Method for detecting substance to be detected in specimen, and method for suppressing false negatives in immunochromatographic method for inspecting food material

Abstract

The present invention provides a method for suppressing false negatives in an immunochromatographic method or the like.　Provided are: a method for detecting a substance to be detected in a specimen, wherein the method comprises treating the specimen or a liquid extract of the specimen using a compound having a functional group having an acid dissociation constant (pKa) of 3 or lower in water at 25°C or a salt of said compound; and a method for suppressing false negatives in an immunochromatographic method or a nucleic acid chromatographic method for detecting a substance to be detected in a specimen, wherein the method comprises treating the specimen or a liquid extract of the specimen using the aforementioned compound or a salt thereof.

Description

Method for detecting a target substance in a specimen and method for suppressing false negatives in immunochromatography for food inspection

[0001] The present invention relates to a method for testing food materials for target substances, and in particular to a method for detecting target substances in food materials. The present invention also relates to a method for suppressing false negatives in immunochromatography or nucleic acid chromatography. In particular, the present invention relates to a technology for suppressing false negatives in immunochromatography or nucleic acid chromatography for detecting target substances in meat or seafood.

[0002] Immunological assays such as immunochromatography and latex assays are important for quickly and reliably determining the presence of infectious diseases or immune disorders. However, in the analysis of samples actually collected from patients, false negative results, in which the target substance is judged to be negative even though it is present in the sample, are a problem. False negatives provide incorrect information about the disease, which not only delays the identification of the cause but can also lead to inappropriate measures, resulting in serious consequences such as a more severe condition. Therefore, reducing false negatives is an extremely important issue given the main purpose of using simple testing methods.

[0003] For example, Patent Document 1 reports a method for detecting influenza viruses from specimens derived from biological mucosa, such as nasopharyngeal mucosa and saliva, in which the specimen is treated with piperazine-1,4-bis(2-ethanesulfonic acid (PIPES)) or dextran sulfate sodium in order to suppress false negatives due to biodefense components contained in the specimen. However, the method of Patent Document 1 is characterized by blocking biodefense components such as IgA with PIPES or the like, and does not disclose the applicability of the method to specimens other than those derived from biological mucosa.

[0004] Patent Document 2 reports a method for suppressing the sedimentation of colloidal gold particles bound to antibodies or antigens using dextran sulfate. This method solves the problem that colloidal gold particles tend to sediment, which requires uniform concentration of colloidal gold particles during immunoassay, and is prone to cause measurement errors. However, Patent Document 2 also makes no mention of the effect of dextran sulfate in suppressing false negatives. Furthermore, the method targets biological samples such as blood, urine, feces, and cerebrospinal fluid.

[0005] Patent Document 3 relates to a method of adding an agglutination promoter such as dextran sulfate or chondroitin sulfate to a measurement method based on immune agglutination as the measurement principle. Patent Document 3 is characterized by background correction, in which only measurement interfering substances contained in a stool sample are subjected to an agglutination reaction with microparticles, and the resulting background value is subtracted. However, Patent Document 3 also does not describe at all the effect of dextran sulfate or the like in suppressing false negatives.

[0006] A "polyanion" has two or more anionic groups per molecule, resulting in a negative charge for the molecule as a whole. As mentioned above, polyanions are widely used in immunoassays to prevent nonspecific adsorption and inhibit the sedimentation of gold colloid particles, but there have been no reports of their application to the analysis of analytes in foodstuffs. Furthermore, foodstuffs contain various proteins, some of which may adversely affect antigen-antibody reactions. However, no reports have been published on the relationship between polyanions and techniques for suppressing false negatives caused by proteins in foodstuffs.

[0007] Patent No. 6116268 Patent No. 5442179 Patent No. 5323328

[0008] Certain microorganisms in foodstuffs, particularly meat and seafood, can cause food poisoning. Therefore, technology for accurately and quickly detecting the presence or absence of microorganisms and their sanitary conditions is important, but there is a problem that proteins in foodstuffs can cause false negatives. In particular, when testing meat, seafood, and other foods for detectable substances using immunochromatography or the like, there is a problem that proteins in the meat or seafood can clog the sample pad, resulting in false negatives. Therefore, an object of the present invention is to develop a method for accurately and quickly detecting detectable substances in foodstuffs while suppressing false negatives, and in particular, an object of the present invention is to suppress false negatives in testing meat, seafood, and other foods for detectable substances using immunochromatography or the like.

[0009] The present inventors conducted extensive research to solve the above-mentioned problems and found that pre-treating a sample with a polyanion in immunochromatographic or other methods to test for a substance to be detected in foodstuffs can suppress false-negative results caused by cationic proteins or their aggregates inherent in the foodstuff, thereby enabling rapid and accurate detection of the substance to be detected in the sample. In particular, the present inventors discovered that pre-treating a sample with a polyanion in immunochromatographic or other methods to test for a substance to be detected in foodstuffs can eliminate clogging of the sample pad and suppress false-negative results. They speculated that clogging of the sample pad occurs when inherent cationic proteins or their aggregates present in samples such as meat and seafood bind to negatively charged labeled antibodies, such as gold colloid particles, and that this clogging is the main cause of false-negative results. They further hypothesized that if the positive charge of the cationic protein aggregates could be neutralized in advance, binding of the cationic protein aggregates to the gold colloid particles could be suppressed. Based on this assumption, the sample was pre-treated with a polyanion capable of neutralizing the positive charge, and as a result, false negatives were successfully suppressed, leading to the completion of the present invention.

[0010] That is, the present invention provides the following: [1] A method for detecting an analyte in a sample, comprising treating the sample or an extract of the sample with a compound having a functional group whose acid dissociation constant (pKa) in water at 25°C is 3 or less, or a salt thereof. [2] A method for suppressing false negatives in immunochromatography or nucleic acid chromatography for detecting an analyte in a sample, comprising treating the sample or an extract of the sample with a compound having a functional group whose acid dissociation constant (pKa) in water at 25°C is 3 or less, or a salt thereof. [3] The method according to [2], wherein the false negative is caused by protein aggregates derived from the sample. [4] The method according to [2], wherein the false negative is caused by clogging of the sample-derived protein aggregates at a site upstream of the detection section when the sample is added to an immunochromatographic test strip (10) including a membrane having at least a sample pad (2), a development section (3), and a detection section (4) in this order from upstream to downstream. [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 residues containing protein. [6] The method according to any one of [1] to [5], wherein the sample is meat or seafood. [7] The method according to [6], wherein the meat is selected from the group consisting of chicken, pork, beef, horse meat, mutton, venison, and whale meat, and the seafood is selected from the group consisting of tuna, salmon, sea bass, shrimp, squid, octopus, and shellfish. [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. [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.

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

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

[10] , wherein the compound or salt thereof is polyphosphoric acid or a polyphosphate (degree of phosphoric acid polymerization: 60 to 100), and the concentration of polyphosphoric acid or the polyphosphate in a specimen treatment solution obtained after treating the specimen or an extract of the specimen with the compound or a salt thereof is in the range of 0.25 to 2.5% (w / v).

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

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

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

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

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

[13] , wherein the analyte is selected from the group consisting of an allergen; a component of a virus, bacterium, fungus, or yeast, or a substance secreted by any one of these; a toxin; histamine; an antibiotic; a pesticide residue; and a parasite antigen.

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

[14] , wherein the substance to be detected is a bacterial component or a substance secreted by bacteria.

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

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

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

[16] , wherein the substance to be detected is a ribosomal protein of bacteria.

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

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

[0011] According to the present invention, in the testing of meat, seafood, and other analytes by immunochromatography or the like, clogging of the sample pad or other upstream portion of the detection unit can be eliminated, thereby suppressing false negatives. Furthermore, by suppressing false negatives, the analyte in the sample can be detected quickly and accurately.

[0012] FIG. 1 is a schematic diagram of a test strip of an immunochromatography device used in the method of the present invention. FIG. 2 is a diagram showing a surface image (300x) of the sample pad of the test strip before the addition of a sample. FIG. 3 is a diagram showing a surface image (300x) of the sample pad after treating the evaluation bacterial solution with reaction solution B prepared in Example 5. FIG. 4 is a diagram showing a surface image (5000x) of the sample pad after treating the evaluation bacterial solution with reaction solution B prepared in Example 5. FIG. 5 is a diagram showing a surface image (300x) of the sample pad after treating the evaluation bacterial solution with reaction solution A prepared in Example 5. FIG. 6 is a diagram showing a surface image (5000x) of the sample pad after treating the evaluation bacterial solution with reaction solution A prepared in Example 5.

[0013] The present invention will be described in detail below with reference to specific embodiments. However, the present invention is not limited to the following embodiments and can be practiced with appropriate modifications.

[0014] A first embodiment of the present invention relates to a method for detecting an analyte in a sample, the method comprising the step of treating the sample or an extract of the sample with a compound or a salt thereof having a functional group whose acid dissociation constant (pKa) in water at 25°C is 3 or less. The detection method of this first embodiment is not particularly limited, and examples thereof include immunochemical methods and spectroscopy, and the device used in the detection method is also not particularly limited. In particular, the detection method of this first embodiment can be used to suppress false negatives in immunochromatography or nucleic acid chromatography for detecting an analyte in a sample. A second embodiment of the present invention relates to a method for suppressing false negatives in immunochromatography or nucleic acid chromatography for detecting an analyte in a sample, the method comprising the step of treating the sample or an extract of the sample with a compound or a salt thereof having a functional group whose acid dissociation constant (pKa) in water at 25°C is 3 or less. Both the method according to the first embodiment and the method according to the second embodiment have the effect of suppressing false negatives caused by cationic proteins or their aggregates inherent in the specimen by treating the specimen with a "compound having a functional group whose acid dissociation constant (pKa) in water at 25°C is 3 or less" or a salt thereof.

[0015] A "false negative" refers to the failure to generate a positive signal in an assay despite the presence of a substance to be detected in the sample. For example, in immunochromatography, when a substance to be detected is present in the sample, a complex of the immobilized substance, the substance to be detected, and the labeled reagent is formed on the solid support. However, in a false negative, the complex does not form or its formation is suppressed, thereby inhibiting the generation of a signal from the labeled reagent. In the present invention, this false negative can be caused by aggregates of proteins derived from the sample, particularly proteins suspected to be cationic proteins. In particular, this false negative can occur when a sample is added to an immunochromatographic test strip (10) ( FIG. 1 ) including a membrane having at least a sample pad (2), a development section (3), and a detection section (4) arranged in this order from upstream to downstream, due to clogging by aggregates of protein derived from the sample in a region upstream of the detection section, such as the sample pad and / or the development section.

[0016] In the methods according to the first and second embodiments, the "specimen" is not particularly limited, but may be, for example, a food sample containing protein, preferably meat or seafood. Meat and seafood contain myofibrillar proteins, which are primarily composed of salt-soluble proteins, myosin or actomyosin. These proteins are eluted by the addition of salts, initiating an agglutination reaction. Environmental samples that may contain food residues containing protein are also within the scope of the present invention. Proteins contained in food residues are preferably derived from meat, seafood, or the like. In the method of the present invention, "cationic protein" refers to a protein at least a portion of which is positively charged. "Cationic protein" also includes aggregates formed by changes in its three-dimensional structure. Therefore, the entity of the "cationic protein" in the present invention is presumed to be primarily myosin or actomyosin.

[0017] Examples of meat include chicken, pork, beef, horse, lamb, goat, venison, whale, rabbit, wild boar, duck, quail, duck, pheasant, turkey, sparrow, ostrich, frog, soft-shelled turtle, locust, and bee meat. Seafood is not particularly limited as long as it is aquatic. Examples include tuna, salmon, sea bass, salmon, trout, horse mackerel, eel, conger eel, sweetfish, monkfish, grunt, sardine, bonito, flounder, filefish, whiting, crucian carp, mackerel, shark, Spanish mackerel, saury, capelin, sea bream, cod, loach, herring, conger eel, pufferfish, crucian carp, yellowtail, smelt, jellyfish, mantis shrimp, sea cucumber, sea squirt, and sea urchin. Also included are fish eggs such as salmon roe, herring roe, and caviar.

[0018] Environmental samples that may contain food residues refer to any sample obtained from an environment that comes into contact with food ingredients. The type of environmental sample is not particularly limited, and examples include samples obtained by wiping the surfaces of hands, work clothes, work shoes, nail brushes, cutting boards, knives, handles, conveyor belts, packaging materials, work desks, faucets, etc. in food and beverage manufacturing facilities, food and beverage service sites, etc. with a sampling tool (swab) such as clean cotton or clean cloth impregnated with a liquid medium (water, physiological isotonic solution, ethanol, etc.), as well as liquid samples such as tap water and well water.

[0019] Extraction of the specimen can be performed by crushing and homogenizing the specimen, adding an aqueous solution containing a salt such as physiological saline, and rubbing or shaking the mixture. The specimen can also be obtained by wiping the surface of a food material or environment with a swab, or by dissolving the swab in a buffer solution.

[0020] The obtained sample extract and a solution (referred to as "reaction solution" in Example 2 below) of "a compound having a functional group whose acid dissociation constant (pKa) in water at 25°C is 3 or less" (hereinafter referred to as "the compound of the present invention") can be mixed in an appropriate ratio to prepare a "sample treatment solution." If necessary, the reaction solution may contain a compound with bacteriolytic activity, a surfactant (Tween (registered trademark) 20, Tween (registered trademark) 80, Triton (registered trademark) X-100, Nonidet P-40, etc.), etc.

[0021] Examples of "functional groups having an acid dissociation constant (pKa) of 3 or less in water at 25°C" include a phosphate group (first acid dissociation constant: 2.15), a sulfite group (first acid dissociation constant: 1.86), and a sulfate group (1.99) (Science Chronology 2013 (Desktop Edition), National Institutes of Natural Sciences, National Astronomical Observatory, Maruzen Publishing Co., Ltd., p. 509, 2012). The compound of the present invention is not particularly limited as long as it has a functional group selected from a phosphate group, a sulfite group, and a sulfate group. The number of 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. Salts of the compound of the present invention include sodium salts and potassium salts. Hereinafter, when the term "compound of the present invention" is used, it includes salts thereof unless otherwise specified.

[0022] The compound of the present invention may be either monomeric or polymeric as long as it has three or more of the above functional groups. Examples of the monomeric compound include polyhydric alcohols having three or more hydroxy groups (e.g., glycerin) and monosaccharides.

[0023] The polymeric compound is a polymer containing a repeating unit containing the functional group (hereinafter, sometimes referred to as a "polyanion" or "additive"). Examples of such polyanions include polyphosphate (average degree of phosphate polymerization of approximately 60), heparin, dextran sulfate, and salts thereof. Examples of polyanion salts include sodium polyphosphate, sodium heparin, and sodium dextran sulfate.

[0024] The molecular weight of these polyanions varies depending on the degree of polymerization, but those with a molecular weight of 5,000 to 500,000 are typically preferred. Specifically, sodium polyphosphate may be used as long as it has a degree of phosphate polymerization of 3 or more (three or more phosphate groups), with those with a degree of phosphate polymerization of 60 to 100 and a molecular weight of 20,000 to 300,000 being preferred. Both dextran sulfate sodium and heparin sodium are nearly 100% sulfonated, and therefore have three or more sulfate groups regardless of their degree of polymerization. Commercially available dextran sulfate sodium products include those with an average molecular weight of approximately 5,000 (molecular weight 1,000 to 9,000; number of sulfate groups: approximately 30), those with an average molecular weight of 6,500 to 10,000 (number of sulfate groups: approximately 55), those with an average molecular weight of 9,000 to 20,000 (number of sulfate groups: approximately 94), and those with an average molecular weight of approximately 500,000 (number of sulfate groups: approximately 3,253). All of these products are suitable for use. Although low molecular weight heparin (molecular weight 4,000 to 6,000) can be used as heparin sodium, heparin with an average molecular weight of 10,000 to 15,000 is preferred. The higher the molecular weight of the polyanion, the more likely it is to suppress false negatives even at low concentrations, so it is preferable to use commercially available polyanions with a higher molecular weight.

[0025] In addition, PIPES (piperazine-1,4-bis(2-ethanesulfonic acid)) having two sulfate groups and HEPES (2-[4-(2-hydroxymethyl)piperazin-1-yl]ethanesulfonic acid) having one sulfate group are not preferred in the method of the present invention in terms of the degree of sulfation.

[0026] The concentration of the compound of the present invention in the sample treatment solution varies depending on the type thereof, but is preferably in the following ranges. When the compound of the present invention is polyphosphate or a polyphosphate salt (preferably sodium polyphosphate), the concentration of polyphosphate or polyphosphate salt in the sample treatment 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). When the compound of the present invention is heparin or a heparin salt (preferably sodium heparin), the concentration of heparin or heparin salt in the sample treatment 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). When the compound of the present invention is dextran sulfate or a dextran sulfate salt (preferably, dextran sulfate sodium), the concentration of dextran sulfate or a dextran sulfate salt in the sample treatment 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). The sample treatment solution adjusted to this concentration is added to the sample pad of the test strip of the immunochromatography device shown in Figure 1 below.

[0027] The pH of the sample treatment solution is preferably 6.5 to 8.5, more preferably 6.5 to 8.0, even more preferably 7.0 to 7.5, and even more preferably 7.4 to 7.5.

[0028] Examples of substances to be detected include allergens; microorganisms (for example, components of viruses, bacteria, fungi, or yeasts) or substances secreted by these; toxins; histamine; antibiotics; residual pesticides; and parasite antigens.

[0029] In the present invention, "allergen" refers to a substance (antigen) that primarily causes food allergies. Examples of causative ingredients include chicken, beef, pork, salmon, mackerel, shrimp, crab, abalone, squid, and salmon roe (food labeling standards based on Article 4, Paragraph 1 of the Food Labeling Act). Examples of viruses include norovirus, hepatitis E virus, and hepatitis A virus. Examples of bacteria include Salmonella, Campylobacter, Staphylococcus aureus, enterohemorrhagic Escherichia coli, pathogenic Escherichia coli other than enterohemorrhagic Escherichia coli, Clostridium perfringens, Vibrio parahaemolyticus, Clostridium botulinum, Bacillus cereus, Listeria, Shigella, Yersinia enterocolitica, Vibrio vulnificus, and Cronobacter sakazakii. Examples of fungi include Mucor plumbeus, Aspergillus flavus, Aspergillus niger, Paecilomyces variotii, Fusarium oxysporum, Rhizopus stolonifer, Penicillium roqueforti, etc. Examples of yeast include baker's yeast, brewer's yeast, sake yeast, and spoilage yeasts such as those of the genus Saccharomyces, Debaryomyces, Wickerhamomyces, and Zygosaccharomyces. Toxins include enterotoxins, endotoxins, botulinum toxin A, tetrodotoxin, etc. Antibiotics include penicillins, cephalosporins, tetracyclines, sulfonamides, aminoglycosides, aminocyclitols, macrolides, quinolones, ionophores, carbadox, nitrofurans, etc.

[0030] In the present invention, the term "residual pesticide" refers to a pesticide that remains in feed or the like and is concentrated in the bodies of animals or fish that eat it. Examples include dicarboximide pesticides such as iprodione; dithiolane pesticides such as isoprothiolane; acid amide pesticides such as flutolanil; triazole pesticides such as myclobutanil and bitertanol; imazalil and triflumizole; organohalogen pesticides such as chlorfenapyr and chlorothalonil; carbamate pesticides such as carbaryl; macrolide pesticides such as emamectin; neonicotinoid pesticides such as imidacloprid and acetamiprid; and organophosphorus pesticides such as malathion and isoxathion.

[0031] Parasites include Anisakis, Nybelinia, Tentacularia, Philometroides seriolae, Pennella, Didymozoidae, Toxoplasma gondii, Sarcocystis hominis, S. suihominis, S. fayeri, and Trichinella spp.

[0032] The method of the present invention targets, among these substances to be detected, bacterial components or substances secreted by bacteria, preferably bacterial intracellular antigens, more preferably bacterial ribosomal proteins, and even more preferably ribosomal protein L7 / L12. Ribosomal protein L7 / L12 has high detection sensitivity due to the presence of multiple copies within cells. Furthermore, as described in International Publication WO 2000 / 06603, it is possible to actually obtain antibodies capable of distinguishing specific bacteria that cause food poisoning from other bacteria by species or genus.

[0033] The method of the present invention can be carried out, for example, using a test strip for an immunochromatography device (hereinafter simply referred to as "immunochromatographic test strip") as shown in Figure 1. The immunochromatographic test strip (10) generally includes a porous membrane equipped with at least a sample pad (2), a development section (3), and a detection section (4). A labeled antibody application section (1) for a labeled antibody that forms a complex with the analyte is located upstream of the sample pad. The labeled antibody is elutable and retained at the development start site of the development section. The detection section is located downstream of the development section and has a capture antibody immobilized thereon. Furthermore, the sample pad, development section, and detection section are fixed in a line on a solid-phase support (6) such as polyethylene. A labeled control antibody is added to the labeled antibody application section in addition to the labeled antibody, and a primary antibody for the control antibody that binds to the labeled control antibody can also be added to the detection section. The labeled antibody application section may be located between the sample pad and the development section, or it may be located independently of the test strip in a container for adding a sample treatment solution. If the labeled antibody application section is located between the sample pad and the development section, the above-mentioned clogging by the sample (false negative) may occur upstream of the detection section. When the sample treatment solution is dripped onto the sample pad, if the sample contains an analyte, the analyte specifically binds to the labeled antibody in the development section to form a complex. This complex develops downstream in the development section and binds to the capture antibody in the detection section. In this way, a sandwich complex is formed between the labeled antibody, the analyte, and the capture antibody in the detection section, and the analyte can be analyzed qualitatively or quantitatively by detecting this complex. An example of a label that constitutes a labeled antibody is gold colloid particles, which enable qualitative detection through a color reaction caused by the gold colloid particles. Furthermore, quantitative detection of the analyte in the sample is also possible based on the degree of coloration. For details of immunochromatographic test strips, see, for example, Japanese Patent No. 5,693,938.

[0034] The immunochromatography device can also be provided as a kit that combines a container (for example, a microtube) that contains the immunochromatographic test strip, a polyanion, and, optionally, a compound having a bacteriolytic activity.

[0035] Immunochromatography is broadly divided into a lateral flow type, in which the immunochromatographic test strip is arranged horizontally and utilizes the movement of liquid due to capillary action (capillary force), and a flow-through type, in which the immunochromatographic test strip is arranged vertically and liquid passes from top to bottom due to gravity and capillary force. In the present invention, either the lateral flow type or the flow-through type may be used, but the lateral flow type is more preferred.

[0036] The nucleic acid chromatography method involves extracting a target nucleic acid from a specimen, using this target nucleic acid as a template to carry out a PCR amplification reaction with special tagged primers, developing the amplified target nucleic acid on a dedicated strip (nucleic acid chromatography chip), and converting the presence or absence of target DNA into the presence or absence of a band, which can then be visually determined (see, for example, JP 2014-82977 A).

[0037] The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples in any way.

[0038] Example 1 Materials and Preparation Methods 1. Preparation of Labeling Antibodies and Capture Antibodies Staphylococcus aureus was used as the immunogen bacterium. Antibodies against S. aureus ribosomal protein L7 / L12 were prepared according to the method described in International Publication No. WO 2000 / 06603. Specifically, Escherichia coli transformed with an expression vector incorporating DNA encoding the full-length amino acid sequence of S. aureus ribosomal protein L7 / L12 were cultured in LB medium and purified as a fusion protein using an affinity column tag sequence derived from the expression vector. Using this full-length L7 / L12 protein as an immunogen, mice were immunized four times with 50 μg of immunogen per dose, according to standard hybridoma isolation methods, with PBS adjusted to a concentration of 0.4 mg / mL. An equal volume of Freund's adjuvant was added. After confirming an increase in serum antibody titer by blood sampling, spleen cells were removed from the mice. Excised mouse spleen cells were fused with myeloma cells to obtain various hybridomas. The obtained hybridomas were cultured in HAT medium and screened using antibodies in the culture supernatant. Screening was performed by ELISA, and two hybridomas producing antibodies specifically reactive with Staphylococcus aureus were selected. Following standard methods for monoclonal antibody production, each selected hybridoma was cultured in TIL Media I medium supplemented with 10% fetal bovine serum (FBS) and administered intraperitoneally to mice, and ascites fluid was collected. The collected ascites fluid was centrifuged to separate floating matter and red blood cells, and then filtered through a 0.45 μm filter. The obtained filtrate was passed through a Protein G column to adsorb the antibodies, and the resulting antibodies were purified from the mouse ascites.

[0039] 2. Preparation of Immunochromato Test Strips Immunochromato test strips were prepared using the obtained antibodies as the following capture antibodies and labeling antibodies.

[0040] (1) Immunochromatographic Development Membrane Carrier A solution was prepared by mixing the capture antibodies in a 100 mM sodium phosphate buffer solution at a concentration of 1.5 mg / mL, and adding 3% (v / v) trehalose. The resulting solution was applied to a commercially available nitrocellulose membrane cut to a width of 2.5 cm and a length of 15 cm, with 1 cm of the solution being applied. ２ The solution was applied in an amount of 1 μL per sample to form a single line, and then dried to prepare a membrane carrier for immunochromatographic development.

[0041] (2) Preparation of Colloidal Gold Labeling Antibody and Colloidal Gold Labeling Antibody-Attached Member A 1 / 10 volume of the labeling antibody was added to a commercially available colloidal gold solution (particle size 60 nm) and mixed to prepare a solution with an antibody concentration of 0.1 mg / mL. This solution was left to stand at room temperature for 30 minutes to allow the antibody to bind to the surface of the colloidal gold particles. A BSA solution was then added to the colloidal gold solution to a final concentration of 0.1% for blocking, preparing a colloidal gold labeling antibody solution. This antibody solution was impregnated into a commercially available glass fiber sheet and then dried to prepare a colloidal gold labeling antibody-attached member.

[0042] (3) Preparation of immunochromatographic test strips In addition to the immunochromatographic development membrane carrier prepared in (1) and the gold colloid-labeled antibody-attached member prepared in (2), a polyester fiber nonwoven fabric was prepared as a member for adding specimens, and filter paper was prepared as an absorption member. These members were then attached to a commercially available polyethylene substrate, which was then cut to a width of 5 mm to prepare immunochromatographic test strips with the same configuration as shown in Figure 1.

[0043] Example 2 Inhibition of Colloidal Gold Clogging with Various Reaction Solution Compositions (1) Preparation of Bacterial Solution for Evaluation Beef (minced meat), pork (minced meat), chicken (minced meat), tuna (sashimi), and salmon (sashimi) were selected as food ingredients, and 5 g of each food ingredient was weighed out and placed in a stomacher bag containing 45 ml of physiological saline and kneaded for 60 seconds to obtain each food ingredient extract. Staphylococcus aureus was selected as the bacterium to be detected, and 1 × 10 ５ cfu / ml.

[0044] (2) Preparation of Various Reaction Solutions 5 μg / ml of lysostaphin was added to 0.1 M Tris-HCl (pH 7.4) buffer for lysis of Staphylococcus aureus, and 0.2% Tween (registered trademark) 20 was added for immunochromatographic development to prepare a mother solution. To this was added each of the additives shown in Table 1 to prepare various reaction solutions under the conditions shown in Table 1. For comparison, Table 1 also shows examples using sodium citrate (condition (1-6)), PIPES (condition (1-7)), and HEPES (condition (1-8)). The concentration of each additive was 0.5% (w / v).

[0045]

[0046] (3) Evaluation with immunochromatographic test strip The above-mentioned bacterial solution for evaluation and the above-mentioned various reaction solutions were mixed in a 1:1 ratio, and the immunochromatographic test strip prepared in Example 1 was inserted into this mixture to evaluate the presence or absence of detection by antigen-antibody reaction. The evaluation was performed by visually judging the degree of redness caused by the gold colloid particles in the test line area. A visually positive result was indicated as "+", a weakly visually positive result as "±", and a visually negative result with clogging of the gold colloid particles in the sample pad as "-". The results are shown in Table 2.

[0047]

[0048] In the reaction solution condition (1-1) using only the mother liquor, when an immunochromatographic test strip was inserted into various food ingredient extracts, the sample pad became clogged with gold colloid particles, resulting in a visually negative result. This is presumably due to protein aggregates, presumably cationic proteins, in the food ingredient extract inhibiting the movement of the gold colloid particles in the liquid. On the other hand, in the conditions (1-2) to (1-4) in which various polyanions were added, no clogging of the gold colloid particles in the sample pad was observed in the various food ingredient extracts, resulting in a visually positive result. Furthermore, in the condition (1-5) in which a polyanion having a carboxymethyl group was added, the condition (1-6) in which citric acid was added, or the conditions (1-7) to (1-8) in which a compound having two or less sulfate groups was added, the sample pad became clogged with gold colloid particles, resulting in a visually negative result, similar to the reaction solution condition (1-1) using only the mother liquor.

[0049] These results demonstrate that the use of specific polyanions can suppress false negatives caused by proteins suspected to be cationic proteins.

[0050] Example 3: Study of polyanion concentration (1) Preparation of bacterial solution for evaluation As ingredients, beef (minced meat), pork (minced meat), chicken (minced meat), tuna (sashimi), and salmon (sashimi) were selected, and 5 g of each ingredient was weighed out and placed in a stomacher bag containing 45 ml of physiological saline and kneaded for 60 seconds to obtain various ingredient extracts. Staphylococcus aureus was selected as the bacterium to be detected, and 1 × 10 ５ The culture medium was prepared using various food extracts or physiological saline to give a concentration of cfu / ml.

[0051] (2) Preparation of Various Reaction Solutions Using a 0.1 M Tris-HCl (pH 7.4) buffer solution containing 5 μg / ml lysostaphin for lysis of Staphylococcus aureus and 0.2% Tween (registered trademark) 20 for immunochromatographic development as a mother solution, various reaction solutions containing various additives at 0.0001 to 5% (w / v) were prepared. Note that sodium dextran sulfate and sodium heparin were prepared in the range of 0.0001 to 1% (w / v), and sodium polyphosphate and carboxymethyldextran were prepared in the range of 0.0001 to 5% (w / v).

[0052] (3) Evaluation with immunochromatographic test strips The above-mentioned bacterial solution for evaluation and the above-mentioned various reaction solutions were mixed in a 1:1 ratio, and an immunochromatographic test strip was inserted into this mixture to evaluate the presence or absence of detection by antigen-antibody reaction. The evaluation was performed by visually judging the degree of redness caused by the gold colloid particles in the test line area. A visually positive result was indicated as "+", a weakly visually positive result as "±", and a visually negative result with clogging of the gold colloid particles in the sample pad as "-". The results are shown in Table 3.

[0053]

[0054] When dextran sodium sulfate was used as an additive, a sufficient effect of inhibiting clogging of colloidal gold particles was confirmed when the additive concentration in the sample treatment solution was 0.0005% (w / v) or higher, resulting in a sufficiently high test line strength due to the antigen-antibody reaction. On the other hand, when the additive concentration in the sample treatment solution was 0.5% (w / v) or higher, the test line strength was confirmed to be reduced (visually weakly positive) despite no clogging of colloidal gold particles. Therefore, it was confirmed that a dextran sodium sulfate concentration in the sample treatment solution ranging from 0.0005 to 0.5% (w / v) is suitable.

[0055] When sodium polyphosphate was used as an additive, a sufficient effect of suppressing clogging of colloidal gold particles was confirmed at an additive concentration of 0.025% (w / v) or higher in the sample treatment solution, and the test line strength due to the antigen-antibody reaction was sufficiently high. On the other hand, when the additive concentration in the sample treatment solution was 2.5% (w / v) or higher, the test line strength decreased (visually weakly positive) even though clogging of colloidal gold particles did not occur. Therefore, it was confirmed that a sodium polyphosphate concentration in the sample treatment solution in the range of 0.25 to 2.5% (w / v) is suitable.

[0056] When heparin sodium was used as an additive, a sufficient effect of suppressing clogging of colloidal gold particles was confirmed at an additive concentration of 0.0005% (w / v) or higher in the sample treatment solution, and the test line strength due to the antigen-antibody reaction was sufficiently high. On the other hand, when the additive concentration in the sample treatment solution was 0.5% (w / v) or higher, the test line strength decreased (visually weak positive) even though clogging of colloidal gold particles did not occur. Therefore, it was confirmed that a heparin sodium concentration in the sample treatment solution in the range of 0.0005 to 0.5% (w / v) is suitable.

[0057] When carboxymethyldextran was used as an additive, clogging of the gold colloid particles occurred under all additive concentration conditions, resulting in visually negative results.

[0058] Example 4: Study of pH of polyanions (1) Preparation of bacterial solutions for evaluation As ingredients, beef (minced meat), pork (minced meat), chicken (minced meat), tuna (sashimi), and salmon (sashimi) were selected, and 5 g of each ingredient was weighed out and placed in a stomacher bag containing 45 ml of physiological saline and kneaded for 60 seconds to obtain various ingredient extracts. Staphylococcus aureus was selected as the bacterium to be detected, and 1 × 10 ５ The culture medium was prepared using various food extracts or physiological saline to give a concentration of cfu / ml.

[0059] (2) Preparation of various reaction solutions 5 μg / ml of lysostaphin was added to 0.1 M Tris-HCl (or MES-NaOH) buffer to lyse Staphylococcus aureus, and 0.2% Tween (registered trademark) 20 was added to prepare a mother solution. Various additives were adjusted to the desired concentrations, and the pH was then adjusted to prepare reaction solutions with different pHs (5.5 to 8.5). The concentrations of dextran sulfate sodium and heparin sodium were adjusted to 0.01% (w / v), and sodium polyphosphate was adjusted to 1% (w / v).

[0060] (3) Evaluation with Immunochromatographic Test Strips The above-mentioned bacterial solution for evaluation and the above-mentioned various reaction solutions were mixed in a 1:1 ratio, and an immunochromatographic test strip was inserted into this mixture to evaluate the presence or absence of detection by antigen-antibody reaction. The evaluation was performed by visually judging the degree of redness caused by the gold colloid particles in the test line area. A visually positive result was indicated as "+", a weakly positive result as "±", a visually negative result with the occurrence of clogging of the gold colloid particles in the sample pad as "-", and a visually negative result with no clogging of the gold colloid particles in the sample pad as "- -". The results are shown in Table 4.

[0061]

[0062] The pH values ​​in Table 4 refer to the pH values ​​of the sample treatment solution. Table 4 confirms that under conditions (3-8) to (3-28) in which a polyanion was added, the effect of suppressing clogging of colloidal gold particles was sufficiently achieved by setting the pH of the sample treatment solution in the range of 6.5 to 8.5. On the other hand, under conditions (3-1) to (3-7) in which no polyanion was added, clogging of colloidal gold particles occurred in various food extracts under all pH conditions.

[0063] Under conditions where the pH was above 8.5, the effect of suppressing clogging of gold colloid particles was confirmed even without adding polyanions, but the detection sensitivity tended to decrease.

[0064] Example 5: Observation of the surface of a sample pad clogged with gold colloid particles (1) Preparation of bacterial solution for evaluation: 5 g of minced chicken was selected as the food material, and the food material was weighed out and placed in a stomacher bag containing 45 ml of physiological saline and kneaded for 60 seconds to obtain a food material extract. Staphylococcus aureus was selected as the bacterium to be detected, and 1 × 10 ５ The concentration was adjusted to cfu / ml using food extract.

[0065] (2) Preparation of Various Reaction Solutions A mother solution was prepared by adding 5 μg / ml of lysostaphin to 0.1 M Tris-HCl (pH 7.4) buffer solution for lysis of Staphylococcus aureus and 0.2% Tween (registered trademark) 20 for immunochromatographic development. To this mother solution, 0.5% (w / v) of dextran sodium sulfate was added to prepare "reaction solution A," and "reaction solution B," which consisted of the mother solution alone.

[0066] (3) Observation of the surface of the sample pad after evaluation of the immunochromatographic test strip The above-mentioned bacterial solution for evaluation and the above-mentioned various reaction solutions were mixed in a 1:1 ratio, and the immunochromatographic test strip was inserted into this mixed solution. After that, the sample pad was cut out at the position in contact with the liquid surface (indicated by 7 in Figure 1), and the surface of the sample pad was observed with a scanning electron microscope (FlexSEM1000, Hitachi High-Tech Corporation).

[0067] When the evaluation bacterial solution was treated with reaction solution A, no clogging of the sample pad with colloidal gold particles was observed. On the other hand, when the evaluation bacterial solution was treated with reaction solution B, reddish aggregates were observed on the sample pad, confirming clogging of the colloidal gold particles. Figure 2 shows a surface image of the sample pad before the addition of the specimen, and Figure 3 shows a surface image of the sample pad after the evaluation bacterial solution was treated with reaction solution B (both at 300x magnification). As can be seen from the figures, when the evaluation bacterial solution was treated with reaction solution B, aggregate-like substances were found to be clogging the fibers of the sample pad compared to before the addition of the specimen. Figure 4 shows a surface image of the sample pad at a higher magnification (5000x magnification) after the evaluation bacterial solution was treated with reaction solution B. From the figure, particulate matter was observed adhering to the surface of the aggregates. These aggregates were presumed to be aggregates of a protein, presumed to be cationic proteins, and these particulate matter was presumed to be colloidal gold particles.

[0068] 5 and 6 show surface images of the sample pad after the bacterial solution for evaluation was treated with reaction solution A. From the figures, it was observed that what appeared to be aggregates were lodged between the fibers of the sample pad (however, the amount of aggregation was reduced compared to when treated with reaction solution B), but it was confirmed that no particulate matter was attached to the surface of the aggregates.

[0069] Therefore, when the bacterial solution for evaluation is treated with a polyanion such as dextran sulfate, (i) the polyanion reduces the formation of secondary aggregation of aggregates derived from proteins presumed to be cationic proteins, and (ii) the polyanion masks the aggregates of proteins presumed to be cationic proteins that were already present, thereby neutralizing the positive charge of the protein aggregates and blocking the binding of the protein aggregates to negatively charged gold colloid particles, suggesting that clogging of the sample pad is eliminated. This elimination of clogging also suggests that the desired antigen-antibody reaction complex is formed, suppressing false negatives.

[0070] The method of the present invention can be used for rapid and accurate testing of target substances in meat, seafood, etc.

[0071] 10 Test strip of immunochromatography device 1 Labeled antibody application section 2 Sample pad 3 Development section 4 Detection section 5 Absorption pad 6 Solid phase support 7 Liquid level

Claims

1. A method for detecting a substance to be detected in a sample, the method comprising treating the sample or an extract of the sample with a compound or a salt thereof having a functional group whose acid dissociation constant (pKa) in water at 25°C is 3 or less.

2. A method for suppressing false negatives in an immunochromatography or nucleic acid chromatography method for detecting a substance to be detected in a sample, the method comprising treating the sample or an extract of the sample with a compound or a salt thereof having a functional group whose acid dissociation constant (pKa) in water at 25°C is 3 or less.

3. The method of claim 2, wherein the false negatives are due to protein aggregates derived from the sample.

4. The method according to claim 2, wherein the false negative is due to clogging of the membrane with protein aggregates derived from the sample when the sample is added to an immunochromatographic test strip (10) including at least a sample pad (2), a development section (3), and a detection section (4) in that order from upstream to downstream.

5. The method of claim 1 or 2, wherein the sample is a foodstuff sample containing protein or an environmental sample containing foodstuff residues containing protein.

6. The method according to claim 1 or 2, wherein the specimen is meat or seafood.

7. The method of claim 6, wherein the meat is selected from the group consisting of chicken, pork, beef, horse, lamb, venison, and whale, and the seafood is selected from the group consisting of tuna, salmon, sea bass, shrimp, squid, octopus, and shellfish.

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

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

10. The method of claim 1 or 2, wherein the compound or a salt thereof is polyphosphate, heparin or dextran sulfate, or a salt thereof.

11. The method according to claim 1 or 2, wherein the compound or its salt is polyphosphoric acid or a polyphosphate (degree of phosphoric acid polymerization: 60 to 100), and the concentration of polyphosphoric acid or the polyphosphate in a specimen treatment solution obtained after treating the specimen or an extract of the specimen 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 a heparin salt, and the concentration of heparin or a heparin salt in a specimen treatment solution after treating the specimen or an extract of the specimen 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 a dextran sulfate salt, and the concentration of dextran sulfate or the dextran sulfate salt in a specimen treatment solution after treating the specimen or an extract of the specimen 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 substance to be detected is selected from the group consisting of allergens; components of viruses, bacteria, fungi, or yeasts or substances secreted by them; toxins; histamine; antibiotics; pesticide residues; and parasitic antigens.

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

16. The method according to claim 1 or 2, wherein the substance to be detected is an intracellular antigen of a bacteria.

17. The method according to claim 1 or 2, wherein the substance to be detected is a bacterial ribosomal protein.

18. The method according to claim 1 or 2, wherein the substance to be detected is ribosomal protein L7 / L12.

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