Test reagents that improve specificity by suppressing false negatives

Abstract

The present application relates to a specimen extraction solution and a specimen extraction method, and an inspection reagent using the same, the specimen extraction solution containing a component capable of inhibiting false negative reactions that cannot be completely inhibited by conventional methods when detecting antigens such as viruses, bacteria, and proteins to be detected from specimens from body fluids such as a nasal swab specimen, a nasal aspirate specimen, a nasal lavage specimen, a nasal discharge specimen, a throat swab specimen, a saliva specimen, a fecal specimen, a serum specimen, a plasma specimen, a urine specimen, and the like using an inspection reagent that utilizes antigen-antibody reactions or reactions of substances having interactions with each other. The specimen extraction solution of the inspection reagent, or a component that comes into contact with a specimen in a process before a detection reaction of a detection object or a process performed simultaneously with the detection reaction, and the like contain a water-soluble compound having a phenyl group, a benzyl group, a tolyl group, or a xylyl group in the structure, and at least a functional group including a carboxyl group or a carboxyl group methylated or ethylated atom group or a hydroxyl group bonded to the above group.

Description

Technical Field

[0001] This invention relates to a technology for detecting viruses, bacteria, and proteins in bodily fluid samples such as nasal swabs, nasal aspirates, nasal irrigation samples, blown nasal mucus, pharyngeal swabs, saliva, fecal samples, serum samples, plasma samples, and urine samples using a detection reagent that utilizes antigen-antibody reactions or the binding reaction of interacting substances. This technology effectively suppresses false-negative reactions that cannot be completely suppressed by existing methods by using compounds with specific chemical structures in the sample extraction solution and in components that come into contact with the sample during or before the detection reaction. Background Technology

[0002] In recent years, various diagnostic reagents and kits have been developed that utilize antigen-antibody reactions and the binding reactions of interacting substances to detect infections such as viruses and bacteria, or to determine the presence or absence of pregnancy. All diagnostic reagents include a pretreatment process to create suitable conditions for the detection reaction after specimen collection from the patient; this process is crucial for obtaining accurate results. In particular, simplified diagnostic reagents are characterized by their lack of need for special equipment, ease of operation, and low cost. They are widely used not only in large hospitals and medical testing centers but also in general hospitals and clinics, and are frequently used by users other than medical professionals. Therefore, high diagnostic accuracy is extremely important. Examples of simplified diagnostic reagents currently on the market include those for detecting pathogen infections and those for diagnosing pregnancy. These reagents are often administered at the medical facility where the patient first visits, allowing for on-site assessment of infection or pregnancy based on the specimen collected from the patient, enabling early intervention. Therefore, the importance of simplified diagnostic reagents in healthcare continues to grow. Furthermore, with the increased use of simplified testing reagents, users are demanding higher reproducibility and greater accuracy in the test results.

[0003] Currently, as representative reagents for simple testing methods, immunoassays, especially immunochromatography, are generally known to favor antigen-antibody reactions. Immunochromatography detects (measures or quantifies) the analyte by forming a complex on a membrane containing a trap (capture substance) that specifically binds to the analyte and a label that also specifically binds to the analyte. Immunochromatography is widely used for the detection of various analytes due to its simple apparatus and relatively low cost.

[0004] In one method of immunochromatography, a sample containing the analyte is dropped onto an inspection device to form a analyte-label complex. The complex is then spread and captured by a detection unit for detection or quantitative labeling. The inspection device comprises: a detection unit having an antibody specifically binding to the analyte immobilized on a membrane strip such as nitrocellulose as a capture substance; and a labeling unit containing a label specifically binding to the analyte.

[0005] In recent years, clinical practice has demanded higher reliability of diagnostic results for clinical diagnostic drugs, including immunochromatography, making the improvement of reagent reliability a key issue. Highly reliable reagents are those with high sensitivity and specificity, minimizing the risk of misdiagnosis. Regarding specificity, a persistent technical challenge has been how to design reagents to address the diversity of specimen composition caused by individual patient background differences. Effectively eliminating nonspecific reactions is crucial for simplified diagnostic methods. To address these challenges, it has been reported that contacting the specimen with basic amino acids such as arginine and lysine, inorganic salts, glycine ethyl ester, surfactants, animal-derived immunoglobulins, sulfated surfactants and polymers, and surfactants with quaternary ammonium ions has some effect on improving specificity (see Patent Documents 1, 2, 3, 4, and 5), but the effects are limited, and uncontrollable nonspecific reactions still exist. Therefore, a technique that more effectively improves specificity without reducing sensitivity is desired.

[0006] Existing technical documents

[0007] Patent documents

[0008] Patent Document 1: Japanese Patent Application Publication No. 2003-279577

[0009] Patent Document 2: Japanese Patent Application Publication No. 2005-24323

[0010] Patent Document 3: Japanese Patent Application Publication No. 2004-301684

[0011] Patent Document 4: Japanese Patent No. 6116268

[0012] Patent Document 5: Japanese Patent No. 6601932 Summary of the Invention

[0013] When detecting viruses, bacteria, or proteins from bodily fluids such as nasal swabs, nasal aspirates, nasal irrigation fluid, nasal mucus, pharyngeal swabs, saliva, fecal samples, serum, plasma, and urine using test reagents that utilize antigen-antibody reactions or the binding reactions of interacting substances, false positive or false negative reactions still occur, which cannot be completely suppressed by existing methods. This is one of the reasons that hinder accurate diagnosis. In this invention, a test reagent is provided that uses a specimen extraction solution containing components capable of suppressing false negative reactions without reducing sensitivity, as well as a specimen extraction method.

[0014] The inventors have conducted in-depth research on a method to more effectively suppress non-specific reactions when using specimens derived from bodily fluids, such as nasal swabs, nasal aspirates, nasal irrigation samples, blown nasal mucus, pharyngeal swabs, saliva samples, fecal samples, serum samples, plasma samples, and urine samples, as test samples. As a result, they discovered a component that can suppress non-specific reactions more significantly than existing methods. Furthermore, they found that by adding this component to the specimen extraction solution, to components that come into contact with the specimen in processes prior to or concurrent with the detection reaction, they can suppress false negative reactions that cannot be detected by existing methods, thus completing this invention.

[0015] That is, the present invention has the following composition.

[0016] [1] A test kit that detects a substance in a specimen by means of an antigen-antibody reaction or the binding reaction of interacting substances, characterized in that the test kit contains a specimen extract containing a non-specific reaction inhibitor that inhibits false negatives, wherein the non-specific reaction inhibitor is a compound having a phenyl, benzyl, tolyl or xylyl group and a carboxyl, methoxycarboxyl or ethoxycarboxyl group, optionally having a hydroxyl group, and having a water-soluble molecular weight of less than 6000 Da.

[0017] [2] The test reagent according to [1] is characterized in that the test reagent is an immunochromatographic test reagent, comprising a specimen extract containing a nonspecific reaction inhibitory component that inhibits false negatives, wherein the nonspecific reaction inhibitory component is a compound having a phenyl, benzyl, tolyl or xylyl group and a carboxyl group, a methoxycarboxyl group or an ethoxycarboxyl group, optionally having a hydroxyl group, and having a water-soluble molecular weight of less than 6000 Da.

[0018] [3] The test reagent according to [1] or [2] is characterized in that the test reagent is an immunochromatographic test device, the immunochromatographic test device comprising a portion impregnated with a specimen extract containing a nonspecific reaction inhibitory component that inhibits false negatives, the nonspecific reaction inhibitory component being a compound having a phenyl, benzyl, tolyl or xylyl group and a carboxyl, methoxycarboxyl or ethoxycarboxyl group, optionally having a hydroxyl group, and having a water-soluble molecular weight of less than 6000 Da.

[0019] [4] The test reagent according to any one of [1] to [3] is characterized in that the specimen extract contains 0.1 to 10 (w / v)% of a nonspecific reaction inhibitor that inhibits false negatives.

[0020] [5] The test reagent according to any one of [1] to [4] is characterized in that the compound having a phenyl, benzyl, tolyl or xylyl group and a carboxyl, methoxycarboxyl or ethoxycarboxyl group, optionally having a hydroxyl group, and having a water-soluble molecular weight of 6000 Da or less is a compound represented by any one of the following general formulas (I) to (V) or tryptophan:

[0021] [Chemical Formula 1]

[0022]

[0023] In general formula (I), R1 is H, OH, =O, NH2, COOH, N HC O- C N H2-C-COOH or CH3, R2 is H, CH3, C2H5, Li, Na, K, Rb, Cs or Fr, n is 0 or 1, m is 0, 1, 2, 3 or 4;

[0024] [Chemical Formula 2]

[0025]

[0026] In general formula (II), R2 is H, CH3, C2H5, Li, Na, K, Rb, Cs or Fr, and R3 is COOH, COOLi, COONa, COOK, COORb, COOCs, COOFr, COOCH3, COOC2H5, OCOH or CH3. R3 and COOR2 are located in the ortho, meta or para position of the benzene ring.

[0027] [Chemical Formula 3]

[0028]

[0029] 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;

[0030] [Chemical Formula 4]

[0031]

[0032] In general formula (IV), R5 is a 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.

[0033] [Chemical Formula 5]

[0034]

[0035] In the 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.

[0036] [6] The test reagent according to any one of [1] to [5] is characterized in that the compound having a phenyl, benzyl, tolyl or xylyl group and a carboxyl, methoxycarboxyl or ethoxycarboxyl group, optionally having a hydroxyl group, and having a water-soluble molecular weight of 6000 Da or less is selected from aspartame, phenylalanine, phenylalanine methyl ester, mandelic acid, 2-phenylpropionic acid, 3-phenylpropionic acid, phenylglycine, phenylglycine methyl ester, phenylglycine ethyl ester, phenyllactic acid, phenylpyruvic acid, benzoic acid, phthalic acid, acetylsalicylic acid, hippuric acid, N-toluylglycine, N-benzyloxycarbonyl 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.

[0037] [7] The test reagent according to any one of [1] to [6] is characterized in that the specimen extract further comprises 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.

[0038] [8] The test reagent according to any one of [1] to [7] is characterized in that the specimen extract further comprises a halide selected from the group consisting of lithium chloride, sodium chloride, potassium chloride, sodium bromide, potassium bromide, sodium iodide and potassium iodide.

[0039] [9] A method for detecting a substance selected from the group consisting of viral antigens, bacterial antigens, and protein antigens in a specimen by utilizing the antigen-antibody reaction or the reaction between interacting substances contained in a specimen extract, wherein the specimen is selected from the group consisting of pharyngeal swab specimens, nasal swab specimens, nasal aspirate specimens, pharyngeal irrigation specimens, nasal irrigation specimens, blown nasal mucus specimens, saliva specimens, serum specimens, plasma specimens, whole blood specimens, fecal specimens, fecal suspension specimens, and urine specimens, characterized in that the method suppresses false negative reactions for detection by pre-contaminating the specimen with a non-specific reaction inhibitory component that suppresses false negative reactions, wherein the non-specific reaction inhibitory component is a compound having a phenyl, benzyl, tolyl, or xylyl group and a carboxyl, methoxycarboxyl, or ethoxycarboxyl group, optionally having a hydroxyl group, and having a water-soluble molecular weight of less than 6000 Da.

[0040]

[10] According to the method described in [9], the method for detecting the substance to be detected is immunochromatography, wherein the specimen is placed in a specimen extract containing a nonspecific reaction inhibitory component that inhibits false negatives, and the specimen extract is added to an immunochromatographic testing device, wherein the nonspecific reaction inhibitory component is a compound having a phenyl, benzyl, tolyl or xylyl group and a carboxyl, methoxycarboxyl or ethoxycarboxyl group, optionally having a hydroxyl group, and having a water-soluble molecular weight of less than 6000 Da.

[0041]

[11] The method according to [9] is characterized in that the method for detecting the substance to be detected is immunochromatography, wherein the specimen is added to an immunochromatographic testing device comprising a portion of a specimen extract containing a nonspecific reaction inhibitory component that inhibits false negatives, wherein the nonspecific reaction inhibitory component is a compound having a molecular weight of 6000 Da or less that is phenyl, benzyl, tolyl or xylyl and has a carboxyl, methoxycarboxyl or ethoxycarboxyl group, optionally having a hydroxyl group, and is water-soluble.

[0042]

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

[11] is characterized in that the specimen extract contains 0.1 to 10 (w / v)% of a nonspecific reaction inhibitor that inhibits false negatives.

[0043]

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

[12] , wherein the compound having a phenyl, benzyl, tolyl or xylyl group and a carboxyl, methoxycarboxyl or ethoxycarboxyl group, optionally having a hydroxyl group, and having a water-soluble molecular weight of 6000 Da or less is a compound represented by any one of the following general formulas (I) to (V) or tryptophan:

[0044] [Chemical Formula 6]

[0045]

[0046] In general formula (I), R1 is H, OH, =O, NH2, COOH, N H - C O- C N H2-C-COOH or CH3, R2 is H, CH3, C2H5, Li, Na, K, Rb, Cs or Fr, n is 0 or 1, m is 0, 1, 2, 3 or 4;

[0047] [Chemical Formula 7]

[0048]

[0049] In general formula (II), R2 is H, CH3, C2H5, Li, Na, K, Rb, Cs or Fr, and R3 is COOH, COOLi, COONa, COOK, COORb, COOCs, COOFr, COOCH3, COOC2H5, OCOH or CH3. R3 and COOR2 are located in the ortho, meta or para position of the benzene ring.

[0050] [Chemical Formula 8]

[0051]

[0052] 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;

[0053] [Chemical Formula 9]

[0054]

[0055] In general formula (IV), R5 is a 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.

[0056] [Chemical Formula 10]

[0057]

[0058] In the 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.

[0059]

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

[13] is characterized in that the compound having phenyl, benzyl, tolyl or xylyl and having carboxyl, methoxycarboxyl or ethoxycarboxyl, optionally having hydroxyl, and having a water-soluble molecular weight of 6000 Da or less is selected from aspartame, phenylalanine, phenylalanine methyl ester, mandelic acid, 2-phenylpropionic acid, 3-phenylpropionic acid, phenylglycine, phenylglycine methyl ester, phenylglycine ethyl ester, phenyllactic acid, phenylpyruvic acid, benzoic acid, phthalic acid, acetylsalicylic acid, hippuric acid, N-toluylglycine, N-benzyloxycarbonyl 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.

[0060]

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

[14] is characterized in that the specimen extract further comprises 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.

[0061]

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

[15] is characterized in that the specimen extract further comprises a halide selected from the group consisting of lithium chloride, sodium chloride, potassium chloride, sodium bromide, potassium bromide, sodium iodide and potassium iodide.

[0062] This specification includes the disclosure of Japanese Patent Application No. 2020-150525, which forms the basis of the priority claim of this application.

[0063] Invention Effects

[0064] According to the present invention, a detection reagent can be provided that can detect specific viruses, bacteria, proteins, low-molecular-weight compounds in specimens derived from bodily fluids, such as nasal swabs, nasal aspirates, nasal irrigation fluid, blown nasal mucus, pharyngeal swabs, saliva, fecal samples, serum, plasma, and urine, by utilizing antigen-antibody reactions or the binding reactions of interacting substances. The detection reagent can more effectively suppress false negative reactions caused by sample contamination and exhibits high reproducibility and high accuracy. Furthermore, it can further prevent erroneous clinical diagnoses due to nonspecific reactions, which is beneficial to patients, as well as to doctors, laboratory technicians, and nurses using the reagent. Attached Figure Description

[0065] Figure 1 This is a diagram illustrating the structure of the inspection device used in this invention. Detailed Implementation

[0066] The present invention will now be described in detail.

[0067] This invention relates to a method for suppressing false negative reactions and preventing signal degradation, i.e., preventing reduced sensitivity, when detecting a substance in a sample by means of a detection reagent that utilizes an antigen-antibody reaction or the binding reaction of interacting substances.

[0068] In this invention, antibodies also include antigen-binding fragments of antibodies.

[0069] (specimen)

[0070] There are no restrictions on the types of specimens used. Examples of specimens include: pharyngeal swabs, nasal swabs, nasal aspirates, pharyngeal irrigation fluid, nasal irrigation fluid, blown nasal mucus, saliva, serum, plasma, whole blood, fecal suspension, urine, and culture medium. These are referred to as pharyngeal swab specimens, nasal swab specimens, nasal aspirates specimens, pharyngeal irrigation specimens, nasal irrigation specimens, blown nasal mucus specimens, saliva specimens, serum specimens, plasma specimens, whole blood specimens, fecal specimens, fecal suspension specimens, urine specimens, and culture medium specimens. Specimens can be used after dilution with buffer solution or undiluted.

[0071] (Substance to be tested)

[0072] The substances being tested are not restricted in any way and can be any substance that can be tested. Specific examples include: viral antigens such as influenza virus, adenovirus, respiratory syncytial virus (RSV), human metapneumovirus (hMPV), hepatitis A virus (HAV), hepatitis B virus (HBV), human immunodeficiency virus (HIV), norovirus, SARS-CoV, MERS-CoV, and SARS-CoV2 coronaviruses; bacterial antigens such as methicillin-resistant Staphylococcus aureus (MRSA), group A streptococci, group B streptococci, and Legionella; toxins produced by bacteria; mycoplasma antigens; chlamydia antigens such as Chlamydia trachomatis; protozoan antigens; fungal antigens; hormones such as human chorionic gonadotropin (hCG); proteins such as C-reactive protein, myoglobin, cardiac troponin, and procalcitonin; various tumor markers; and antigens such as pesticides and environmental hormones. Antibodies against the above-mentioned bacteria and viruses can also be listed.

[0073] (Specimen collection)

[0074] There are no restrictions on the methods of specimen collection. Examples include: using specimen collection instruments such as cotton swabs to collect pharyngeal swabs, nasal swabs, nasal aspirate specimens, pharyngeal irrigation specimens, blown nasal mucus specimens, saliva specimens, serum specimens, plasma specimens, whole blood specimens, fecal specimens, fecal suspension specimens, urine specimens, culture medium specimens, and other specimens from body fluids and excretions; collecting the above specimens by aspirating with a suction device; and collecting the above specimens using blood collection tubes.

[0075] (The non-specific reaction inhibitory component that suppresses false negatives comes into contact with the collected specimen.)

[0076] In the method of this invention, the specimen is contacted with a non-specific reaction inhibitor that suppresses false negatives. By contacting the specimen with this non-specific reaction inhibitor, false negatives can be suppressed during specimen testing. It should be noted that since the detected substance comes into contact with the non-specific reaction inhibitor when the specimen is contacted, the method of this invention is also referred to as contacting the detected substance with the non-specific reaction inhibitor. Alternatively, contacting the specimen with the non-specific reaction inhibitor may also be referred to as treating the specimen with the non-specific reaction inhibitor.

[0077] In addition, specimen extraction solution refers to a liquid that makes the substances to be tested in the specimen float and thus easier to measure. For example, it can be used to extract specific substances to be tested from cells or other sources without dissolving them. It can also be simply referred to as specimen processing solution, specimen dilution solution, specimen flotation solution, etc.

[0078] In this invention, the specimen and the non-specific reaction-inhibiting component that suppresses false negatives need to be pre-contaminated before the test. "Before the test" means before the detectable substance in the specimen reacts with an antibody or antigen targeting that substance, or before the detectable substance in the specimen reacts with a substance that interacts with it. The reaction with an antibody or antigen refers to the binding of the substance to the antibody or antigen, and the reaction between the detectable substance in the specimen and a substance that interacts with it refers to the binding of the substance to the interacting substance.

[0079] Examples of methods for contacting the non-specific reaction inhibitory component for suppressing false negatives according to the present invention with the specimen include: placing the specimen in a solution containing the non-specific reaction inhibitory component for suppressing false negatives and mixing to bring them into contact; and using an examination device for testing that pre-contains a specimen extract containing the non-specific reaction inhibitory component for suppressing false negatives, and adding the specimen to the examination device for testing, thereby bringing the specimen into contact with the non-specific reaction inhibitory component for suppressing false negatives.

[0080] As a specific example of a method for mixing and contacting a specimen with a solution containing a non-specific reaction inhibitor that suppresses false negatives, the following method can be cited: A specimen extract, obtained by suspending and dispersing the collected specimen, is pre-contains a non-specific reaction inhibitor that suppresses false negatives. When the specimen is added to the specimen extract and mixed, the specimen comes into contact with the non-specific reaction inhibitor that suppresses false negatives. For example, when using a specimen extract containing a non-specific reaction inhibitor that suppresses false negatives, if the specimen is a nasal swab, a cotton swab can be used to collect the nasal swab. The cotton swab, soaked in the collected specimen, is placed in the specimen extract to allow the specimen to suspend and disperse for extraction, thereby bringing the specimen into contact with the non-specific reaction inhibitor that suppresses false negatives.

[0081] A specific example of a method for using an examination device for measurement, which pre-contains a specimen extract containing a non-specific reaction inhibitory component that suppresses false negatives, and then adds the specimen to the examination device to bring the specimen into contact with the non-specific reaction inhibitory component that suppresses false negatives, can be listed as follows: The specimen extract, which pre-contains a non-specific reaction inhibitory component that suppresses false negatives, is applied to a fibrous or porous substrate such as a pad or filter sheet made of non-woven fabric, textile, or sponge, through impregnation, coating, etc. After the collected specimen is added to the examination device, the specimen comes into contact with the non-specific reaction inhibitory component that suppresses false negatives pre-contained in the fibrous or porous substrate. An example of such an examination device is an immunochromatographic device described later.

[0082] The material of the porous substrate for the testing device is not limited in any way, and examples include: pulp, cotton, wool, polyester, polypropylene, nylon, acrylic fiberglass, nitrocellulose, etc. When a specimen extract containing a non-specific reaction inhibitor that suppresses false negatives is pre-contained in the testing device used for measurement, and the specimen is added to the testing device so that the specimen comes into contact with the non-specific reaction inhibitor that suppresses false negatives, for example, the specimen extract containing the non-specific reaction inhibitor that suppresses false negatives is pre-impregnated in the porous substrate and dried. The specimen comes into contact with the porous substrate in a step before or simultaneously with the reaction on the testing device that is used to detect the analyte. For example, when a specimen is added to the testing device, the specimen spreads on the testing device, reaches the reaction site on the device, and produces a reaction such as an antibody-antigen reaction. By pre-positioning a porous substrate containing a specimen extract containing a non-specific reaction inhibitor that suppresses false negatives at a site on the testing device before the reaction site, the specimen comes into contact with the non-specific reaction inhibitor that suppresses false negatives before the reaction. For example, a filter can be used as a porous substrate, and a filter containing a specimen extract containing a non-specific reaction inhibitor that suppresses false negatives can be placed at the specimen addition site. In this case, when using nasal swabs as specimens, nasal swabs are collected using a cotton swab. The cotton swab, which has been soaked in the collected specimen, is placed in a specimen extract of any composition that does not contain a non-specific reaction inhibitor that suppresses false negatives. After the specimen is dispersed and dissolved, the specimen extract is pre-impregnated in a filter containing a non-specific reaction inhibitor that suppresses false negatives, which is a component of the examination device. This allows the specimen to come into contact with the non-specific reaction inhibitor that suppresses false negatives.

[0083] (Inhibiting components and concentrations of non-specific reaction inhibitors that suppress false negatives)

[0084] The non-specific reaction inhibitory component for suppressing false negatives in this invention refers to a compound having a phenyl, benzyl, tolyl, or xylyl group and a carboxyl, methoxycarboxyl, or ethoxycarboxyl group, optionally a hydroxyl group, and having a water-soluble molecular weight of 6000 Da or less. Additionally, it also includes metal salts, optical isomers, geometric isomers, structural isomers, stereoisomers, and positional isomers of these compounds.

[0085] These compounds are compounds or tryptophan represented by any of the following general formulas (I) to (V).

[0086] [Chemical Formula 11]

[0087]

[0088] 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.

[0089] [Chemical Formula 12]

[0090]

[0091] In general formula (II), R2 is H, CH3, C2H5, Li, Na, K, Rb, Cs or Fr, and R3 is COOH, COOLi, COONa, COOK, COORb, COOCs, COOFr, COOCH3, COOC2H5, OCOH or CH3. R3 and COOR2 are located at the ortho, meta or para position of the benzene ring.

[0092] [Chemical Formula 13]

[0093]

[0094] 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.

[0095] [Chemical Formula 14]

[0096]

[0097] In general formula (IV), R5 is a 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.

[0098] [Chemical Formula 15]

[0099]

[0100] In the 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.

[0101] Examples of compounds may be listed, but are not limited to, those consisting of, for example, aspartame, phenylalanine, phenylalanine methyl ester (including hydrochloride), mandelic acid, 2-phenylpropionic acid, 3-phenylpropionic acid, phenylglycine, phenylglycine methyl ester, phenylglycine ethyl ester, phenyllactic acid, phenylpyruvic acid, benzoic acid, phthalic acid, acetylsalicylic acid, hippuric acid, N-toluylglycine, N-benzyloxycarbonyl amino acid, N-phenylglycine, phenoxyacetic acid, tryptophan, and their metal salts, as well as optical isomers, geometric isomers, structural isomers, stereoisomers, and positional isomers. In this invention, references to aspartame, phenylalanine, phenylalanine methyl ester (including hydrochloride), mandelic acid, 2-phenylpropionic acid, 3-phenylpropionic acid, phenylglycine, phenylglycine methyl ester, phenylglycine ethyl ester, phenyllactic acid, phenylpyruvic acid, benzoic acid, phthalic acid, acetylsalicylic acid, hippuric acid, N-toluylglycine, N-benzyloxycarbonyl amino acid, N-phenylglycine, phenoxyacetic acid, and tryptophan include their metal salts, further including optical isomers, geometric isomers, structural isomers, stereoisomers, and positional isomers. Examples of metal salts include, for example, Li salts, Na salts, K salts, Rb salts, Cs salts, and Fr salts.

[0102] 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.

[0103] Examples of compounds represented by general formula (II) include phthalic acid and acetylsalicylic acid.

[0104] Examples of compounds represented by general formula (III) include: hippuric acid and N-toluylglycine. Examples of compounds represented by general formula (IV) include: N-benzyloxycarbonyl amino acids. Examples of compounds represented by general formula (V) include: N-phenylglycine and phenoxyacetic acid.

[0105] The above-mentioned non-specific reaction inhibitors that suppress false negatives are contained in the specimen extract or in components such as filter sheets that come into contact with the specimen during the pre-detection process. The concentration is preferably 0.001% (w / v) or more, more preferably 0.1% (w / v) or more, and most preferably 1% (w / v) or more. Alternatively, multiple of these non-specific reaction inhibitors may be used simultaneously; in this case, the concentration is also preferably 0.001% (w / v) or more, more preferably 0.1% (w / v) or more, and most preferably 1% (w / v) or more. The upper limit of the concentration is not limited; for example, it may be 10% (w / v) or less or 5% (w / v) or less. It should be noted that when a porous substrate such as a filter sheet contains a specimen extract containing non-specific reaction inhibitors that suppress false negatives, it is sufficient that the porous substrate contains the above-mentioned concentration of non-specific reaction inhibitors that suppress false negatives.

[0106] (Components other than those that inhibit non-specific reactions that suppress false negatives)

[0107] In the specimen extraction solution or the solution impregnated in components such as porous substrates that come into contact with the specimen in the process prior to the detection reaction in this invention, in addition to non-specific reaction inhibitory components that suppress false negatives, known substances or surfactants capable of reducing non-specific reactions, pH buffering components, various proteins, salts, and sugars may also be included. Examples of components capable of reducing non-specific reactions include: arginine, arginine ethyl ester, arginine methyl ester, glycine ethyl ester, glycine methyl ester, lysine, and various isomers of the above compounds. Furthermore, examples of surfactants include: nonionic surfactants such as polyethylene glycol monoisooctylphenyl ether and polyoxyethylene sorbitan monolaurate; amphoteric surfactants such as CHAPS and lauryl sulfobetaine; anionic surfactants such as sodium dodecyl sulfate; and cationic surfactants such as dodecyl trimethylammonium chloride. The concentration of surfactant in the specimen extract is preferably 0.5-5 (w / v)%, more preferably 1-3 (w / v)%, and even more preferably 1.5-2.5 (w / v)%.

[0108] Examples of buffering agents include: phosphate buffer, Tris buffer, Good's buffer, etc. 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.

[0109] (Detection Method)

[0110] In the method of the present invention, detection is performed by utilizing an antigen-antibody reaction or a binding reaction of interacting substances. Examples of combinations of interacting substances include: combinations of ligands and receptors, combinations of receptors and receptors, combinations of biotin and avidin or streptavidin, etc.

[0111] Any detection method that utilizes antigen-antibody reactions or the binding reaction of interacting substances is not particularly limited and can include: immunochromatography, latex agglutination, immunoturbidimetry, chemiluminescent enzyme immunoassay (CLEIA), enzyme-linked immunosorbent assay (EIA), and enzyme-linked immunosorbent assay (ELISA). Immunochromatography is particularly preferred. Most of these are immunological methods that utilize antigen-antibody reactions, but reactions of interacting substances can also be used instead of antigen-antibody reactions. Among these methods, sandwich methods are preferred. In a typical sandwich method, a first substance that binds to the analyte is pre-immobilized on a specific carrier as the analyte capture substance, allowing the analyte to bind to the carrier. Then, a labeled second substance that binds to the analyte binds to the analyte, forming a complex of "first substance bound to analyte - analyte - labeled second substance bound to analyte" ("-" indicates binding). The analyte is detected by measuring the signal emitted by the labeled substance. The first substance bound to the test substance and the second substance bound to the test substance can be the same substance. The test substance and the substance bound to it can be an antigen and antibody, or an antibody and an antigen, or substances that interact with each other. As the solid phase on which the first substance bound to the test substance is immobilized, any material capable of immobilizing substances such as proteins using known techniques can be used; any known material can be selected, such as a porous membrane with capillary action, particulate matter, test tubes, resin plates, etc. Furthermore, as the material used to label the second substance bound to the test substance, enzymes, radioactive isotopes, fluorescent substances, luminescent substances, colored particles, colloidal particles, etc., can be used.

[0112] In the sandwich method, from the perspective of simplicity and speed of clinical examination, the membrane cross-flow immunoassay, i.e., immunochromatography, is particularly preferred.

[0113] The following describes a conventional immunochromatographic method that utilizes antigen-antibody reactions. The immunochromatographic assay device is shown below. Figure 1 .

[0114] Figure 1A is a top view, and B is a cross-sectional view. The inspection device has various parts stacked on the nitrocellulose membrane 1 that is laminated on the plastic plate 6. In the specific example shown in the figure, the plastic plate 6 is laminated with the following parts: the nitrocellulose membrane 1 with two detection parts 3 formed by the detection substance such as antibodies and the capture substance, the absorbent pad part 5 formed by filter paper, the marker part 2, and the sample addition part 4 formed by glass fiber filter.

[0115] Furthermore, as shown in the figure, one end region of the absorbent pad 5, one end region of the nitrocellulose membrane 1, another end region of the nitrocellulose membrane 1, one end region of the marker 2, another end region of the marker 2, and one end region of the sample addition 4 are respectively superimposed to form a continuous crossflow channel.

[0116] The labeling section 2 contains a label formed by the chemical or physical binding of a labeling substance and an antibody or other detectable substance. Examples of labeling substances include: gold colloidal particles, platinum colloidal particles, colored latex particles, magnetic particles, enzymes, quantum dots, fluorescent dyes, and phosphors. The labeling section is composed of a porous substrate containing the aforementioned label, and the substrate material can be commonly used such as gas fiber or non-woven fabric. The porous substrate impregnated with and dried with the label is also referred to as a stabilized and dried labeling pad. In other words, the labeling section includes the aforementioned stabilized and dried labeling pad containing an antibody that binds to the detectable substance through an antigen-antibody reaction and is labeled with colored latex particles.

[0117] In addition, the detection unit 3 uses a strip-shaped solid phase containing antibodies that bind to the substance being detected through an antigen-antibody reaction as the site for capturing the substance.

[0118] Examples of components that come into contact with the sample in a process preceding or simultaneously with the detection reaction of the substance being tested include the components described above (e.g., 124), but any component that comes into contact with the sample in a process preceding or simultaneously with the detection reaction of the substance being tested is acceptable and is not limited to this. After the sample is added to the sample addition section 4, the sample flows from the sample addition section 4 to the absorbent pad section 5. When the flow from the sample addition section 4 to the absorbent pad section 5 is described as a flow from upstream to downstream, the component that comes into contact with the sample in a process preceding or simultaneously with the detection reaction of the substance being tested can be described as existing upstream of the site where the detection reaction occurs.

[0119] Next, the immunoassay using this testing device will be explained. First, the sample is floated in the sample extraction solution to prepare a sample sample after extraction of the analyte. Next, the sample sample is added dropwise to the sample addition section 4 of the testing device. The sample sample containing the analyte moves horizontally on the membrane while being immersed in the label section 2, where the label dissolves and unfolds. If the analyte is present in the sample sample, a analyte-label complex is formed. After this complex reaches the detection section 3, a capture antibody-analyte-label complex is formed on the band. By detecting the presence of the complex using the signal emitted from the label in the complex, it is possible to determine whether the analyte is present in the sample. Other components that do not participate in the reaction are absorbed by the absorption pad section 5. It should be noted that, although in Figure 1 In the example shown, there are two detection units 3, but this is to capture two different substances, such as influenza A virus and influenza B virus, respectively. By setting up multiple such detection units 3, multiple substances can be detected simultaneously through immunoassay.

[0120] In the above-mentioned immunochromatographic method, the non-specific reaction inhibitory component for inhibiting false negatives may be pre-contained in the specimen extract used to mix with the specimen to extract the substance to be detected, or the non-specific reaction inhibitory component for inhibiting false negatives may be pre-contained in the nitrocellulose membrane 1, the labeling part 2 and / or the sample addition part 4 of the immunochromatographic testing device.

[0121] This invention includes test reagents that utilize antigen-antibody reactions or the binding reaction of interacting substances. The test reagent may also be referred to as the test device itself, or as a test kit comprising the test device and other reagents. The test reagents of this invention include, for example, test kits comprising an immunochromatographic test device and a specimen extract containing a non-specific reaction inhibitory component that inhibits false negatives. Additionally, the test reagents of this invention include immunochromatographic test devices having a portion comprising a specimen extract containing a non-specific reaction inhibitory component that inhibits false negatives.

[0122] Example

[0123] The present invention will be specifically described through the following embodiments, but the present invention is not limited to these embodiments.

[0124] In the following examples, examples are described in which non-specific reactions in specimens were suppressed in immunochromatographic kits for detecting influenza virus, RS virus, adenovirus, and mycoplasma when using the specimen extraction solution of the present invention.

[0125] Detection of influenza virus antigens by immunochromatography

[0126] 1. Preparation of anti-influenza virus monoclonal antibodies

[0127] (1) Anti-influenza A virus NP (nucleoprotein) antibody

[0128] BALB / c mice were immunized with influenza A virus antigen. Spleens were harvested from mice after a period of rearing and fused with mouse myeloma cells (P3×63) using the method of Kohler et al. (Nature, vol. 256, p495-497 (1975)). The resulting fused cells (hybridomas) were incubated at 37°C. The antibody activity of the supernatant was confirmed by ELISA using a plate immobilized with influenza A virus NP antigen, and the cells were purified (monoclonalized). The two cell lines obtained were intraperitoneally administered to BALB / c mice treated with norpheline. Approximately two weeks later, antibody-containing ascites fluid was collected. IgG was purified from the ascites fluid using affinity chromatography with a protein A column, yielding two purified anti-influenza A virus NP antibodies.

[0129] (2) Anti-influenza B virus NP antibody

[0130] Using influenza B virus antigen, two purified anti-influenza B virus NP antibodies were obtained by the same method as (1).

[0131] 2. Making the marking pad

[0132] One type of antibody was used for purifying anti-influenza A virus NP antibodies and another for purifying anti-influenza B virus NP antibodies. Anti-influenza A virus antibodies were covalently bound to red latex particles, suspended in a flotation solution, and subjected to ultrasonic treatment to prepare a well-dispersed anti-A latex flotation. Similarly, an anti-B latex flotation was prepared by covalently binding anti-influenza B virus antibodies to blue latex particles. The anti-A and anti-B latex flotations were mixed, coated onto glass fibers measuring 20 cm × 1 cm, and thoroughly dried in warm air to create a labeled pad containing the dried mixture.

[0133] 3. Preparation of sample addition pads

[0134] Use fiberglass measuring 2.0cm x 20cm.

[0135] 4. Inspect the fabrication of the components.

[0136] Inspection device usage and Figure 1The devices shown have the same composition. A nitrocellulose membrane is cut to a size of 2cm × 20cm and supported with a plastic sheet with adhesive. A 20cm layer of anti-influenza A virus antibody (a different antibody than the one described above) and anti-influenza B virus antibody (a different antibody than the one described above) solutions, approximately 1mm wide, are coated at positions 0.8cm and 1.0cm from the bottom, respectively. The membrane is then thoroughly dried in warm air to solidify the antibody (detection section). Next, a 3cm × 20cm filter paper is stacked 5mm above the nitrocellulose membrane to form an absorption pad. A label pad is then stacked 2mm below the nitrocellulose membrane to form a label pad. A sample addition pad is then aligned and stacked 7mm above the label pad to form a sample addition section. Finally, the membrane is cut into strips 5mm wide to create an integrated testing device.

[0137] Detection of RS virus, adenovirus, and Mycoplasma pneumoniae antigens by immunochromatography

[0138] 1. Preparation of monoclonal antibodies against RS virus, adenovirus, and Mycoplasma pneumoniae

[0139] BALB / c mice were independently immunized with RS virus antigen, adenovirus antigen, or Mycoplasma pneumoniae antigen, respectively. Spleens were harvested from the mice after a period of rearing and fused with mouse myeloma cells (P3×63) using the method of Kohler et al. (Nature, vol. 256, p495-497 (1975)). The resulting fused cells (hybridomas) were incubated at 37°C, and cell purification (monoclonalization) was performed while confirming antibody activity in the supernatant using ELISA on plates immobilized with the aforementioned antigens. Two cell lines were intraperitoneally administered to BALB / c mice treated with norpheline. Approximately two weeks later, antibody-containing ascites fluid was collected. IgG was purified from the ascites fluid using affinity chromatography on a protein A column, yielding two purified anti-immunogen antibodies for each immunogen.

[0140] 2. Making the marking pad

[0141] Purified anti-immunogen antibodies were covalently bound to red latex particles, suspended in a flotation solution, and subjected to ultrasonic treatment to prepare well-dispersed anti-RS virus latex flotations, anti-adenovirus latex flotations, and anti-Mycoplasma pneumoniae latex flotations. These latex flotations were then coated onto 20cm × 1cm glass fibers and thoroughly dried in warm air to create labeled pads containing the dried mixture.

[0142] 3. Preparation of sample addition pads

[0143] Use fiberglass measuring 2.0cm x 20cm.

[0144] 4. Inspect the fabrication of the components.

[0145] Inspection device usage and Figure 1 The device shown has the same composition. A nitrocellulose membrane is cut to a size of 2cm × 20cm and supported with a plastic sheet with adhesive. Approximately 1mm wide amounts of anti-RS virus antibody (a different antibody from the above) or anti-adenovirus antibody (a different antibody from the above), or anti-Mycoplasma pneumoniae antibody (20cm) are coated at positions 0.8cm and 1.0cm from the bottom edge, respectively. The membrane is then thoroughly dried in warm air to solidify the antibody (detection section). Next, a 3cm × 20cm filter paper is stacked 5mm above the nitrocellulose membrane to form an absorption pad. A label pad is then stacked 2mm below the nitrocellulose membrane to form a label pad. A sample addition pad is then aligned and stacked 7mm above the label pad to form a sample addition section. Finally, the membrane is cut into 5mm wide strips to fabricate an integrated testing device.

[0146] 5. Preparation of specimen extraction solution

[0147] Each of the following test cases is recorded.

[0148] Experimental Example 1

[0149] Inhibition of false negative reactions in extracts containing L-phenylalanine (L-Phe)

[0150] 1-1. Preparation of specimen extract

[0151] A mixture containing 50 mM Tris buffer (pH 8.0), 2 (w / v)% polyoxyethylene octylphenyl ether, and 5 (w / v)% L-arginine was prepared as the sample extraction solution 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, 2.0 (w / v)% L-Phe, and 400 mM sodium bromide was prepared as the sample extraction solution for Experiment 1.

[0152] 1-2. Test Methods

[0153] Saliva samples collected with cotton swabs were floated and dispersed in the extracts of control 1 and test 3, and then filtered through a filter. Additionally, 6.8 × 10⁻⁶ mg of [agent / material] was added and mixed. 2A sample was prepared by inactivating influenza A virus at a concentration of PFU / mL. The sample was then added to the prepared testing device, and the colorimetric intensity of the detection section was measured after 5 minutes. Simultaneously, samples were prepared from the extracts of Control 1 and Test 1 without saliva and with saliva added only and mixed at a concentration of 6.8 × 10⁻⁶ PFU / mL. 2 The experiment was conducted using a sample of inactivated influenza A virus at a concentration of PFU / mL. For colorimetric intensity determination, a red sample scored on a 10-point scale was used.

[0154] 1-3. Results of Color Development Intensity Test

[0155] The results are shown in Table 1. It should be noted that the color intensity values ​​increase sequentially in the order of 0, 1+, 2+...10+, where 0 indicates no color development and the larger the value, the stronger the false negative reaction.

[0156] In the specimen extract of control 1, the signal intensity decreased by 2 levels compared to the sample with saliva. On the other hand, in the specimen extract of test 3, no difference in signal intensity was observed between the sample with and without saliva. Therefore, it can be concluded that the present invention can suppress false negative reactions.

[0157] [Table 1]

[0158]

[0159] Experimental Example 2

[0160] The effect of L-Phe-containing sample extract on the false negative inhibition of RS virus detection by immunochromatography

[0161] 2-1. Preparation of specimen extract

[0162] A mixture containing 50 mM Tris buffer (pH 8.0), 2 (w / v) % polyoxyethylene octylphenyl ether, and 5 (w / v) % L-arginine was prepared as the sample extraction solution for Control 1. Next, a mixture containing 1.5 (w / v) % L-Phe, 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, and 150 mM sodium bromide was prepared as the sample extraction solution for Experiment 4.

[0163] 2-2. Test Methods

[0164] Saliva samples were collected from the oral cavity using cotton swabs. The saliva samples were then floated and dispersed in the extracts of the control and experimental samples (sample 4). The goal was to achieve a concentration of 3.5 × 10⁻⁶. 4 TCID 50Inactivated RS virus was added and mixed separately at a rate of / mL, and used as the sample for this experiment. Additionally, only samples reaching 3.5 × 10⁻⁶ were tested. 4 TCID 50 Inactivated RS virus was added to the extract of the control and test samples (sample 4) at a rate of / mL, without adding saliva, to prepare samples. These samples were then added to the prepared testing device, and the color intensity of the detection section was measured after 5 minutes. For the color intensity measurement, red and blue were measured using color samples scored at 10 levels of color intensity.

[0165] 2-3. Results of color intensity test

[0166] The results are shown in Table 2. It should be noted that the color intensity values ​​increase sequentially in the order of 0, 1+, 2+...10+, where 0 indicates no color is observed, and the higher the value, the stronger the signal.

[0167] In the control sample extract, the false negative reaction caused a decrease in color intensity because no saliva sample was added. However, in the sample extract of test 4, the color intensity did not change at all even without the addition of saliva. This shows that the sample extract containing the components of the present invention inhibits false negatives caused by saliva.

[0168] [Table 2]

[0169] The effect of L-Phe sample extract in RS virus detection on false negative inhibition

[0170]

[0171] Experimental Example 3

[0172] Comparison of sensitivity between L-Phe-containing specimen extracts and existing specimen extracts

[0173] 3-1. Preparation of specimen extract

[0174] A mixture containing 50 mM Tris buffer (pH 8.0), 2 (w / v)% polyoxyethylene octylphenyl ether, and 5 (w / v)% L-arginine was prepared as the sample extraction solution 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 as the sample extraction solution for Experiment 1.

[0175] 3-2. Test Methods

[0176] Add a final concentration of 6.8 × 10⁻⁶ to the extracts of control 1 and test 1, respectively. 2 PFU / mL, 3.4×10 2 PFU / mL, 1.7×10 2 Samples were prepared by inactivating influenza A virus at a concentration of PFU / mL. Similarly, samples were prepared to achieve a final concentration of 4.0 × 10⁻⁶ PFU / mL for inactivated influenza B virus. 2 PFU / mL, 2.0×10 2 PFU / mL, 1.0×10 2 A sample of PFU / mL was prepared. Then, 50 μL of this sample was added to the prepared testing device, and the color intensity of the detection section was measured after 5 minutes. For the color intensity measurement, red and blue samples were measured using color samples scored at 10 levels of color intensity.

[0177] 3-3. Results of Color Development Intensity Test

[0178] The results are shown in Table 3. It should be noted that the color rendering intensity values ​​increase sequentially in the order of 0, 0.5+, 1+, 2+...10+, where 0 indicates no color development, and higher values ​​indicate a stronger signal. The results are expressed as "color rendering intensity of type A / color rendering intensity of type B".

[0179] The sensitivity was the same in both the sample extract of Control 1 and the sample extract of Test 1. The signal intensity was higher in Test 1. In summary, using the sample extract used in this invention improves specificity without reducing sensitivity.

[0180] [Table 3]

[0181] Comparison of sensitivity between L-Phe-containing specimen extracts and existing specimen extracts

[0182]

[0183] Test Example 4

[0184] False negative inhibition effect of specimens containing L-Phe analogues

[0185] 4-1. Preparation of specimen extraction solution

[0186] A mixture containing 50 mM Tris buffer (pH 8.0), 2 (w / v)% polyoxyethylene octylphenyl ether, and 2 (w / v)% L-arginine was prepared as the control (no additives) sample extract. Next, a sample extract containing 50 mM Tris buffer (pH 8.0), 2 (w / v)% polyoxyethylene octylphenyl ether, and 2 (w / v)% L-arginine, plus one compound from Table 4 (final concentration in parentheses), was prepared as the experimental sample extract.

[0187] [Table 4]

[0188] List of additives in specimen extracts

[0189] Additive name (final concentration) Abbreviation L-Phenylalanine (2.0%) L-Phe D-Phenylalanine (2.0%) D-Phe Aspartame (1.0%) - hippuric acid (2.0%) - ±Mandelic acid (2.0%) - L-Tryptophan (1.0%) L-Trp L-Phenylalanine Methyl Ester (2.0%) L-PheME N-Phenylglycine (2.0%) N-PG DL-2-Phenylglycine (0.1%) 2-PG Sodium benzoate (2.0%) - 3-Phenylacetic acid (1.5%) 3PPA N-(m-Toluyl)glycine (1.5%) NmTG Acetylsalicylic acid (1.5%) AcSA

[0190] 4-2. Test Methods

[0191] Saliva samples showing false negative reactions, influenza virus negativity, and RSV negativity in the control specimen flotation solution were collected using cotton swabs. These samples were then flotated and dispersed in the control and other test specimen extracts, and used as the samples for this test. A final concentration of 6.8 × 10⁻⁶ was then added and mixed. 2 Influenza A virus at a final concentration of 4.0 × 10⁻⁶ PFU / mL or influenza B virus at a final concentration of 3.5 × 10⁻⁶ PFU / mL. 4 TCID 50 / mL of RS virus antigen. Next, these were added to each of the above-prepared examination devices, and after 5 minutes, the colorimetric intensity of the detection section was measured. During the colorimetric intensity measurement, a color sample scored on a 10-level scale was used to determine the degree of suppression of false negatives.

[0192] 4-3. Results of Color Development Intensity Test

[0193] The results are shown in Tables 5 and 6. In the description of the results, "×" indicates no effect, "△" indicates a slight improvement, "〇" indicates a significant improvement, and "◎" indicates a strong improvement. It should be noted that in the influenza virus test kit, "Influenza A / Influenza B" is used for description.

[0194] In RS virus test kits, almost all tested compounds were confirmed to inhibit false negatives, and even in influenza virus test kits, multiple compounds were confirmed to inhibit false negatives.

[0195] [Table 5]

[0196] The inhibitory effect of L-Phe analogue-containing specimen extracts in RS virus detection reagents on false negatives caused by saliva.

[0197] condition False negative inhibition effect Comparison (no additives) -(Causing false negatives) L-Phenylalanine (2.0%) △ D-Phenylalanine (2.0%) △ Aspartame (1.0%) △ hippuric acid (2.0%) 〇 ±Mandelic acid (2.0%) 〇 L-Tryptophan (1.0%) △ L-PheME (2.0%) △ N-Phenylglycine (2.0%) ◎ DL-2-Phenylglycine (0.1%) 〇 Sodium benzoate (2.0%) 〇 3-PPA (1.5%) ◎ NmTG (1.5%) ◎ Acetylsalicylic acid (1.5%) △

[0198] "×" indicates no effect, and "△" indicates a slight improvement.

[0199] “〇” indicates a visible and significant improvement effect, and “◎” indicates a visible and powerful improvement effect.

[0200] [Table 6]

[0201] The inhibitory effect of specimen extracts containing L-Phe analogs in influenza virus test kits on false negatives caused by saliva.

[0202] condition False negative inhibition effect Comparison (no additives) False negatives L-Phenylalanine (2.0%) 〇 / 〇 D-Phenylalanine (2.0%) △ / △ Aspartame (1.0%) × / × hippuric acid (2.0%) 〇 / 〇 ±Mandelic acid (2.0%) ◎ / ◎ L-Tryptophan (1.0%) △ / △ L-PheME (2.0%) × / × N-Phenylglycine (2.0%) ◎ / ◎ DL-2-Phenylglycine (0.1%) × / × Sodium benzoate (2.0%) × / × 3-PPA (1.5%) 〇 / ◎ NmTG (1.5%) 〇 / 〇 Acetylsalicylic acid (1.5%) × / ×

[0203] The description is in the form of "Influenza A / Influenza B".

[0204] "×" indicates no effect, and "△" indicates a slight improvement.

[0205] “〇” indicates a visible and significant improvement effect, and “◎” indicates a visible and powerful improvement effect.

[0206] Industrial applicability

[0207] The method of this invention can be used to accurately detect various substances.

[0208] Explanation of reference numerals in the attached figures

[0209] 1: Nitrocellulose membrane; 2: Labeling section; 3: Detection section; 4: Sample addition section; 5: Absorbent pad section; 6: Plastic plate.

[0210] All publications, patents and patent applications cited in this specification are incorporated herein by reference.

Claims

1. A testing reagent kit that uses an antigen-antibody reaction to detect a analyte in a specimen. The specimens selected were pharyngeal swabs, nasal swabs, nasal aspirates, nasal irrigation fluid, blown nasal mucus, and saliva. The substances to be detected are selected from the group consisting of influenza virus antigen; adenovirus antigen; human respiratory syncytial virus (RSV) antigen; human metapneumovirus (hMPV) antigen; coronavirus antigens including SARS-CoV, MERS-CoV, or SARS-CoV2; group A streptococci; group B streptococci; and mycoplasma antigens. The test reagent comprises a specimen extract containing 0.1-10 (w / v)% of a non-specific reaction inhibitor that inhibits false negatives. The non-specific reaction inhibitor that inhibits false negatives is selected from the group consisting of L-phenylalanine, D-phenylalanine, mandelic acid, N-phenylglycine, and 3-phenylpropionic acid.

2. The testing reagent according to claim 1, wherein, The specimen extract further contains amino acids or amino acid derivatives selected from the group consisting of arginine, lysine, arginine ethyl ester, arginine methyl ester, glycine ethyl ester and glycine methyl ester, as well as optical isomers, geometric isomers, structural isomers and stereoisomers of these compounds.

3. The testing reagent according to claim 1 or 2, wherein, The specimen extract further contains halides selected from the group consisting of lithium chloride, sodium chloride, potassium chloride, sodium bromide, potassium bromide, sodium iodide, and potassium iodide.

4. A method for suppressing false negative reactions when using antigen-antibody reactions to detect a substance in a specimen, the method suppressing false negative reactions by pre-contaminating the specimen with a specimen extract, wherein the substance to be detected in the specimen is selected from the group consisting of influenza virus antigen; adenovirus antigen; human respiratory syncytial virus (RSV) antigen; human metapneumovirus (hMPV) antigen; coronavirus antigens containing SARS-CoV, MERS-CoV, or SARS-CoV2; group A streptococci; group B streptococci; and mycoplasma antigens, wherein the specimen is selected from the group consisting of pharyngeal swab specimens, nasal swab specimens, nasal aspirate specimens, nasal irrigation specimens, blown nasal mucus specimens, and saliva. The specimen extract contains 0.1-10 (w / v)% of a non-specific reaction inhibitor that inhibits false negatives, wherein the non-specific reaction inhibitor that inhibits false negatives is selected from the group consisting of L-phenylalanine, D-phenylalanine, mandelic acid, N-phenylglycine and 3-phenylpropionic acid.

5. The method according to claim 4, wherein, The method for detecting the substance being tested is immunochromatography.

6. The method according to claim 4 or 5, wherein, The specimen extract further contains amino acids or amino acid derivatives selected from arginine, lysine, arginine ethyl ester, arginine methyl ester, glycine ethyl ester and glycine methyl ester, as well as optical isomers, geometric isomers, structural isomers and stereoisomers of these compounds.

7. The method according to claim 4 or 5, wherein, The specimen extract further contains halides selected from the group consisting of lithium chloride, sodium chloride, potassium chloride, sodium bromide, potassium bromide, sodium iodide, and potassium iodide.

Images