Test Kit with Improved Specificity by Suppressing False Positives

Abstract

To provide a specimen extraction liquid that contains a component capable of strongly suppressing false positive reactions which, when detecting, with an inspection reagent that utilizes antigen antibody reaction or mutual reaction of substances having mutual action, antigens such as viruses, bacteria, proteins to be detected, etc., from specimens derived from body fluids such as nasal muddy specimens, nasal suction specimens, nasal cleaning specimens, nasal chinose specimens, studyngeal specimens, saliva specimens, excrement specimens, serum specimens and urine specimens, could not fully be suppressed by conventional methods, and a specimen extraction method and an inspection reagent using said specimen extraction liquid and method.SOLUTION: A water-soluble chemical compound having, in a structure, a phenyl group or a benzyl group or a tolyl group or a xylyl group, to which at least a carboxyl group or a functional group including its methylated or ethylated atom group or a hydroxyl group is bound, is included in the specimen extraction liquid of an inspection reagent having the specimen extraction liquid in a structure or a member, etc., that comes into contact with a specimen in a process preceding the detection reaction of the object to be detected or a simultaneous process as the detection reaction.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a technique for strongly suppressing false positive reactions that could not be completely suppressed by conventional methods by using a compound having a specific chemical structure in a specimen extract, a member that comes into contact with the specimen in a step before the detection reaction or a step simultaneous with the detection reaction, when detecting a detected substance such as a virus, a bacterium, or a protein to be detected from a specimen derived from a body fluid such as a nasal swab specimen, a nasal aspirate specimen, a nasal wash specimen, a nasal mucus specimen, a throat swab specimen, a saliva specimen, a fecal specimen, a serum specimen, a plasma specimen, a urine specimen, etc., using a detection reagent that utilizes an antigen-antibody reaction or a binding reaction between substances having an interaction.

Background Art

[0002] In recent years, various test reagents and kits for detecting the presence or absence of pathogen infections such as viruses and bacteria, the presence or absence of pregnancy, etc., using an antigen-antibody reaction or a binding reaction between substances having an interaction have been developed one after another. All test reagents include a pretreatment step for creating conditions suitable for the detection reaction after collecting a specimen from a patient, and this step is important for obtaining accurate results. In particular, many simple test reagents are characterized by not requiring special equipment, being easy to operate, and being inexpensive, and are widely used not only in large hospitals and medical testing centers but also in general hospitals and clinics, and are often used by users other than medical testing experts. Therefore, it is very important that the test accuracy of the reagent is high. Examples of simple test reagents currently on the market include simple test reagents for detecting pathogen infections and simple test reagents for pregnancy diagnosis. These test reagents are often carried out at the medical institutions where patients first visit, and the presence or absence of infection or pregnancy can be determined on the spot for the specimens collected from the patients, and treatment measures etc. can be taken at an early stage, so the importance of simple test reagents in medicine is increasing more and more. And with the increasing use of simple test reagents, users are demanding higher reproducibility of test results and test accuracy as the performance of the reagents.

[0003] Currently, as a typical reagent for simple inspection methods, immunoassay methods that utilize antigen-antibody reactions, particularly immunochromatography, are generally known. In the immunochromatography method, a complex of a capturer (capturing substance) that specifically binds to the substance to be detected and a label that specifically binds to the substance to be detected is formed on a membrane, and the label is detected / quantified to detect (measure or quantify) the substance to be detected. The immunochromatography method is widely used for detecting a wide variety of substances to be detected because the measuring device is simple and it is also excellent in terms of cost.

[0004] In one form of the immunochromatography method, a detection unit in which an antibody that specifically binds to the substance to be detected is immobilized as a capturing substance on a membrane strip such as nitrocellulose, and a labeling unit containing a label that specifically binds to the substance to be detected are provided. A sample solution containing the substance to be detected is dropped onto the inspection device, and while forming a complex of the substance to be detected-label, it is developed and the complex is captured by the detection unit to detect or quantify the label.

[0005] In recent years, regarding clinical diagnostic reagents including the immunochromatography method, it has been desired from the clinical field that the diagnostic results be more reliable, and further improvement in the reliability of the reagents has become an issue. A highly reliable inspection reagent is an inspection reagent with high sensitivity and specificity that is less likely to cause misjudgment. In particular, regarding specificity, there has always been a technical issue of how to deal with the diversity of sample components derived from differences in the background of each patient in reagent design, and more effective elimination of non-specific reactions is an extremely important issue in simple inspection methods. In order to solve these problems, it has been reported that contacting the sample with basic amino acids such as arginine and lysine, inorganic salts, glycine ethyl ester, surfactants, animal-derived immunoglobulins, etc. has a certain effect on improving specificity (see Patent Documents 1, 2, and 3), but all of them have limited effects and non-specific reactions that cannot be suppressed still exist. Therefore, a technique that more strongly improves specificity and does not reduce sensitivity is desired.

Prior Art Documents

Patent Documents

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2003-279577 [Patent Document 2] Japanese Patent Application Laid-Open No. 2005-24323 [Patent Document 3] Japanese Patent Application Laid-Open No. 2004-301684 [Summary of the Invention] [Problems to be Solved by the Invention]

[0007] When detecting a substance to be detected such as a virus, a bacterium, or a protein to be detected from a sample derived from a body fluid such as a nasal swab sample, a nasal aspiration sample, a nasal lavage sample, a nasal mucus sample, a pharyngeal swab sample, a saliva sample, a fecal sample, a serum sample, a plasma sample, or a urine sample using a detection reagent that utilizes an antigen-antibody reaction or a binding reaction between substances having an interaction, false positive reactions and false negative reactions that could not be completely suppressed by conventional methods still exist, which is one of the factors hindering accurate diagnosis. In the present invention, there is provided a test reagent using a sample extraction solution or a sample extraction method containing a component capable of strongly suppressing false positive reactions without reducing sensitivity. [Means for Solving the Problems]

[0008] As a result of intensively searching for a method of more strongly suppressing non-specific reactions that occur when samples derived from body fluids such as nasal swab samples, nasal aspiration samples, nasal lavage samples, nasal mucus samples, pharyngeal swab samples, saliva samples, fecal samples, serum samples, plasma samples, and urine samples are used as test samples, the present inventors have found a component that can suppress false positive reactions significantly more than conventional methods. Furthermore, by adding this component to a sample extraction solution or a member that comes into contact with the sample in a step before the detection reaction or in a step simultaneous with the detection reaction, it has been found that false positive reactions detected by the conventional method can be suppressed, and the present invention has been completed.

[0009] That is, the present invention has the following configuration. [1] An inspection reagent including a specimen extract containing a non-specific reaction suppressing component that suppresses false positives, which is a water-soluble compound having a phenyl group, a benzyl group, a tolyl group, or a xylyl group, further having a carboxyl group, a methoxycarbonyl group, or an ethoxycarbonyl group, and optionally further having a hydroxyl group, and having a molecular weight of 6000 Da or less, for detecting a substance to be detected in a specimen by utilizing a binding reaction between substances having an antigen-antibody reaction or interaction. [2] An inspection reagent for immunochromatography, which is the inspection reagent of [1], including a specimen extract containing a non-specific reaction suppressing component that suppresses false positives, which is a water-soluble compound having a phenyl group, a benzyl group, a tolyl group, or a xylyl group, further having a carboxyl group, a methoxycarbonyl group, or an ethoxycarbonyl group, and optionally further having a hydroxyl group, and having a molecular weight of 6000 Da or less, and being for an immunochromatography inspection device. [3] An immunochromatography inspection device including a site impregnated with a specimen extract containing a non-specific reaction suppressing component that suppresses false positives, which is a water-soluble compound having a phenyl group, a benzyl group, a tolyl group, or a xylyl group, further having a carboxyl group, a methoxycarbonyl group, or an ethoxycarbonyl group, and optionally further having a hydroxyl group, and having a molecular weight of 6000 Da or less, and being the inspection reagent of [1] or [2]. [4] The inspection reagent of any one of [1] to [3], wherein the specimen extract contains a non-specific reaction suppressing component that suppresses false positives at 0.1 to 10 (w / v)%.

[0010] [5] The inspection reagent of any one of [1] to [4], which is a compound represented by any of the following general formulas (I) to (V) or tryptophan: [Chemical formula] [In general formula (I), R1 is H, OH, =O, NH2, COOH, NH-CO-CNH2-C-COOH, or CH3; R2 is H, CH3, C2H5, Li, Na, K, Rb, Cs, or Fr; n is 0 or 1; and m is 0, 1, 2, 3, or 4.] [Chemistry] [In general formula (II), R2 is H, CH3, C2H5, Li, Na, K, Rb, Cs or Fr, R3 is COOH, COOLi, COONa, COOK, COORb, COOCs, COOFr, COOCH3, COOC2H5, OCOH or CH3, and R3 and COOR2 are located ortho, meta or para to the benzene ring.] [Chemistry] [In general formula (III), R2 is H, CH3, C2H5, Li, Na, K, Rb, Cs or Fr, R4 is H or CH3, and n is 0 or 1.] [Chemistry] [In general formula (IV), R5 is the side chain of glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, asparagine, glutamine, phenylalanine, tyrosine, tryptophan, glutamic acid, aspartic acid, arginine, lysine or histidine.] [Chemistry] [In general formula (V), R2 is H, CH3, C2H5, Li, Na, K, Rb, Cs or Fr, R6 is NH or O, and n is 0, 1, 2, 3 or 4.].

[0011] [6] A compound having a phenyl group, benzyl group, tolyl group or xylyl group, further having a carboxyl group, methoxycarbonyl group or ethoxycarbonyl group, and optionally further having a hydroxyl group, having water solubility and a molecular weight of 6000 Da or less, is a compound selected from the group consisting of aspartame, phenylalanine, phenylalanine methyl ester, mandelic acid, 2-phenylpropionic acid, 3-phenylpropionic acid, phenylglycine, phenylglycine methyl ester, phenylglycine ethyl ester, phenyl lactic acid, phenylpyruvic acid, benzoic acid, phthalic acid, acetylsalicylic acid, hippuric acid, N-toluoyl glycine, N-carbobenzyloxy amino acid, N-phenylglycine, phenoxyacetic acid, tryptophan, and metal salts of these compounds, as well as optical isomers, geometric isomers, structural isomers, stereoisomers and positional isomers of these compounds, and is a test reagent according to any one of [1] to [5]. [7] A test reagent according to any one of [1] to [6], wherein the sample extract further contains an amino acid or amino acid derivative selected from the group consisting of arginine, lysine, arginine ethyl ester, arginine methyl ester, glycine ethyl ester and glycine methyl ester, and optical isomers, geometric isomers, structural isomers and stereoisomers of these compounds. [8] A test reagent according to any one of [1] to [7], wherein the sample extract further contains a halide selected from the group consisting of lithium chloride, sodium chloride, potassium chloride, sodium bromide, potassium bromide, sodium iodide and potassium iodide.

[0012] [9] In a method for detecting a substance to be detected selected from the group consisting of viral antigens, bacterial antigens, and protein antigens in a specimen selected from the group consisting of throat swab specimens, nasal swab specimens, nasal aspirate specimens, throat wash specimens, nasal wash specimens, nasal mucus specimens, saliva specimens, serum specimens, plasma specimens, whole blood specimens, fecal specimens, fecal suspension specimens, and urine specimens, by utilizing the reaction between substances having an antigen-antibody reaction or interaction contained in a specimen extract, the specimen is preliminarily contacted with a non-specific reaction suppressing component that is a water-soluble compound having a phenyl group, benzyl group, tolyl group, or xylyl group, further having a carboxyl group, methoxycarbonyl group, or ethoxycarbonyl group, and optionally further having a hydroxyl group, and having a molecular weight of 6000 Da or less, to suppress false positive reactions and perform detection.

[10] The method for detecting a substance to be detected is an immunochromatographic method, wherein the specimen is placed in a specimen extract containing a non-specific reaction suppressing component that is a water-soluble compound having a phenyl group, benzyl group, tolyl group, or xylyl group, further having a carboxyl group, methoxycarbonyl group, or ethoxycarbonyl group, and optionally further having a hydroxyl group, and having a molecular weight of 6000 Da or less to suppress false positive reactions, and the specimen extract is added to an immunochromatographic test device. The method according to [9].

[11] The method for detecting a substance to be detected is an immunochromatographic method, wherein the specimen is added to an immunochromatographic test device containing a site impregnated with a specimen extract containing a non-specific reaction suppressing component that is a water-soluble compound having a phenyl group, benzyl group, tolyl group, or xylyl group, further having a carboxyl group, methoxycarbonyl group, or ethoxycarbonyl group, and optionally further having a hydroxyl group, and having a molecular weight of 6000 Da or less to suppress false positive reactions. The method according to [9].

[12] The method according to any one of [9] to

[11] , wherein the specimen extract contains a non-specific reaction suppressing component that suppresses false positive reactions at 0.1 to 10 (w / v)%.

[0013]

[13] A compound having a phenyl group, a benzyl group, a tolyl group or a xylyl group, further having a carboxyl group, a methoxycarbonyl group or an ethoxycarbonyl group, and optionally further having a hydroxyl group, having water solubility and a molecular weight of 6000 Da or less, is a compound represented by any of the following general formulas (I) to (V) or tryptophan, by any of the methods of [9] to

[12] : [Chemical formula] [In general formula (I), R1 is H, OH, =O, NH2, COOH, NH-CO-CNH2-C-COOH or CH3, R2 is H, CH3, C2H5, Li, Na, K, Rb, Cs or Fr, n is 0 or 1, and m is 0, 1, 2, 3 or 4.] [Chemical formula] [In general formula (II), R2 is H, CH3, C2H5, Li, Na, K, Rb, Cs or Fr, R3 is COOH, COOLi, COONa, COOK, COORb, COOCs, COOFr, COOCH3, COOC2H5, OCOH or CH_{3}, and R3 and COOR2 are located ortho, meta or para to the benzene ring.] [Chemical formula] [In general formula (III), R2 is H, CH3, C2H5, Li, Na, K, Rb, Cs or Fr, R4 is H or CH3, and n is 0 or 1.] [Chemical formula] [In general formula (IV), R5 is the side chain of glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, asparagine, glutamine, phenylalanine, tyrosine, tryptophan, glutamic acid, aspartic acid, arginine, lysine or histidine.] [Chemical formula] [In general formula (V), R2 is H, CH3, C2H5, Li, Na, K, Rb, Cs or Fr, R6 is NH or O, and n is 0, 1, 2, 3 or 4.].

[0014]

[14] A compound having a phenyl group, benzyl group, tolyl group or xylyl group, further having a carboxyl group, methoxycarbonyl group or ethoxycarbonyl group, and optionally further having a hydroxyl group, having water solubility and a molecular weight of 6000 Da or less is selected from the group consisting of aspartame, phenylalanine, phenylalanine methyl ester, mandelic acid, 2-phenylpropionic acid, 3-phenylpropionic acid, phenylglycine, phenylglycine methyl ester, phenylglycine ethyl ester, phenyl lactic acid, phenylpyruvic acid, benzoic acid, phthalic acid, acetylsalicylic acid, hippuric acid, N-toluoyl glycine, N-carbobenzyloxy amino acid, N-phenylglycine, phenoxyacetic acid, tryptophan, and metal salts of these compounds, and optical isomers, geometric isomers, structural isomers, stereoisomers and positional isomers of these compounds. The method according to any one of [9] to

[13] .

[15] The method according to any one of [9] to

[14] , wherein the sample extract further contains an amino acid or amino acid derivative selected from the group consisting of arginine, lysine, arginine ethyl ester, arginine methyl ester, glycine ethyl ester and glycine methyl ester, and optical isomers, geometric isomers, structural isomers and stereoisomers of these compounds.

[16] The method according to any one of [9] to

[15] , wherein the sample extract further contains a halide selected from the group consisting of lithium chloride, sodium chloride, potassium chloride, sodium bromide, potassium bromide, sodium iodide and potassium iodide. [Advantages of the Invention]

[0015] According to the present invention, a detection reagent for detecting specific viruses, bacteria, proteins, low-molecular-weight compounds, etc., utilizing antigen-antibody reactions or binding reactions between interacting substances in samples derived from bodily fluids such as nasal swab samples, nasal aspirate samples, nasal wash samples, nasal mucus samples, throat swab samples, saliva samples, stool samples, serum samples, plasma samples, and urine samples, can be provided that more effectively suppresses false positive reactions caused by sample contamination, resulting in highly reproducible and highly accurate test reagents.Furthermore, it is possible to more effectively prevent erroneous clinical diagnoses due to non-specific reactions, which is beneficial for both patients and users such as doctors, laboratory technicians, and nurses. [Brief description of the drawings]

[0016]

Figure 1

[0017] The present invention will be described in detail below. The present invention provides a method for detecting a substance to be detected in a sample using a detection reagent that utilizes an antigen-antibody reaction or a binding reaction between interacting substances, by contacting the sample with a compound, thereby suppressing false positive reactions and preventing a decrease in signal, i.e., a decrease in sensitivity. In the present invention, antibodies also include antigen-binding fragments of antibodies.

[0018] (Specimen) The specimen to be used is not limited. For example, specimens include throat swabs, nasal swabs, nasal aspirates, throat washes, nasal washes, blown nasal secretions, saliva, serum, plasma, whole blood, fecal suspensions, urine, culture fluid, etc. These specimens are called throat swab specimens, nasal swab specimens, nasal aspirates, throat wash specimens, nasal wash specimens, blown nasal secretions, saliva specimens, serum specimens, plasma specimens, whole blood specimens, fecal suspension specimens, urine specimens, culture fluid specimens, etc. They can be used after dilution with a buffer solution, or as is.

[0019] (Substances to be detected) The substance to be detected is not limited in any way and can be any substance to be detected. Specific examples include viral antigens such as influenza virus, adenovirus, respiratory syncytial (RS) virus, human metapneumovirus (hMPV), hepatitis A virus (HAV), hepatitis B virus (HBV), human immunodeficiency virus (HIV), norovirus, and coronaviruses such as SARS-CoV, MERS-CoV, and SARS-CoV-2; bacterial antigens such as methicillin-resistant Staphylococcus aureus (MRSA), group A streptococcus, group B streptococcus, and Legionella bacteria; toxins produced by bacteria and the like; mycoplasma antigens; chlamydia antigens such as Chlamydia trachomatis; antigens of protozoa; antigens of fungi; hormones such as human chorionic gonadotropin; proteins such as C-reactive protein, myoglobin, cardiac troponin, and procalcitonin; various tumor markers; antigens such as pesticides and environmental hormones. Furthermore, antibodies against the above-mentioned bacteria, viruses, etc. can be mentioned.

[0020] (Sampling of Specimen) The method of sampling the specimen is not limited in any way. Examples include methods of sampling specimens derived from body fluids and excreta such as throat swab specimens, nasal swab specimens, nasal aspirate specimens, throat wash specimens, nasal mucus specimens, saliva specimens, serum specimens, plasma specimens, whole blood specimens, fecal specimens, fecal suspension specimens, urine specimens, and culture fluid specimens using a specimen sampling instrument such as a cotton swab, methods of sampling using suction by a suction device, methods of sampling using a blood collection tube, etc.

[0021] (Contact between the non-specific reaction suppressing component for suppressing false positives and the collected specimen) In the method of the present invention, a sample is brought into contact with a non-specific reaction suppressing component that suppresses false positives. Here, by bringing the sample into contact with the non-specific reaction suppressing component that suppresses false positives, false positives can be suppressed when the sample is measured. In addition, when the sample is brought into contact with the non-specific reaction suppressing component that suppresses false positives, the substance to be detected and the non-specific reaction suppressing component that suppresses false positives come into contact with each other. Therefore, in the method of the present invention, it is also said that the substance to be detected is brought into contact with the non-specific reaction suppressing component that suppresses false positives. Further, bringing the sample into contact with the non-specific reaction suppressing component that suppresses false positives is sometimes referred to as treating the sample with the non-specific reaction suppressing component that suppresses false positives.

[0022] In addition, the sample extract refers to a liquid that suspends the substance to be detected in the sample to make it easier to measure. For example, it is not necessary to extract a specific substance to be detected from cells or the like by dissolution or the like, and it can simply be referred to as a sample treatment solution, a sample dilution solution, a sample suspension solution, or the like.

[0023] In the present invention, it is necessary to contact the sample with the non-specific reaction suppressing component that suppresses false positives in advance before subjecting them to a test. Here, "before subjecting them to a test" refers to before the substance to be detected in the sample reacts with an antibody or antigen against it, or before the substance to be detected in the sample reacts with a substance having an interaction. The reaction with an antibody or antigen refers to the binding with an antibody or antigen, and the reaction between the substance to be detected in the sample and a substance having an interaction refers to the binding with a substance having an interaction.

[0024] As a method of contacting the non-specific reaction suppressing component that suppresses false positives of the present invention with the sample, there are a method of putting the sample into a solution containing the non-specific reaction suppressing component that suppresses false positives and mixing them to bring them into contact, and a method of including a sample extract containing the non-specific reaction suppressing component that suppresses false positives in a test device used for measurement, and adding the sample to the test device used for the test to bring the sample into contact with the non-specific reaction suppressing component that suppresses false positives.

[0025] As a specific example of a method of mixing and contacting a sample with a solution containing a non-specific reaction inhibitory component that suppresses false positives, a non-specific reaction inhibitory component that suppresses false positives is included in a sample extract that suspends and disperses the collected sample, and when the sample is added to and mixed with the sample extract, the sample is brought into contact with the non-specific reaction inhibitory component that suppresses false positives. For example, when using a sample extract containing a non-specific reaction inhibitory component that suppresses false positives, if the sample is a nasal swab, use a cotton swab to collect the nasal swab, and place the cotton swab soaked with the collected sample into the sample extract to suspend and disperse the sample for extraction, whereby the sample can be brought into contact with the non-specific reaction inhibitory component that suppresses false positives.

[0026] As a specific example of a method of including a sample extract containing a non-specific reaction inhibitory component that suppresses false positives in a test device used for measurement and bringing the sample into contact with the non-specific reaction inhibitory component that suppresses false positives by adding the sample to the test device, a sample extract containing a non-specific reaction inhibitory component that suppresses false positives is impregnated or coated on a fibrous or porous substrate such as a pad or a filter made of non-woven fabric, woven fabric, sponge, etc. that the test device has, and when the collected sample is added to the test device, the sample is brought into contact with the non-specific reaction inhibitory component that suppresses false positives included in the fibrous or porous substrate. As such a test device, a device for an immunochromatographic method described later can be mentioned.

[0027] The material of the porous substrate of the test device is not limited, and examples include pulp, cotton, wool, polyester, polypropylene, nylon, acrylic glass fiber, nitrocellulose, etc. When the test device used for measurement is impregnated with a specimen extract containing a nonspecific reaction inhibitor that suppresses false positives, and the specimen is added to the test device to contact the specimen with the nonspecific reaction inhibitor that suppresses false positives, for example, the specimen extract containing the nonspecific reaction inhibitor that suppresses false positives is impregnated into the porous substrate and dried, and the specimen is contacted with the porous substrate in a step before or simultaneously with the reaction to detect the analyte on the test device. For example, when the specimen is added to the test device, the specimen spreads on the test device and reaches the reaction site on the device where a reaction such as an antibody-antigen reaction occurs. By providing a porous substrate impregnated with a specimen extract containing a nonspecific reaction inhibitor that suppresses false positives at a site on the test device before the reaction site, the specimen comes into contact with the nonspecific reaction inhibitor that suppresses false positives before the reaction. For example, a filter may be used as the porous substrate, and a filter containing a specimen extract containing a non-specific reaction inhibitor that suppresses false positives may be provided at the site where the specimen is added. In this case, when a nasal swab is used as the specimen, the nasal swab may be collected using a cotton swab, and the cotton swab soaked with the collected specimen may be placed in a specimen extract of any composition that does not contain a non-specific reaction inhibitor that suppresses false positives, thereby dispersing and dissolving the specimen. After that, a filter containing a non-specific reaction inhibitor that suppresses false positives, which is a component of the test device, may be impregnated with the specimen extract, thereby bringing the specimen into contact with the non-specific reaction inhibitor that suppresses false positives.

[0028] (Non-specific reaction inhibitors and concentrations that suppress false positives) The nonspecific reaction suppressing component of the present invention that suppresses false positives refers to a water-soluble compound having a molecular weight of 6000 Da or less, which has a phenyl group, a benzyl group, a tolyl group, or a xylyl group, and further has a carboxyl group, a methoxycarboxyl group, or an ethoxycarboxyl group, and optionally further has a hydroxyl group. It also includes optical isomers, geometric isomers, structural isomers, stereoisomers, and positional isomers, including metal salts, of these compounds.

[0029] These compounds are compounds represented by any one of the following general formulas (I) to (V) or tryptophan.

[0030] [ka] [In general formula (I), R1 is H, OH, =O, NH2, COOH, NH-CO-CNH2-C-COOH or CH3, R2 is H, CH3, C2H5, Li, Na, K, Rb, Cs or Fr, n is 0 or 1, and m is 0, 1, 2, 3 or 4.]

[0031] [ka] [In general formula (II), R2 is H, CH3, C2H5, Li, Na, K, Rb, Cs, or Fr, R3 is COOH, COOLi, COONa, COOK, COORb, COOCs, COOFr, COOCH3, COOC2H5, OCOH, or CH3, and R3 and COOR2 are located at the ortho, meta, or para positions of the benzene ring.]

[0032] [ka] [In general formula (III), R2 is H, CH3, C2H5, Li, Na, K, Rb, Cs, or Fr, R4 is H or CH3, and n is 0 or 1.]

[0033] [ka] [In general formula (IV), R5 is the side chain of glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, asparagine, glutamine, phenylalanine, tyrosine, tryptophan, glutamic acid, aspartic acid, arginine, lysine or histidine.]

[0034] [Chemical formula] [In general formula (V), R2 is H, CH3, C2H5, Li, Na, K, Rb, Cs or Fr, R6 is NH or O, and n is 0, 1, 2, 3 or 4.]

[0035] Although not limited, for example, aspartame, phenylalanine, phenylalanine methyl ester (including hydrochloride), mandelic acid, 2-phenylpropionic acid, 3-phenylpropionic acid, phenylglycine, phenylglycine methyl ester, phenylglycine ethyl ester, phenyl lactic acid, phenylpyruvic acid, benzoic acid, phthalic acid, acetylsalicylic acid, hippuric acid, N-toluoyl glycine, N-carbobenzyloxy amino acid, N-phenylglycine, phenoxyacetic acid, tryptophan, and metal salts thereof are included. Compounds selected from the group consisting of optical isomers, geometric isomers, structural isomers, stereoisomers and positional isomers are included. In the present invention, when referring to aspartame, phenylalanine, phenylalanine methyl ester (including hydrochloride), mandelic acid, 2-phenylpropionic acid, 3-phenylpropionic acid, phenylglycine, phenylglycine methyl ester, phenylglycine ethyl ester, phenyl lactic acid, phenylpyruvic acid, benzoic acid, phthalic acid, acetylsalicylic acid, hippuric acid, N-toluoyl glycine, N-carbobenzyloxy amino acid, N-phenylglycine, phenoxyacetic acid, tryptophan, their metal salts are included, and further optical isomers, geometric isomers, structural isomers, stereoisomers and positional isomers are also included. Examples of the metal salts include Li salts, Na salts, K salts, Rb salts, Cs salts, Fr salts, etc.]

[0036] Examples of compounds represented by the above general formula (I) include aspartame, phenylalanine, phenylalanine methyl ester, mandelic acid, 2-phenylpropionic acid, 3-phenylpropionic acid, phenylglycine, phenylglycine methyl ester, phenylglycine ethyl ester, phenyllactic acid, phenylpyruvic acid, and benzoic acid. Compounds represented by general formula (II) include phthalic acid and acetylsalicylic acid. Examples of the compound represented by general formula (III) include hippuric acid and N-toluoylglycine. An example of the compound represented by general formula (IV) is N-carbobenzyloxyamino acid. Examples of the compound represented by general formula (V) include N-phenylglycine and phenoxyacetic acid.

[0037] Nonspecific reaction inhibitors that suppress these false positives are contained in components such as sample extracts and filter filters that come into contact with samples prior to the detection reaction. The concentration is preferably 0.001 (w / v)% or higher, more preferably 0.1 (w / v)% or higher, and most preferably 1 (w / v)% or higher. Multiple types of nonspecific reaction inhibitors can also be used simultaneously, in which case the concentration is preferably 0.001 (w / v)% or higher, more preferably 0.1 (w / v)% or higher, and most preferably 1 (w / v)% or higher. There is no need to set an upper limit for the concentration, but it can be, for example, 10 (w / v)% or lower, or 5 (w / v)% or lower. When a sample extract containing a nonspecific reaction inhibitor that suppresses false positives is contained in a porous substrate such as a filter, the porous substrate can also contain the nonspecific reaction inhibitor at the above-mentioned concentration.

[0038] (Other ingredients other than the non-specific reaction inhibitors that suppress false positives) In the solution to be impregnated into members such as a porous substrate that comes into contact with the specimen in the specimen extract or in a process prior to the detection reaction in the present invention, in addition to the non-specific reaction inhibitory component that suppresses false positives, known substances that can reduce non-specific reactions, surfactants, pH buffering components, various proteins, salts, and saccharides may be included. For example, as components that can reduce non-specific reactions, arginine, arginine ethyl ester, arginine methyl ester, glycine ethyl ester, glycine methyl ester, lysine, and various isomers of the above-mentioned compounds can be mentioned. Examples of surfactants include nonionic surfactants such as polyethylene glycol mono-p-isooctylphenyl ether and polyoxyethylene sorbitan monolaurate, zwitterionic surfactants such as CHAPS and laurylamidobetaine, anionic surfactants such as sodium dodecyl sulfate, and cationic surfactants such as dodecyltrimethylammonium chloride. The concentration of the surfactant in the specimen extract is preferably 0.5 to 5 (w / v)%, more preferably 1 to 3 (w / v)%, and even more preferably 1.5 to 2.5 (w / v)%.

[0039] Examples of buffering components include phosphate buffer, Tris buffer, and Good buffer. Examples of protein components include BSA (bovine serum albumin), casein, gelatin, IgG, etc. Examples of salts include lithium chloride, sodium chloride, potassium chloride, sodium bromide, potassium bromide, sodium iodide, potassium iodide, etc.

[0040] (Detection method) In the method of the present invention, detection is performed by a method using an antigen-antibody reaction or a binding reaction between substances having an interaction. Examples of combinations of substances having an interaction include combinations of a ligand and a receptor, combinations of a receptor and a receptor, combinations of biotin and avidin or streptavidin, etc.

[0041] The detection method is not particularly limited as long as it utilizes substances having antigen-antibody reactions or interactions. Examples include immunochromatography, latex agglutination, immunoturbidimetry, immunogel filtration, chemiluminescent enzyme immunoassay (CLEIA), enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), etc. Immunochromatography is particularly preferred. Many of these are immunological techniques that utilize antigen-antibody reactions, but reactions between substances having interactions can also be utilized instead of antigen-antibody reactions. Among these methods, the sandwich method is preferred. In a typical sandwich method, a first substance that binds to the substance to be detected is immobilized on a specific carrier as a substance for capturing the substance to be detected, and the substance to be detected is bound to this substance. Further, a second substance that binds to the substance to be detected and is labeled is bound to the substance to be detected, and a complex of "a first substance that binds to the substance to be detected - the substance to be detected - a second substance that binds to the substance to be detected and is labeled" ("-" indicates binding) is formed, and the substance to be detected is measured by measuring the signal emitted from the labeled substance. The first substance that binds to the substance to be detected and the second substance that binds to the substance to be detected may be the same substance. The substance that binds to the substance to be detected and the substance to be detected may be an antigen and an antibody or an antibody and an antigen, or may be substances having interactions with each other. As the solid phase on which the first substance that binds to the substance to be detected is immobilized, any substance such as a protein that can be immobilized by known techniques can be used. For example, a porous thin film (membrane) having capillary action, particulate matter, a test tube, a resin plate, etc., which are known, can be arbitrarily selected. Further, as the substance for labeling the second substance that binds to the substance to be detected, an enzyme, a radioisotope, a fluorescent substance, a luminescent substance, colored particles, colloidal particles, etc. can be used.

[0042] Among the sandwich methods, from the viewpoints of simplicity and rapidity of clinical tests, immunochromatography, which is a lateral flow type immunoassay using a membrane, is particularly preferred.

[0043] Hereinafter, a general immunochromatography method using an antigen-antibody reaction will be described. The test device for immunochromatography is shown in FIG. 1.

[0044] The top of Fig. 1 is a top view, and the bottom is a cross-sectional view. In the inspection device, various parts are laminated on a nitrocellulose membrane (a) laminated on a plastic plate (f). In the specific example of the figure, on the plastic plate (f), a nitrocellulose membrane (a) on which two detection parts (c) are formed with a substance to capture a substance to be detected such as an antibody, an absorption pad part (e) formed of filter paper, a label part (g), and a sample addition part (h) formed of a glass fiber filter are laminated respectively.

[0045] And, as shown in the figure, one end region of the absorption pad part (e), one end region of the nitrocellulose membrane (a), the other end region of the nitrocellulose membrane (a) and one end region of the label part (g), the other end region of the label part (g) and one end region of the sample addition part (h) are respectively overlapped, whereby a continuous lateral flow channel is formed.

[0046] The label part (g) contains a label in which a labeling substance is chemically or physically bound to a substance to capture a substance to be detected such as an antibody. Examples of the labeling substance include gold colloid particles, platinum colloid particles, color latex particles, magnetic particles, enzymes, quantum dots, fluorescent dyes, and phosphors. The label part is composed of a porous substrate containing the above label, and as the material of the substrate, commonly used glass fiber (glass fiber), non-woven fabric, etc. can be used. The porous substrate impregnated with the label and dried is also called a stabilized dry label pad. That is, the label part is a part containing the above stabilized dry label pad containing an antibody that binds to a substance to be detected by an antigen-antibody reaction and is labeled with colored latex particles.

[0047] In addition, the detection part (c) is a part where an antibody that binds to a substance to be detected by an antigen-antibody reaction is solid-phased linearly as a capture substance.

[0048] Examples of members that come into contact with the specimen in the steps before the detection reaction of the substance to be detected or in steps simultaneous with the detection reaction include the above (a), (b), and (c), etc. However, it is not limited to these as long as it is a member that comes into contact with the specimen in the steps before the detection reaction of the substance to be detected or in steps simultaneous with the detection reaction. When the specimen is added to the sample addition part (c), the specimen flows from the sample addition part (c) toward the absorption pad part (f). When the flow from the sample addition part (c) to the absorption pad part (f) is expressed as a flow from upstream to downstream, it can be said that the member that comes into contact with the specimen in the steps before the detection reaction of the substance to be detected or in steps simultaneous with the detection reaction exists upstream of the site where the detection reaction occurs.

[0049] Next, an immunoassay method using this test device will be described. First, a specimen is suspended in a specimen extraction solution to prepare a specimen sample from which the substance to be detected has been extracted. Next, the specimen sample is dropped onto the sample addition part (c) of the test device. The specimen sample containing the substance to be detected moves horizontally on the membrane and is impregnated into the labeled body part (b) to dissolve and develop the labeled body. If the substance to be detected is present in the specimen sample, a complex of the substance to be detected - labeled body is formed. When this complex reaches the detection part (d), a complex of the capture antibody - substance to be detected - labeled body is formed on the line thereof. The presence or absence of the substance to be detected in the specimen can be determined by detecting the presence of the complex based on the signal emitted from the labeling substance in this complex. Other components and the like that did not participate in the reaction are absorbed by the absorption pad part (f). In the example shown in FIG. 1, there are two detection parts (d), which are for capturing two types of substances to be detected, such as influenza A virus and influenza B virus, respectively. By providing a plurality of such detection parts (d), it is possible to simultaneously immunoassay a plurality of types of substances to be detected.

[0050] In the above-mentioned immunochromatography method, the sample extract, which is mixed with the sample to extract the substance to be detected, may contain a non-specific reaction inhibitor to suppress false positives, or the nitrocellulose membrane (a), label portion (b), and / or sample addition portion (d) of the immunochromatography test device may contain a non-specific reaction inhibitor to suppress false positives.

[0051] The present invention includes test reagents that utilize antigen-antibody reactions or binding reactions between interacting substances. The test reagent may refer to the test device itself, or it may refer to a test kit that includes a test device and other reagents. The test reagent of the present invention includes, for example, a test kit that includes an immunochromatographic test device and a specimen extract containing a nonspecific reaction inhibitor that suppresses false positives. The test reagent of the present invention also includes an immunochromatographic test device equipped with a portion containing a specimen extract containing a nonspecific reaction inhibitor that suppresses false positives. [Example]

[0052] The present invention will be specifically explained by the following examples, but the present invention is not limited to these examples.

[0053] In the following examples, examples will be described in which non-specific reactions in specimens were suppressed in immunochromatography kits for detecting influenza virus, RS virus, adenovirus, and mycoplasma when the specimen extract of the present invention was used.

[0054] Detection of influenza virus antigens by immunochromatography 1. Preparation of anti-influenza virus monoclonal antibodies (1) Anti-influenza A virus NP (each protein) antibody BALB / c mice were immunized with the influenza A virus antigen, and the spleen was removed from the mice reared for a certain period. The spleen was fused with mouse myeloma cells (P3×63) by the method of Kohler et al. (Kohler et al., Nature, vol. 256, p495-497 (1975)). The obtained fused cells (hybridomas) were maintained in a 37°C incubator, and cell purification (single cloning) was performed while confirming the antibody activity in the supernatant by ELISA using a plate immobilized with the influenza A virus NP antigen. Two strains of the obtained cells were intraperitoneally administered to pristane-treated BALB / c mice, and about two weeks later, ascites containing antibodies was collected. IgG was purified from the obtained ascites by affinity chromatography using a protein A column, and two types of purified anti-influenza A virus NP antibodies were obtained. (2) Anti-influenza B virus NP antibody Using the influenza B virus antigen, two types of purified anti-influenza B virus NP antibodies were obtained in the same manner as in (1).

[0055] 2. Preparation of the labeled body pad One type each of the purified anti-influenza A virus NP antibody and the purified anti-influenza B virus NP antibody were used. The anti-influenza A virus antibody was covalently bound to red latex particles, suspended in a suspension, and sonicated to prepare a well-dispersed anti-influenza A latex suspension. Similarly, an anti-influenza B latex suspension in which the anti-influenza B virus antibody was covalently bound to blue latex particles was prepared. The anti-influenza A latex suspension and the anti-influenza B latex suspension were mixed, applied to a glass fiber with a size of 20 cm × 1 cm, and dried well under warm air to prepare a labeled body pad forming a dry mixture.

[0056] 3. Preparation of the sample addition pad Glass fiber with a size of 2.0 cm × 20 cm was used.

[0057] 4. Preparation of the test device The test device used was of the same configuration as that shown in Fig. 1. A nitrocellulose membrane was cut to a size of 2 cm × 20 cm and backed with an adhesive plastic plate. An amount of anti-influenza A virus antibody (a different antibody from the above) solution with a width of about 1 mm was applied at positions 0.8 cm and 1.0 cm from the lower end, and an amount of anti-influenza B virus antibody (a different antibody from the above) solution was each applied for 20 cm. It was dried well under warm air to immobilize the antibody (detection part). Next, a filter paper with a size of 3 cm × 20 cm was overlapped with the upper end of the nitrocellulose membrane by 5 mm to provide an absorption pad part. Further, a label pad was overlapped with the lower end of the nitrocellulose membrane by 2 mm to provide a label part. Furthermore, a sample addition pad was overlapped at a position 7 mm away from the upper end of the label pad to provide a sample addition part. Then, a test device integrated by cutting into strips with a width of 5 mm using a cutter was produced.

[0058] Detection of RS virus, adenovirus, and Mycoplasma pneumoniae antigen by immunochromatography 1. Preparation of anti-RS virus, anti-adenovirus, and anti-Mycoplasma pneumoniae monoclonal antibodies RS virus antigen, or adenovirus antigen, or Mycoplasma pneumoniae antigen was independently immunized into BALB / c mice, and the spleen was removed from the mice bred for a certain period. It was fused with mouse myeloma cells (P3×63) by the method of Kohler et al. (Kohler et al., Nature, vol, 256, p495 - 497(1975)). The obtained fused cells (hybridomas) were maintained in a 37°C incubator, and the purification (single cloning) of the cells was performed while confirming the antibody activity of the supernatant by ELISA using plates solid-phased with the above respective antigens. Two strains of the obtained cells were each intraperitoneally administered to pristane-treated BALB / c mice, and about 2 weeks later, antibody-containing ascites were collected. IgG was purified from the obtained ascites by affinity chromatography using a protein A column, and two types of purified anti-immunogen antibodies were obtained for each immunogen.

[0059] 2. Preparation of the label pad Each purified anti-immunogen antibody was covalently bonded to red latex particles, suspended in a suspension, and sonicated to prepare an anti-RS virus latex suspension, an anti-adenovirus latex suspension, and an anti-pneumonia mycoplasma latex suspension that were sufficiently dispersed and suspended. These latex suspensions were each applied to glass fibers sized 20 cm × 1 cm, and dried well under warm air to form a labeled body pad that was a dried mixture.

[0060] 3. Preparation of Sample Addition Pad Glass fibers sized 2.0 cm × 20 cm were used.

[0061] 4. Preparation of Test Device As the test device, one with the same configuration as shown in FIG. 1 was used. A nitrocellulose membrane was cut to a size of 2 cm × 20 cm and backed with an adhesive plastic plate. An anti-RS virus antibody (a different antibody from the above)-containing solution, an anti-adenovirus antibody (a different antibody from the above), or an anti-pneumonia mycoplasma antibody in an amount forming a width of about 1 mm was applied at positions 0.8 cm and 1.0 cm from the lower end, and dried well under warm air to immobilize the antibody (detection part). Next, a filter paper sized 3 cm × 20 cm was overlapped 5 mm on the upper end of the nitrocellulose membrane to provide an absorption pad part. Further, the labeled body pad was overlapped 2 mm on the lower end of the nitrocellulose membrane to provide a labeled body part. Furthermore, the sample addition pad was overlapped at a position 7 mm away from the upper end of the labeled body pad to provide a sample addition part. Then, a test device integrated by cutting into strips 5 mm wide with a cutter was prepared.

[0062] 5. Preparation of Specimen Extract It is described in each of the following test examples. Test Example 1 False Positive Suppression Effect of Specimen Extract Containing L-Phenylalanine (L-Phe) in the Detection of Influenza Virus by Immunochromatography 1-1. Preparation of Specimen Extract A mixed solution containing 50 mM Tris buffer (pH 8.0), 2 (w / v)% polyoxyethylene octyl phenyl ether, and 5 (w / v)% L-arginine was prepared and used as the sample extract for Control 1. Next, a mixed solution containing 50 mM Tris buffer (pH 8.0), 1.25 (w / v)% polyoxyethylene alkyl ether, 0.75 (w / v)% polyoxyethylene octyl phenyl ether, 4 (w / v)% glycine ethyl ester, 1.5 (w / v)% L-Phe, and 400 mM sodium bromide was prepared and used as the sample extract for Test 1.

[0063] 1-2. Test method Frozen and stored influenza virus-negative nasal aspirates in which false positive reactions were observed with the control sample extract were thawed, and samples were collected with cotton swabs. These samples were suspended and dispersed in the control and Test 1 sample extracts to obtain the samples for this test. Next, these samples were dropped onto the test device prepared above, and the color development intensity of the detection part was measured after 5 minutes. The measurement of the color development intensity was performed using a color scale with scores assigned to 10 levels of color development intensity for each of red and blue.

[0064] 1-3. Results of color development intensity test The results are shown in Table 1. The numerical values of the color development intensity increase in the order of 0, 1+, 2+ ··· 10+, where 0 indicates that no color development was observed, and the higher the numerical value, the stronger the signal. The results are expressed as "Color development intensity of influenza A / Color development intensity of influenza B". False positive reactions were observed with the control sample extract, but false positive reactions were suppressed in all samples with the Test 1 sample extract containing L-phenylalanine.

[0065]

Table 1

[0066] Test Example 2 Verification of the false positive suppression effect of L-Phe alone 2-1. Preparation of sample extract A mixed solution containing 50 mM Tris buffer (pH 8.0), 1.25 (w / v)% polyoxyethylene alkyl ether, 0.75 (w / v)% polyoxyethylene octylphenyl ether, 4 (w / v)% glycine ethyl ether, and 400 mM sodium bromide was prepared and used as the sample extract for Control 2. Next, a mixed solution containing 50 mM Tris buffer (pH 8.0), 1.25 (w / v)% polyoxyethylene alkyl ether, 0.75 (w / v)% polyoxyethylene octylphenyl ether, 4 (w / v)% glycine ethyl ether, 400 mM sodium bromide, and 1.5% L-Phe was prepared and used as the sample extract for Test 2.

[0067] 2-2. Test method Frozen and stored influenza virus-negative nasal aspirates in which false positive reactions were observed with the sample extract of Control 1 were thawed, and samples were collected with cotton swabs. These samples were suspended and dispersed in the sample extracts of Control 1 and Test 2 to obtain the samples for this test. Next, these samples were dropped onto the test device prepared above, and the color development intensity of the detection part was measured after 5 minutes. The measurement of the color development intensity was performed using a color scale with scores assigned to 10 levels of color development intensity for each of red and blue.

[0068] 2-3. Results of the color development intensity test The results are shown in Table 2. Note that the numerical values of the color development intensity increase in the order of 0, 1+, 2+····10+, where 0 indicates that no color development was observed, and the higher the numerical value, the stronger the signal. The results are expressed as "Color development intensity of type A / Color development intensity of type B". Even in the samples in which false positive reactions were observed with the sample extract of Control 2, the false positive reactions were suppressed with the sample extract containing L-phenylalanine of Test 2.

[0069]

Table 2

[0070] Test Example 3 Sensitivity comparison between the sample extract containing L-Phe and the conventional sample extract 3-1. Preparation of sample extract A mixture containing 50 mM Tris buffer (pH 8.0), 2 (w / v)% polyoxyethylene octylphenyl ether, and 5 (w / v)% L-arginine was prepared and used as the specimen extract for Control 1. Next, a mixture containing 50 mM Tris buffer (pH 8.0), 1.25 (w / v)% polyoxyethylene alkyl ether, 0.75 (w / v)% polyoxyethylene octylphenyl ether, 4 (w / v)% glycine ethyl ester, 1.5 (w / v)% L-Phe, and 150 mM sodium bromide was prepared and used as the specimen extract for Test 1.

[0071] 3-2. Test method Control 1 and Test 1 specimen extracts were diluted to a final concentration of 6.8 x 10 2 PFU / mL, 3.4 x 10 2 PFU / mL, 1.7x10 2 Samples were prepared by adding inactivated influenza A virus at a final concentration of 4.0 x 10 PFU / mL. 2 PFU / mL, 2.0 x 10 2 PFU / mL, 1.0 x 10 2 Samples were prepared so that the PFU / mL concentration was achieved. Next, 50 μL of each sample was dropped onto the test device prepared above, and the color intensity of the detection area was measured after 5 minutes. The color intensity was measured using color samples with scores for red and blue on a 10-point scale.

[0072] 3-3. Coloring strength test results The results are shown in Table 3 The color intensity values increase in the order of 0, 0.5+, 1+, 2+...10+, with 0 indicating no color and higher values indicating stronger signals. The results are expressed as "color intensity of type A / color intensity of type B." The sensitivity was the same between the specimen extract of Control 1 and the specimen extract of Test 1. In terms of signal intensity, Test 1 was higher. From the above, by using the specimen extract prepared using the present invention, the specificity can be improved without reducing the sensitivity.

[0073]

Table 3

[0074] Test Example 4 False positive suppression effect of specimen extraction containing L-Phe analog substances 4-1. Preparation of specimen extract A mixed solution containing 50 mM Tris buffer (pH 8.0), 2 (w / v)% polyoxyethylene octylphenyl ether, and 2 (w / v)% L-arginine was prepared and used as the specimen extract for the control (without additive). Next, a specimen extract containing 50 mM Tris buffer (pH 8.0), 2 (w / v)% polyoxyethylene octylphenyl ether, 2 (w / v)% L-arginine, and containing one kind of the compound in Table 4 (the concentration in parentheses is the final concentration) was prepared and used as the specimen extract for the test.

[0075]

Table 4

[0076] 4-2. Test method Frozen and stored influenza virus-negative nasal aspirates in which a false positive reaction was observed with the control specimen suspension were thawed, and specimens were collected with a cotton swab. These specimens were suspended and dispersed in the control and each test specimen extract to obtain the samples for this test. Next, these samples were dropped onto the test device prepared above, and the color development intensity of the detection part was measured after 5 minutes. The measurement of the color development intensity was performed using a color scale that was scored on a 10-step color development intensity for each of red and blue.

[0077] 4-3. Color development intensity test results The results are shown in Tables 5, 6 and 7. The results are expressed as "Type A / Type B", and additives with a false positive suppression effect are marked with "〇", while additives without a suppression effect are marked with "×". Although the results vary depending on the specimen, false positive reactions that could not be suppressed by L-arginine could be suppressed by the additives described in Table 4.

[0078]

Table 5

[0079]

Table 6

[0080]

Table 7

[0081] Test Example 5 False positive suppression effect of specimen extraction solution containing L-Phe in the immunochromatographic detection of RS virus, adenovirus, and Mycoplasma pneumoniae 5-1. Preparation of specimen extraction solution A mixed solution containing 50 mM Tris buffer (pH 8.0), 2 (w / v)% polyoxyethylene octylphenyl ether, and 5 (w / v)% L-arginine was prepared as the specimen extraction solution for Control 1. Next, a mixed solution containing 50 mM Tris buffer (pH 8.0), 1.25 (w / v)% polyoxyethylene alkyl ether, 0.75 (w / v)% polyoxyethylene octylphenyl ether, 4 (w / v)% glycine ethyl ester, 1.5 (w / v)% L-Phe, and 150 mM sodium bromide was prepared as the specimen extraction solution for Test 4.

[0082] 5-2. Test method Frozen, stored nasal aspirates negative for respiratory syncytial virus (RSV), adenovirus, and Mycoplasma pneumoniae, which had false-positive reactions in the control sample extract, were thawed and collected with a cotton swab. These samples were suspended and dispersed in the control and test sample extracts (Test 4) to prepare the samples for this test. These samples were then dropped into the test device prepared above, and the color intensity of the detection zone was measured after 5 minutes for RSV and adenovirus, and after 15 minutes for Mycoplasma pneumoniae. Color intensity was measured using a color chart with a 10-point scale for red and blue color intensity.

[0083] 5-3. Coloring strength test results The results for RS virus are shown in Table 8, those for adenovirus in Table 9, and those for Mycoplasma pneumoniae in Table 10. The color intensity values increase in the order of 0, 1+, 2+, 10+, with 0 indicating no color development, and higher values indicating a stronger signal. In all results, false positive reactions were observed in the control specimen extract, but false positive reactions were suppressed in all specimens when Test 4 specimen extract containing L-phenylalanine was used.

[0084] [Table 8]

[0085] [Table 9]

[0086] [Table 10] [Industrial Applicability]

[0087] The method of the present invention can be used for the accurate detection of a variety of substances. [Explanation of symbols]

[0088] nitrocellulose membrane b. labeled body part c. detection part d. sample addition part e. absorption pad part

Claims

**Claim 1** An inspection reagent for detecting a substance to be detected selected from the group consisting of influenza virus antigen; adenovirus antigen; RS (respiratory syncytial) virus antigen; human metapneumovirus (hMPV) antigen; coronavirus antigen including SARS-CoV, MERS-Cov or SARS-CoV2; group A streptococcus; group B streptococcus and mycoplasma antigen in a specimen selected from the group consisting of throat swab specimens, nasal swab specimens, nasal aspirate specimens, nasal wash specimens, nasal mucus specimens and saliva specimens, which utilizes the binding reaction of substances having an antigen-antibody reaction or interaction, and contains a specimen extract containing a non-specific reaction suppressing component that suppresses false positives, which is a compound selected from the group consisting of L-phenylalanine, D-phenylalanine, aspartame, hippuric acid, mandelic acid, L-phenylalanine methyl ester, N-phenylglycine, DL-2-phenylglycine, sodium benzoate, 3-phenylpropionic acid, N-(m-toluoyl)glycine, acetylsalicylic acid and L-tryptophan. **Claim 2** The inspection reagent according to claim 1, wherein the specimen extract contains a non-specific reaction suppressing component that suppresses false positives at 0.1 to 10 (w / v)%. **Claim 3** The inspection reagent according to claim 1 or 2, wherein the specimen extract further contains an amino acid or amino acid derivative selected from the group consisting of arginine, lysine, arginine ethyl ester, arginine methyl ester, glycine ethyl ester and glycine methyl ester, and optical isomers, geometric isomers, structural isomers and stereoisomers of these compounds. **Claim 4** The inspection reagent according to any one of claims 1 to 3, wherein the specimen extract further contains a halide selected from the group consisting of lithium chloride, sodium chloride, potassium chloride, sodium bromide, potassium bromide, sodium iodide and potassium iodide. **Claim 5** A method for detecting a substance to be detected selected from the group consisting of an influenza virus antigen, an adenovirus antigen, an RS (respiratory syncytial) virus antigen, a human metapneumovirus (hMPV) antigen, a coronavirus antigen including SARS-CoV, MERS-Cov or SARS-CoV2, group A streptococcus, group B streptococcus, and a mycoplasma antigen in a specimen selected from the group consisting of a throat swab specimen, a nasal swab specimen, a nasal aspirate specimen, a nasal wash specimen, a nasal mucus specimen, and a saliva specimen, which utilizes a reaction between antigen-antibody reactions or substances having an interaction contained in a specimen extract. In this method, the specimen is preliminarily brought into contact with a non-specific reaction suppressing component that is a compound selected from the group consisting of L-phenylalanine, D-phenylalanine, aspartame, hippuric acid, mandelic acid, L-phenylalanine methyl ester, N-phenylglycine, DL-2-phenylglycine, sodium benzoate, 3-phenylpropionic acid, N-(m-toluoyl)glycine, acetylsalicylic acid, and L-tryptophan to suppress false positive reactions and perform detection.

6. The method according to claim 5, wherein the method for detecting the substance to be detected is an immunochromatography method.

7. The method according to claim 5 or 6, wherein the specimen extract contains a non-specific reaction suppressing component that suppresses 0.1 to 10 (w / v)% false positives.

8. The method according to any one of claims 5 to 7, wherein the specimen extract further contains an amino acid or amino acid derivative selected from the group consisting of arginine, lysine, arginine ethyl ester, arginine methyl ester, glycine ethyl ester, and glycine methyl ester, and optical isomers, geometric isomers, structural isomers, and stereoisomers of these compounds.

9. The method according to any one of claims 5 to 8, wherein the specimen extract further contains a halide selected from the group consisting of lithium chloride, sodium chloride, potassium chloride, sodium bromide, potassium bromide, sodium iodide, and potassium iodide.

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