dextran sulfate salt in the sample or the sample extract after treatment with the compound or its salt is in the range of 0.0005 to 0.5% (w / v).

[0035]

[14] The method according to any one of [1] to

[13] , wherein the substance to be detected is selected from the group consisting of allergens; components of viruses, bacteria, fungi or yeast or substances secreted by them; toxins; histamine; antibiotics; pesticide residues; and parasite antigens.

[0036]

[15] The method according to any one of [1] to

[14] , wherein the substance being detected is a component of bacteria or a substance secreted by bacteria.

[0037]

[16] The method according to any one of [1] to

[15] , wherein the substance being detected is an intracellular antigen of bacteria.

[0038]

[17] The method according to any one of [1] to

[16] , wherein the substance being detected is a bacterial ribosomal protein.

[0039]

[18] The method according to any one of [1] to

[17] , wherein the substance being detected is ribosomal protein L7 / L12.

[0040] The effects of the invention

[0041] According to the present invention, in the examination of edible meat, fish, shellfish, and other substances using methods such as immunochromatography, blockage at the upstream side of the detection section, such as the sample pad, can be eliminated, thereby suppressing false negatives. Furthermore, by suppressing false negatives, the analyte in the sample can be detected rapidly and accurately. Attached Figure Description

[0042] Figure 1 This is a schematic diagram of the test strip of the immunochromatographic apparatus used in the method of the present invention.

[0043] Figure 2 This is a 300x magnified image of the sample pad of the test strip before sample addition.

[0044] Figure 3 This is a graph showing a surface image (300x magnification) of the sample pad after treatment with the evaluation bacterial solution using reaction solution B prepared in Example 5.

[0045] Figure 4 This is a graph showing a surface image (5000x magnification) of the sample pad after treatment with the evaluation bacterial solution using reaction solution B prepared in Example 5.

[0046] Figure 5 This is a graph showing a surface image (300x magnification) of the sample pad after treatment with the evaluation bacterial solution using reaction solution A prepared in Example 5.

[0047] Figure 6 This is a graph showing a surface image (5000x magnification) of the sample pad after treatment with the evaluation bacterial solution using reaction solution A prepared in Example 5. Detailed Implementation

[0048] The present invention will now be described in detail with reference to specific embodiments. However, the present invention is not limited to the following embodiments and can be implemented with appropriate modifications.

[0049] The first embodiment of the present invention relates to a method for detecting a analyte in a sample, characterized in that it includes a step of treating the sample or an extract thereof with a compound having a functional group having an acid dissociation constant (pKa) of 3 or less in water at 25°C. The detection method of this first embodiment is not particularly limited, and examples include immunochemical methods, spectrophotometry, etc., and the apparatus used in this detection method is also not particularly limited. In particular, the detection method of this first embodiment can be used to suppress false negatives in immunochromatographic or nucleic acid chromatography methods for detecting the analyte in a sample.

[0050] A second embodiment of the present invention relates to a method for suppressing false negatives in immunochromatography or nucleic acid chromatography for detecting a substance in a sample, characterized in that it includes a step of treating the sample or an extract of the sample with a compound having a functional group having an acid dissociation constant (pKa) of 3 or less in water at 25°C.

[0051] Both the method of the first embodiment and the method of the second embodiment have the effect of suppressing false negatives caused by cationic proteins or aggregates inherent in the sample by treating the sample with a "compound having a functional group having an acid dissociation constant (pKa) of 3 or less in water at 25°C" or its salt.

[0052] A "false negative" refers to a situation where, although the sample contains the substance being tested, no positive signal is generated during analysis. For example, in immunochromatography, when the substance being tested is present in the sample, an immobilized substance-test substance-labeled reagent complex is formed on a solid support. However, in a false negative, the generation of the signal from the labeled reagent may be hindered due to reasons such as the absence of this complex or inhibition of its formation. In this invention, such a false negative may be caused by aggregates of proteins from the sample source, particularly proteins presumed to be cationic. In particular, such a false negative may be caused by blockage of protein aggregates from the sample source, which is caused by the addition of the sample to an immunochromatographic test strip (10) containing at least a sample pad (2), a spreader (3), and a detection section (4) sequentially from upstream to downstream. Figure 1 When this occurs, blockages may occur at the upstream portion of the detection section, such as in the sample pad and / or the expansion section.

[0053] In the methods of the first and second embodiments, the "sample" is not particularly limited, but can be, for example, a food sample containing protein, preferably edible meat or shellfish. Edible meat and / or shellfish contain myofibrillar proteins, which are mainly composed of myosin or actomyosin, which are salt-soluble proteins. These proteins dissolve upon the addition of salts, triggering a coagulation reaction.

[0054] In addition, environmental samples that may contain food residues containing protein are also subject to this invention. The protein in the food residues is preferably derived from edible meat, fish, shellfish, etc.

[0055] In the method of this invention, "cationic protein" refers to a protein in which at least a portion carries a positive charge. "Catic protein" also includes aggregates formed due to changes in its three-dimensional structure, etc. Therefore, it is presumed that the entity of the "cationic protein" in this invention is primarily myosin or actomyosin.

[0056] Examples of meats that can be consumed include chicken, pork, beef, horse meat, mutton, goat meat, venison, whale meat, rabbit meat, wild boar meat, wild duck meat, quail meat, domestic duck meat, pheasant meat, turkey meat, sparrow meat, ostrich meat, frog meat, turtle meat, locust or bee meat, etc.

[0057] As for fish and shellfish, there are no special restrictions as long as they are aquatic animals. Examples include tuna, salmon, sea bass, salmon, trout, horse mackerel, eel, conger eel, ayu, monkfish, rock bass, sardines, skipjack tuna, flounder, filefish, shad, crucian carp, mackerel, shark, mackerel, saury, sea bream, cod, loach, herring, conger eel, pufferfish, crucian carp, yellowtail, shad, jellyfish, mantis shrimp, sea cucumber, sea squirt, and sea urchin. It also includes salmon roe, herring roe, caviar, and other fish eggs.

[0058] Environmental samples that may contain food residue refer to any sample obtained from the environment that has come into contact with the food. There are no particular restrictions on the types of environmental samples. Examples include samples obtained by wiping the surfaces of fingers, work clothes, work shoes, nail brushes, cutting boards, knives, handles, conveyor belts, packaging materials, workbenches, faucets, etc., from food and beverage manufacturing equipment and food service areas using collection tools (swabs) soaked in liquid media (water, isotonic fluid, ethanol, etc.); as well as liquid samples such as tap water and well water.

[0059] Sample extraction can be carried out by crushing and homogenizing the sample, adding an aqueous solution containing salts such as physiological saline, and then kneading and shaking. Alternatively, the substance obtained by wiping the surface of food or the environment with a swab (cotton swab) or the substance obtained by wiping with a swab can be eluted into a buffer solution as a sample.

[0060] The obtained sample extract can be mixed with a solution of a compound having a functional group having an acid dissociation constant (pKa) of 3 or less in water at 25°C (hereinafter referred to as "the compound of the present invention") (referred to as "reaction solution" in Example 2 described below) in an appropriate proportion to prepare a "sample processing solution". Depending on the circumstances, compounds with bacteriolytic activity, surfactants (such as Tween 20, Tween 80, Triton X-100, Nonidet P-40, etc.) can be added to the reaction solution.

[0061] Examples of functional groups that have an acid dissociation constant (pKa) of 3 or less in water at 25°C include phosphate groups (first acid dissociation constant: 2.15), sulfite groups (first acid dissociation constant: 1.86), and sulfate groups (1.99) (Science Chronology 2013 (Desktop Edition); National Astronomical Observatory of the National Institute of Natural Sciences Maruzen Publishing Co., Ltd., p. 509, 2012). There are no particular limitations on the number of functional groups in the compounds of the present invention, as long as they have functional groups selected from phosphate, sulfite, and sulfate groups. The number of these functional groups is preferably 3 or more, more preferably 4 or more, more preferably 5 or more, more preferably 6 or more, more preferably 7 or more, more preferably 8 or more, more preferably 9 or more, more preferably 10 or more, more preferably 20 or more, more preferably 30 or more, more preferably 40 or more, more preferably 50 or more, and even more preferably 100 or more. Furthermore, examples of salts for the compounds of the present invention include sodium salts and potassium salts. In the following text, when referred to as "a compound of the present invention", its salts are included unless otherwise stated.

[0062] The compounds of the present invention can be any compounds having three or more of the above-described functional groups, and can be monomeric or polymeric. Examples of monomeric compounds include polyols (e.g., glycerol) and monosaccharides having three or more hydroxyl groups.

[0063] Polymer compounds are polymers containing repeating units comprising the aforementioned functional groups (hereinafter sometimes referred to as "polyanions" or "additives"). Examples of such polyanions include polyphosphates (average degree of polymerization of phosphate is about 60), heparin, dextran sulfate, and their salts. Examples of salts of polyanions include sodium polyphosphate, sodium heparin, and sodium dextran sulfate.

[0064] The molecular weight of these polyanions varies depending on the degree of polymerization, and is generally preferred to have a molecular weight of 5,000 to 500,000.

[0065] Specifically, regarding sodium polyphosphate, any sodium polyphosphate with a degree of polymerization of phosphate of 3 or higher (a number of phosphates of 3 or higher) is acceptable, preferably sodium polyphosphate with a degree of polymerization of phosphate of 60 to 100 and a molecular weight of 20,000 to 300,000.

[0066] Both sodium dextran sulfate and sodium heparin are almost 100% sulfurized, and therefore, regardless of their degree of polymerization, they all have more than three sulfate groups. Commercially available sodium dextran sulfate includes those with an average molecular weight of approximately 5,000 (molecular weight 1,000–9,000; approximately 30 sulfate groups), 6,500–10,000 (approximately 55 sulfate groups), 9,000–20,000 (approximately 94 sulfate groups), and approximately 500,000 (approximately 3,253 sulfate groups), all of which are preferred. As for sodium heparin, low molecular weight heparin (molecular weight 4,000–6,000) can also be used, but sodium heparin with an average molecular weight of 10,000–15,000 is preferred.

[0067] The higher the molecular weight of the polyanion, the better it can suppress false negatives even at low concentrations. Therefore, it is preferable to use commercially available substances with higher molecular weights.

[0068] It should be noted that, from the perspective of the degree of sulfation, PIPES (piperazine-1,4-bis(2-ethanesulfonic acid)) having two sulfate groups and HEPES (2-[4-(2-hydroxymethyl)piperazine-1-yl]ethanesulfonic acid) having one sulfate group are not preferred in the method of the present invention.

[0069] The concentration of the compounds of the present invention in the sample processing solution varies depending on the type of compound, and is preferably within the following range.

[0070] When the compound of the present invention is polyphosphoric acid or a polyphosphate (preferably sodium polyphosphate), the concentration of polyphosphoric acid or polyphosphate in the sample processing solution is preferably in the range of 0.25 to 2.5% (w / v), more preferably in the range of 0.25 to 2.4% (w / v), and even more preferably in the range of 0.25 to 1.5% (w / v).

[0071] When the compound of the present invention is heparin or a heparin salt (preferably sodium heparin), the concentration of heparin or a heparin salt in the sample processing solution is preferably in the range of 0.0005 to 0.5% (w / v), more preferably in the range of 0.0005 to 0.4% (w / v), and even more preferably in the range of 0.0005 to 0.05% (w / v).

[0072] When the compound of the present invention is dextran sulfate or a dextran sulfate salt (preferably sodium dextran sulfate), the concentration of dextran sulfate or the dextran sulfate salt in the sample processing solution is preferably in the range of 0.0005 to 0.5% (w / v), more preferably in the range of 0.0005 to 0.4% (w / v), and even more preferably in the range of 0.0005 to 0.05% (w / v).

[0073] Add the sample processing solution adjusted to this concentration to the following section. Figure 1 The sample pad of the test strip in the immunochromatographic apparatus shown.

[0074] In addition, the pH of the sample processing solution is preferably 6.5 to 8.5, more preferably 6.5 to 8.0, more preferably 7.0 to 7.5, and even more preferably 7.4 to 7.5.

[0075] Examples of substances that can be tested include allergens; microorganisms (such as components of viruses, bacteria, fungi, or yeast) or substances they secrete; toxins; histamine; antibiotics; pesticide residues; parasite antigens, etc.

[0076] In this invention, "allergen" mainly refers to the causative substance (antigen) that causes food allergies. Examples of causative foods include chicken, beef, pork, salmon, mackerel, shrimp, crab, abalone, squid, and salmon roe (based on the food labeling standards stipulated in Article 4, Paragraph 1 of the Japanese Food Labelling Law).

[0077] Examples of viruses include norovirus, hepatitis E virus, and hepatitis A virus.

[0078] As bacteria, they can include Salmonella, Campylobacter spp., Staphylococcus aureus, enterohemorrhagic Escherichia coli, pathogenic Escherichia coli other than enterohemorrhagic Escherichia coli, Clostridium perfringens, Vibrio enteritidis, Clostridium botulinum, Bacillus cereus, Listeria spp., Shigella, Yersinia enterocolitis, Vibrio vulnificus, Cronobacter sakazakii, etc.

[0079] Examples of fungi include Mucor plumbeus, Aspergillus flavus, Aspergillus niger, Paecilomyces variotii, Fusarium oxysporum, Rhizopus stolonifer, and Penicillium roqueforti.

[0080] Examples of yeasts include baker's yeast, brewer's yeast, sake yeast, yeasts of the genus *Saccharomyces*, yeasts of the genus *Debaryomyces*, yeasts of the genus *Wickerhamomyces*, yeasts of the genus *Zygosaccharomyces*, and other putrefactive yeasts.

[0081] Examples of toxins include enterotoxins, endotoxins, botulinum toxin A, and tetrodotoxin.

[0082] Examples of antibiotics include penicillin, cephalosporins, tetracyclines, sulfonamides, aminoglycosides, aminocyclic alcohols, macrolides, quinolones, ion carriers, carbadox, and nitrofurans.

[0083] In this invention, "residual pesticides" refers to pesticides that remain in feed or other materials and are concentrated in the bodies of animals and fish that consume them. Examples include: dicarboximide pesticides such as isoprothiolane; dithiopentalide pesticides such as isoprothiolane; amide pesticides such as fluopyram; triazole pesticides such as cyproconazole and bifenthrin; imidacloprid and fluopyram pesticides; organohalogen pesticides such as chlorfenapyr and chlorothalonil; carbamate pesticides such as carbaryl; macrolide pesticides such as emamectin benzoate; neonicotinoid pesticides such as imidacloprid and acetamiprid; organophosphorus pesticides such as malathion and oxazolidinium; and so on.

[0084] Examples of parasites include *Anisakis*, *Nybelinia*, *Tentacularia*, *Philometroides seriolae*, *Pennella*, *Didymozoidae*, *Toxoplasma gondii*, *Sarcocystis hominis*, *S. suihominis*, *S. fayeri*, and *Trichinella* spp.

[0085] In the method of the present invention, among the substances to be detected, bacterial components or substances secreted by bacteria are particularly targeted, preferably intracellular antigens of bacteria, more preferably bacterial ribosomal proteins, and even more preferably ribosomal proteins L7 / L12. Ribosomal proteins L7 / L12 exist in multiple copies within cells, thus resulting in high detection sensitivity. Furthermore, as described in International Publication No. 2000 / 06603, antibodies that can practically identify specific bacteria that may cause food poisoning with other bacteria by species or genus can be obtained.

[0086] The method of the present invention can, for example, be used... Figure 1 The test strips of the immunochromatographic apparatus shown are used for implementation (hereinafter referred to as "immunochromatographic test strips").

[0087] An immunochromatographic test strip (10) typically comprises a membrane consisting of a porous body having at least a sample pad (2), a developing section (3), and a detection section (4). An antibody-attached section (1) is located upstream of the sample pad, containing a labeled antibody that forms a complex with the analyte. The labeled antibody is elutably retained at the developing initiation site of the developing section. The detection section is located downstream of the developing section and has an antibody for capture fixed thereon. Furthermore, the sample pad, developing section, and detection section are fixed on a solid support (6) such as polyethylene. In addition to the labeled antibody, a labeled control antibody can be added to the antibody-attached section, and a primary antibody (control antibody bound to the labeled control antibody) can be further added to the detection section. It should be noted that the antibody-attached section can be located between the sample pad and the developing section, or it can be located independently of the test strip in a container for adding sample processing solution. When the antibody-attached section is located between the sample pad and the developing section, blockage (false negative) caused by the sample may occur upstream of the detection section.

[0088] When the sample processing solution is added to the sample pad, if the sample contains the analyte, the analyte specifically binds to the labeled antibody in the developing section, forming a complex. This complex expands the developing section downstream while simultaneously binding to the capture antibody in the detection section. Thus, in the detection section, a sandwich-shaped complex is formed by the labeled antibody, the analyte, and the capture antibody. Detection of this complex allows for qualitative or quantitative analysis of the analyte.

[0089] One example of a labeling agent constituting a labeled antibody is colloidal gold particles, which enable qualitative detection via a colorimetric reaction based on the colloidal gold particles. Furthermore, the degree of colorimetric reaction can also be used for quantitative detection of the analyte in a sample.

[0090] For details regarding the immunochromatographic test strips, please refer to Japanese Patent No. 5693938.

[0091] Immunochromatographic devices can also be provided as kits, which consist of a container (e.g., a microtube) holding the immunochromatographic test strips, and, if necessary, a polyanionic compound with bacteriolytic activity.

[0092] Immunochromatography can be broadly classified into two types: a lateral flow type, in which the immunochromatographic test strip is arranged laterally and the liquid is moved by capillary action (capillary force); and a flow-through type, in which the test strip is arranged longitudinally and the liquid is moved from top to bottom by gravity and capillary force. In this invention, either the lateral flow type or the flow-through type can be used, with the lateral flow type being more preferred.

[0093] Nucleic acid chromatography is a method as follows: target nucleic acid is extracted from the sample, and the target nucleic acid is used as a template to perform PCR amplification reaction using special primers with tags. The amplified target nucleic acid is then developed on a special test strip (nucleic acid chromatography chip), and the presence or absence of target DNA is converted into the presence or absence of a band, which is determined visually (e.g., Japanese Patent Application Publication No. 2014-82977).

[0094] The invention will now be described in detail with reference to specific embodiments, but the invention is not limited to these embodiments in any way.

[0095] Example

[0096] Example 1: Materials and Preparation Method

[0097] 1. Preparation of antibodies for labeling and for capturing

[0098] The bacteria used are Staphylococcus aureus as immunogens. Antibodies against Staphylococcus aureus ribosomal proteins L7 / L12 were prepared according to the method described in International Publication No. 2000 / 06603.

[0099] Specifically, *E. coli* transformed with an expression vector containing the full-length amino acid sequence of the recombinant Staphylococcus aureus ribosomal protein L7 / L12 was cultured in LB medium and purified as a fusion protein using an affinity column with the tag sequence derived from the expression vector. This full-length L7 / L12 protein was used as an immunogen. Following standard methods for obtaining hybridomas, the immunogen concentration was adjusted to 0.4 mg / mL using PBS, and an equal amount of Freund's adjuvant was added. Mice were immunized four times at a dose of 50 μg / immunotherapy. After confirming an increase in serum antibody titers through experimental blood sampling, spleen cells were extracted from the mice. These extracted mouse spleen cells were fused with myeloma cells to obtain various hybridomas.

[0100] The obtained hybridomas were cultured in HAT medium, and the antibodies in the culture supernatant were used for screening. Screening was performed by ELISA, and two hybridomas that produced antibodies that showed a specific reactivity with Staphylococcus aureus were selected.

[0101] Following standard methods for monoclonal antibody production, selected hybridomas were cultured in TIL MediaI medium supplemented with 10% fetal bovine serum (FBS). The culture was then administered intraperitoneally to mice, and the ascites fluid was collected. The collected ascites fluid was centrifuged to separate suspended solids and red blood cells, and then filtered through a 0.45 μm filter. The resulting filtrate was then passed through a Protein G column to adsorb antibodies, thereby purifying the antibodies from the mouse ascites fluid.

[0102] 2. Preparation of Immunochromatographic Test Strips

[0103] The obtained antibodies were used as the capture antibody and labeling antibody to prepare immunochromatographic test strips.

[0104] (1) Membrane carrier for immunochromatographic development

[0105] Antibodies for capture were mixed at a concentration of 1.5 mg / mL in 100 mM sodium phosphate buffer to prepare a solution supplemented with 3% (v / v) trehalose. The solution was then applied to commercially available nitrocellulose membranes cut into 2.5 cm wide and 15 cm long sections, at 1 cm intervals. 2 The resulting solution was coated in 1 μL as a single line, dried, and used as a membrane carrier for immunochromatographic development.

[0106] (2) Fabrication of colloidal gold labeled antibodies and attachment components for colloidal gold labeled antibodies

[0107] A solution with an antibody concentration of 0.1 mg / mL was prepared by adding 1 / 10 of the labeled antibody to a commercially available colloidal gold solution (particle size 60 nm) and mixing. The solution was allowed to stand at room temperature for 30 minutes to allow the antibody to bind to the surface of the colloidal gold particles. Subsequently, BSA solution was added to achieve a final concentration of 0.1% in the colloidal gold solution for blocking, thus preparing the colloidal gold-labeled antibody solution. This antibody solution was then infiltrated into a commercially available glass fiber sheet, followed by drying to create the colloidal gold-labeled antibody attachment component.

[0108] (3) Preparation of immunochromatographic test strips

[0109] In addition to the immunochromatographic development membrane carrier prepared in (1) and the colloidal gold labeling antibody attachment component prepared in (2), polyester nonwoven fabric as a sample addition component and filter paper as an absorption component were further prepared. These components were then attached to a commercially available polyethylene substrate and cut to a width of 5 mm to create a material compatible with... Figure 1 Immunochromatographic test strips with the same structure.

[0110] Example 2: Inhibition of Colloidal Gold Blockage under Various Reaction Solution Compositions

[0111] (1) Evaluation of the preparation of bacterial suspension

[0112] As ingredients, minced beef, minced pork, minced chicken, tuna (sashimi), and salmon (sashimi) were selected. 5g of each ingredient was weighed and placed into a homogenization bag containing 45ml of physiological saline. The mixture was then kneaded for 60 seconds to obtain extracts. Staphylococcus aureus was selected as the bacteria to be tested, and a 1×10⁻⁶ solution was prepared using the extracts from the various ingredients or physiological saline. 5 cfu / ml.

[0113] (2) Preparation of various reaction solutions

[0114] Add 5 μg / ml of lysozyme for lysing Staphylococcus aureus to 0.1M Tris-HCl (pH 7.4) buffer, and add 0.2% Tween (registered trademark) 20 for immunochromatographic development. Use the resulting liquid as the stock solution, and add the additives shown in Table 1 to it to prepare various reaction solutions under the conditions in Table 1.

[0115] It should be noted that, for comparison purposes, Table 1 also shows examples of sodium citrate under conditions (1-6), PIPES under conditions (1-7), and HEPES under conditions (1-8). The concentration of each additive is set to 0.5% (w / v).

[0116] [Table 1]

[0117] (3) Evaluation of immunochromatographic test strips

[0118] The bacterial culture used for evaluation was mixed with the various reaction solutions described above in a 1:1 ratio. The immunochromatographic test strip prepared in Example 1 was then inserted into the mixture to evaluate whether there was a detection based on an antigen-antibody reaction.

[0119] It should be noted that, in the evaluation, the degree of redness based on colloidal gold particles in the test line section was visually determined. Visually positive results were marked as "+", weakly positive results as "±", and blockages in the sample pad with colloidal gold particles that resulted in visually negative results were marked as "-". The results are shown in Table 2.

[0120] [Table 2]

[0121] Under the reaction conditions (1-1) with only the mother liquor, when the immunochromatographic test strip was inserted into various food extracts, colloidal gold particles clogged the sample pad, resulting in a visually negative result. It is presumed that this is because protein aggregates, presumably cationic proteins, in the food extracts hindered the liquid movement of the colloidal gold particles. On the other hand, under the conditions (1-2) to (1-4) with the addition of various polyanions, no clogging of the sample pad with colloidal gold particles was observed in the various food extracts, resulting in a visually positive result. Furthermore, under the conditions (1-5) with the addition of polyanions containing carboxymethyl groups, and under the conditions (1-6) with the addition of citric acid, or under the conditions (1-7) to (1-8) with the addition of compounds having two or fewer sulfate groups, similarly to the reaction conditions (1-1) with only the mother liquor, colloidal gold particles clogged the sample pad, resulting in a visually negative result.

[0122] The results above show that by using specific polyanions, false negatives caused by proteins that are presumed to be cationic can be suppressed.

[0123] Example 3: Concentration Study of Polyanions

[0124] (1) Evaluation of the preparation of bacterial suspension

[0125] As ingredients, minced beef, minced pork, minced chicken, tuna (sashimi), and salmon (sashimi) were selected. 5g of each ingredient was weighed and placed into a homogenization bag containing 45ml of physiological saline. The mixture was kneaded for 60 seconds to obtain extracts from each ingredient. Staphylococcus aureus was selected as the bacteria to be tested, and a 1×10⁻⁶ solution was prepared using the extracts from each ingredient or physiological saline. 5 cfu / ml.

[0126] (2) Preparation of various reaction solutions

[0127] Add 5 μg / ml of lysozyme for lysing Staphylococcus aureus to 0.1M Tris-HCl (pH 7.4) buffer, and add 0.2% Tween 20 for immunochromatographic development. Use the resulting liquid as the stock solution to prepare various reaction solutions containing various additives at 0.0001–5% (w / v).

[0128] It should be noted that sodium dextran sulfate and sodium heparin are prepared in the range of 0.0001 to 1% (w / v), and sodium polyphosphate and carboxymethyl dextran are prepared in the range of 0.0001 to 5% (w / v).

[0129] (3) Evaluation of immunochromatographic test strips

[0130] The bacterial culture used for evaluation was mixed with the various reaction solutions mentioned above in a 1:1 ratio. An immunochromatographic test strip was then inserted into the mixture to evaluate whether there was a detection based on an antigen-antibody reaction.

[0131] It should be noted that, in the evaluation, the degree of redness based on colloidal gold particles in the test line section was visually determined. Visually positive results were marked as "+", weakly positive results as "±", and blockages in the sample pad resulting in colloidal gold particles and visually negative results as "-". The results are shown in Table 3.

[0132] [Table 3]

[0133] When using sodium dextran sulfate as an additive, sufficient inhibition of colloidal gold particle blockage was confirmed when the additive concentration in the sample processing solution was 0.0005% (w / v) or higher, resulting in sufficiently high test line intensity based on antigen-antibody reaction. On the other hand, when the additive concentration in the sample processing solution was 0.5% (w / v) or higher, although no colloidal gold particle blockage occurred, a decrease in test line intensity (visually weak positive) was observed. Therefore, it was confirmed that the suitable concentration of sodium dextran sulfate in the sample processing solution is in the range of 0.0005–0.5% (w / v).

[0134] When sodium polyphosphate is used as an additive, sufficient inhibition of colloidal gold particle blockage is confirmed when the additive concentration in the sample processing solution is 0.025% (w / v) or higher, and the test line intensity based on the antigen-antibody reaction is sufficiently high. On the other hand, when the additive concentration in the sample processing solution is 2.5% (w / v) or higher, although no colloidal gold particle blockage occurs, a decrease in test line intensity (visually weak positive) is confirmed. Therefore, it is confirmed that the suitable concentration of sodium polyphosphate in the sample processing solution is in the range of 0.25% to 2.5% (w / v).

[0135] When using sodium heparin as an additive, sufficient inhibition of colloidal gold particle blockage was confirmed when the additive concentration in the sample processing solution was 0.0005% (w / v) or higher, resulting in sufficiently high test line intensity based on the antigen-antibody reaction. Conversely, when the additive concentration in the sample processing solution was 0.5% (w / v) or higher, although no colloidal gold particle blockage occurred, a decrease in test line intensity (visually weak positive) was observed. Therefore, it was confirmed that the sodium heparin concentration in the sample processing solution is suitable in the range of 0.0005–0.5% (w / v).

[0136] When carboxymethyl dextran is used as an additive, colloidal gold particles cause blockage under all additive concentration conditions, resulting in visually negative results.

[0137] Example 4: pH Study of Polyanionic Polyions

[0138] (1) Evaluation of the preparation of bacterial suspension

[0139] As ingredients, minced beef, minced pork, minced chicken, tuna (sashimi), and salmon (sashimi) were selected. 5g of each ingredient was weighed and placed into a homogenization bag containing 45ml of physiological saline. The mixture was kneaded for 60 seconds to obtain extracts from each ingredient. Staphylococcus aureus was selected as the bacteria to be tested, and a 1×10⁻⁶ solution was prepared using the extracts from each ingredient or physiological saline. 5 cfu / ml.

[0140] (2) Preparation of various reaction solutions

[0141] Add 5 μg / ml of lysozyme for lysing Staphylococcus aureus to 0.1M Tris-HCl (or MES-NaOH) buffer, and add 0.2% Tween 20 for immunochromatographic development. Use the resulting liquid as the stock solution, adjust the various additives to the specified concentrations, and then adjust the pH to prepare reaction solutions with different pH values ​​(5.5–8.5).

[0142] It should be noted that sodium dextran sulfate and sodium heparin are adjusted to 0.01% (w / v), and sodium polyphosphate is adjusted to 1% (w / v).

[0143] (3) Evaluation of immunochromatographic test strips

[0144] The bacterial culture used for evaluation was mixed with the various reaction solutions mentioned above in a 1:1 ratio. An immunochromatographic test strip was then inserted into the mixture to evaluate whether there was a detection based on an antigen-antibody reaction.

[0145] It should be noted that in the evaluation, the degree of redness based on colloidal gold particles in the test line section was visually determined. Visually positive results were marked as "+", weakly positive results as "±", blockages in the sample pad with colloidal gold particles and visually negative results as "-", and blockages in the sample pad without colloidal gold particles and visually negative results as "--". The results are shown in Table 4.

[0146] [Table 4]

[0147] The pH values ​​in Table 4 refer to the pH values ​​of the sample treatment solution.

[0148] As confirmed by Table 4, under the conditions of adding polyanions (3-8) to (3-28), the clogging inhibition effect of colloidal gold particles can be fully utilized by keeping the pH of the sample treatment solution in the range of 6.5 to 8.5.

[0149] On the other hand, under conditions (3-1) to (3-7) without the addition of polyanions, it was confirmed that colloidal gold particles caused blockage in various food extracts under any pH conditions.

[0150] It should be noted that, under conditions where pH exceeds 8.5, the clogging inhibition effect of colloidal gold particles was confirmed even without the addition of polyanions, but a tendency for decreased detection sensitivity was observed.

[0151] Example 5: Surface observation of a sample pad blocked by colloidal gold particles.

[0152] (1) Evaluation of the preparation of bacterial suspension

[0153] Minced chicken was selected as the ingredient, and 5g was weighed out and placed into a homogenization bag containing 45ml of physiological saline. The mixture was kneaded for 60 seconds to obtain the ingredient extract. Staphylococcus aureus was selected as the bacteria to be tested, and a 1×10⁻⁶ solution was prepared using the ingredient extract. 5 cfu / ml.

[0154] (2) Preparation of various reaction solutions

[0155] Add 5 μg / ml of lysozyme for lysing Staphylococcus aureus to 0.1M Tris-HCl (pH 7.4) buffer, and add 0.2% Tween 20 for immunochromatographic development. Use the resulting liquid as the stock solution to prepare "Reaction Solution A" with 0.5% (w / v) sodium dextran sulfate added, and "Reaction Solution B" with only the stock solution.

[0156] (3) Surface observation of the sample pad after evaluation of the immunochromatographic test strip

[0157] The bacterial suspension used for evaluation was mixed with the various reaction solutions described above at a 1:1 ratio. The immunochromatographic test strip was then inserted into the mixture, and the portion in contact with the liquid surface was cut out (in...). Figure 1 The sample pad (represented by 7) was observed on the surface using a scanning electron microscope (FlexSEM1000, Hitachi High-Tech Co., Ltd.).

[0158] When the bacterial culture for evaluation was treated with reaction solution A, no colloidal gold particles were detected blocking the sample pad. However, when the bacterial culture for evaluation was treated with reaction solution B, reddish aggregates were observed on the sample pad, confirming blockage by colloidal gold particles. A surface image of the sample pad before sample addition is shown below. Figure 2 The surface image of the sample pad after treatment with the evaluation bacterial solution using reaction solution B is shown below. Figure 3 (All 300 times). As confirmed by the attached figures, when the bacterial culture for evaluation was treated with reaction solution B, a substance resembling aggregates was found blocking the fibers of the sample pad compared to before sample addition.

[0159] The surface image of the sample pad after treatment with reaction solution B at a higher magnification (5000x) is shown below. Figure 4 As observed in the attached figure, particulate material is attached to the surface of the aggregate. The aggregate is presumed to be a protein aggregate of cationic proteins, and the particulate material is presumed to be colloidal gold particles.

[0160] On the other hand, the surface image of the sample pad after treatment with the evaluation bacterial solution using reaction solution A is shown. Figure 5 , Figure 6 As observed in the attached figures, there are aggregate-like substances blocking the fibers of the sample pad (however, the aggregation is reduced compared to the case treated with reaction solution B), but it was confirmed that no particulate matter was attached to the surface of the aggregate.

[0161] Therefore, it is suggested that treating the bacterial culture for evaluation with polyanions such as sodium dextran sulfate can (i) reduce the formation of secondary aggregates of protein aggregates presumed to be cationic, and (ii) neutralize the positive charge of existing protein aggregates presumed to be cationic by masking them, thus blocking their binding to negatively charged colloidal gold particles, thereby eliminating sample pad blockage. Furthermore, it is suggested that by eliminating this blockage, the desired antigen-antibody reaction complex can be formed, suppressing false negatives.

[0162] Industrial applicability

[0163] The method of the present invention can be used for rapid and accurate detection of substances in edible meat, fish and shellfish.

[0164] Explanation of symbols

[0165] 10. Test strips for immunochromatographic apparatus

[0166] 1. Labeled antibody attachment site

[0167] 2 Sample pad

[0168] 3. Development Section

[0169] 4. Testing Department

[0170] 5 Absorbent Pads

[0171] 6. Solid support

[0172] 7. Liquid level

Claims

1. A method for detecting a substance to be tested in a sample, wherein, The method includes treating the sample or an extract thereof with a compound having a functional group having an acid dissociation constant (pKa) of less than 3 in water at 25°C.

2. A method for suppressing false negatives in immunochromatography or nucleic acid chromatography for detecting a analyte in a sample, wherein, The method includes treating the sample or an extract thereof with a compound having a functional group having an acid dissociation constant (pKa) of less than 3 in water at 25°C.

3. The method according to claim 2, wherein, The false negatives are caused by aggregates of proteins from the sample source.

4. The method according to claim 2, wherein, The false negative is caused by blockage of protein aggregates from the sample source. This blockage occurs upstream of the detection section when the sample is added to an immunochromatographic test strip (10) containing at least a sample pad (2), a spreader (3), and a detection section (4) in sequence from upstream to downstream.

5. The method according to claim 1 or 2, wherein, The sample is a food sample containing protein or an environmental sample containing food residue containing protein.

6. The method according to claim 1 or 2, wherein, The samples were edible meat or fish and shellfish.

7. The method according to claim 6, wherein, The edible meat is selected from the group consisting of chicken, pork, beef, horse meat, mutton, venison, and whale meat, and the fish and shellfish are selected from the group consisting of tuna, salmon, sea bass, shrimp, squid, octopus, and shellfish.

8. The method according to claim 1 or 2, wherein, The compound has three or more phosphate groups, sulfite groups, and / or sulfate groups.

9. The method according to claim 1 or 2, wherein, The molecular weight of the compound is in the range of 5,000 to 500,000.

10. The method according to claim 1 or 2, wherein, The compound or its salt is polyphosphate, heparin or dextran sulfate, or its salt.

11. The method according to claim 1 or 2, wherein, The compound or its salt is a polyphosphoric acid or polyphosphate with a degree of polymerization of 60 to 100, and the concentration of polyphosphoric acid or polyphosphate in the sample or the sample extract after treatment with the compound or its salt is in the range of 0.25% to 2.5% (w / v).

12. The method according to claim 1 or 2, wherein, The compound or its salt is heparin or heparin salt, and the concentration of heparin or heparin salt in the sample or the sample extract after treatment with the compound or its salt is in the range of 0.0005 to 0.5% (w / v).

13. The method according to claim 1 or 2, wherein, The compound or its salt is dextran sulfate or dextran sulfate salt, and the concentration of dextran sulfate or dextran sulfate salt in the sample or the sample extract after treatment with the compound or its salt is in the range of 0.0005 to 0.5% (w / v).

14. The method according to claim 1 or 2, wherein, The substances to be tested are selected from the group consisting of allergens; components of viruses, bacteria, fungi or yeast or substances secreted by them; toxins; histamine; antibiotics; pesticide residues; and parasite antigens.

15. The method according to claim 1 or 2, wherein, The substance being tested is a component of bacteria or a substance secreted by bacteria.

16. The method according to claim 1 or 2, wherein, The substance being tested is an intracellular antigen of bacteria.

17. The method according to claim 1 or 2, wherein, The substance being tested is bacterial ribosomal protein.

18. The method according to claim 1 or 2, wherein, The substance being tested is ribosomal protein L7 / L12.

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