How to Detect Mycoplasma pneumoniae

Abstract

To provide a method for treating an analyte for rapidly and efficiently extracting an antigen from Mycoplasma pneumoniae contained in the analyte and also to provide an inspection method for detecting the extracted antigen by immunoassay.SOLUTION: Provided is a method for detecting Mycoplasma Pneumoniae contained in an analyte, which includes: a step (a) of obtaining an intermediate composition by bringing the analyte into contact with a first reagent containing an ionic surfactant which does not exhibit alkaline properties; a step (b) of obtaining the reaction liquid by bringing the intermediate composition with a second reagent containing a non-ionic surfactant; and a detection step (c) of detecting an antigen by providing the reaction liquid to the immunoassay using an antibody against the antigen of Mycoplasma Pneumoniae.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for detecting Mycoplasma pneumoniae by immunoassay. [Background technology]

[0002] Respiratory infections are diseases caused by a variety of microorganisms and can be broadly classified into viral respiratory infections caused by influenza viruses, adenoviruses, etc., and bacterial respiratory infections caused by Streptococcus pneumoniae, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Bordetella pertussis, etc.

[0003] Mycoplasma pneumoniae is a typical causative bacterium of atypical pneumonia, and is particularly prevalent in children, adolescents, and adults. Mycoplasmal pneumonia is characterized by a prolonged dry, severe cough, and is considered a disease that is difficult to distinguish from a common cold in the early stages. Macrolide antibiotics are effective against Mycoplasma pneumoniae infections, but these drugs are not very effective against other bacterial pneumonias. Conversely, antibiotics such as cephalosporins that are effective against other bacterial pneumonias are not effective against Mycoplasma pneumoniae. Therefore, it is important to diagnose mycoplasmal pneumonia early.

[0004] Tests for Mycoplasma pneumoniae infection include culture tests, genetic tests, antibody tests, and antigen tests. Culture tests are not suitable for rapid diagnosis because Mycoplasma pneumoniae itself only grows in special culture media, and the procedure is difficult and requires skill, so it can only be performed in limited facilities. It takes about three weeks to culture, so it is not suitable for rapid diagnosis. Polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) are known as genetic tests, but they generally take several hours from sample collection to diagnosis and require specialized equipment, so they can only be performed in limited facilities. Furthermore, antibody tests measure specific antibodies against Mycoplasma pneumoniae, but paired sera collected at intervals must be measured to observe the changes in antibody titer, so they are not suitable for rapid diagnosis. Rapid antibody titer measurement kits that do not require paired sera are also commercially available, but they are considered to be unreliable due to many false positives.

[0005] Antigen testing is a testing method that uses a target antigen derived from a microorganism and an antibody that specifically detects it to determine the presence or absence of a microorganism, and examples of such testing include enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, immunoturbidity assay, Western blotting, immunoprecipitation, immunochromatography assay, and flow cytometry assay. Among these, antigen testing using immunochromatography does not require special equipment or procedures and allows for rapid determination.

[0006] Known antigen tests for Mycoplasma pneumoniae using immunochromatography are, for example, those that use intracellular ribosomal protein L7 / L12, heat shock protein DnaK, and adhesion organelle-constituting proteins on the cell membrane, P1 protein, and P30 protein, as target antigens, and are capable of specifically detecting Mycoplasma pneumoniae. Meanwhile, Non-Patent Document 1 reports that when intracellular proteins, particularly ribosomal protein L7 / L12, are used as target antigens, the sensitivity to live bacteria is higher than that to dead bacteria.

[0007] In order to detect intracellular antigens such as ribosomal protein L7 / L12, not only is the antibody highly specific to the antigen to be measured, but also pretreatment and cell disruption are essential. Patent Document 1 discloses a method for disrupting cells to detect intracellular antigens, in which a bacteriolytic agent containing lysostaphin, an ionic surfactant, and a nonionic surfactant is mixed to lyse Staphylococcus aureus present in milk, which is one of the causative bacteria of bovine mastitis. Patent Document 2 further discloses a method for detecting bacteria contained in a specimen, which includes an extraction step of contacting the specimen with an alkaline solution to extract intracellular antigens of bacteria contained in the specimen into the alkaline solution to obtain a specimen extract, a neutralization step of contacting the specimen extract with a neutralizing solution to obtain a neutralized product, and a detection step of subjecting the neutralized product to an immunoassay using an antibody against intracellular antigens of bacteria to detect the intracellular antigens, and the neutralizing solution includes a means for suppressing false positive reactions in the immunoassay. On the other hand, as a method for extracting intracellular antigens from Mycoplasma pneumoniae, Patent Document 3 discloses a method for extracting antigens by contacting Mycoplasma pneumoniae with polyoxyethylene alkyl ether or polyoxyethylene cetyl ether having an HLB value of 12 to 15, and discloses that this method can detect Mycoplasma pneumoniae in a specimen sample with high sensitivity and specificity. However, according to Non-Patent Document 2, the conventional ribosomal protein L7 / L12 antigen test for Mycoplasma pneumoniae requires standing for 2 minutes to extract the antigen, and there is room for improvement in operability. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication WO2015 / 093544 (Patent No. 6314156) [Patent Document 2] JP 2017-207333 A [Patent Document 3] JP 2014-167439 A (Patent No. 6150559) [Non-patent literature]

[0009] [Non-Patent Document 1] Medical Examination vol69(1), 2020 30-35 [Non-Patent Document 2] Medical Science Digest vol40(10),2014 40-44 Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention aims to provide a sample processing method for rapidly and efficiently extracting an antigen from Mycoplasma pneumoniae contained in the sample, and a testing method for detecting the extracted antigen by immunoassay. [Means for solving the problem]

[0011] In order to solve the above problems, the present inventors have discovered that by contacting Mycoplasma pneumoniae in a specimen with a first reagent containing an ionic surfactant and then with a second reagent containing a nonionic surfactant, intracellular antigens can be instantly and efficiently extracted from Mycoplasma pneumoniae in a specimen and detected simply, quickly and with high sensitivity, and have completed the present invention. That is, the present invention provides the following.

[0012] [1] A method for detecting Mycoplasma pneumoniae contained in a specimen, comprising: (a) contacting the sample with a first reagent containing an ionic surfactant that is not alkaline to obtain an intermediate composition; (b) contacting the intermediate composition with a second reagent containing a nonionic surfactant to obtain a reaction solution; and (c) subjecting the reaction solution to an immunoassay using an antibody against an antigen of Mycoplasma pneumoniae to detect the antigen; A method comprising: [2] A method for detecting Mycoplasma pneumoniae contained in a sample, comprising: (a) contacting the sample with a first reagent comprising an ionic surfactant to obtain an intermediate composition; (b) contacting the intermediate composition with a second reagent containing a nonionic surfactant to obtain a reaction solution; and (c) subjecting the reaction solution to an immunoassay using an antibody against an antigen of Mycoplasma pneumoniae to detect the antigen; A method comprising: [3] The method according to [1] or [2], comprising carrying out steps (a), (b) and (c) in this order. [4] The method according to any one of [1] to [3], wherein the ionic surfactant is a cationic surfactant. [5] The method according to [4], wherein the cationic surfactant is a quaternary ammonium salt. [6] The method according to [5], wherein the quaternary ammonium salt is any one selected from the group consisting of didecyldimethylammonium adipate, didecyldimethylammonium chloride, didecyldimethylammonium bromide, decyltrimethylammonium chloride, decyltrimethylammonium bromide, dodecyltrimethylammonium chloride, trimethyltetradecylammonium chloride, hexadecyltrimethylammonium chloride, trimethylstearylammonium chloride, and benzalkonium chloride. [7] The method according to [5] or [6], wherein the quaternary ammonium salt is didecyldimethylammonium adipate. [8] The method according to any one of [1] to [3], wherein the ionic surfactant is an anionic surfactant. [9] The method according to [8], wherein the anionic surfactant is sodium linear alkylbenzene sulfonate or sodium dodecyl sulfate.

[10] The method according to any one of [1] to [3], wherein the ionic surfactant is an amphoteric surfactant.

[11] The method according to

[10] , wherein the amphoteric surfactant is 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxypropanesulfonate, or lauryl dimethylaminoacetic acid betaine.

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

[11] , wherein the nonionic surfactant is any one selected from the group consisting of polyoxyethylene alkyl ethers, polyoxyethylene polyoxypropylene alkyl ethers, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, and polyoxyethylene alkyl phenyl ethers.

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

[12] , wherein the nonionic surfactant has an HLB value of 10.5 to 18.5.

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

[13] , wherein the nonionic surfactant has an HLB value of 12.5 to 16.7.

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

[14] , wherein the nonionic surfactant is any one selected from the group consisting of polyoxyethylene (20) polyoxypropylene (8) cetyl ether, polyoxyethylene (9) alkyl (sec-C11-15) ether, p-tert-octylphenoxypolyethyl alcohol, polyoxyethylene (13) oleyl ether, polyoxyethylene sorbitan monostearate, polyoxyethylene (20) stearyl ether, polyoxyethylene sorbitan monopalmitate, polyoxyethylene (20) cetyl ether, and poly(oxyethylene) sorbitan monolaurate.

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

[15] , wherein the nonionic surfactant is poly(oxyethylene) sorbitan monolaurate.

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

[16] , which satisfies any of the following: The concentration of the ionic surfactant in the first reagent is 0.0025 to 0.10 (w / v)%. The concentration of the ionic surfactant in the reaction solution is 0.0017 to 0.067 (w / v)%.

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

[17] , which satisfies any of the following: The concentration of the nonionic surfactant in the second reagent is 0.5 to 3.0 (w / v)%. The concentration of the nonionic surfactant in the reaction solution is 0.17 to 1.0 (w / v)%.

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

[18] , wherein the intermediate composition is brought into contact with the second reagent by passing the intermediate composition through a filter provided in a sample addition member or a dropping nozzle impregnated with the second reagent.

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

[19] , wherein the antigen is ribosomal protein L7 / L12.

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

[20] , wherein the immunoassay is an enzyme-linked immunosorbent assay (ELISA), an immunofluorescence assay, an immunoturbidity assay, a Western blotting method, an immunoprecipitation method, an immunochromatography assay, or a flow cytometry assay.

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

[21] , wherein the immunoassay is an immunochromatographic assay.

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

[22] , wherein the sample is a throat swab, a nasal swab, a nasal aspirate, or sputum.

[24] A kit for carrying out the method according to any one of [1] to

[23] , comprising: A first reagent comprising an ionic surfactant A second reagent, comprising a non-ionic surfactant Effect of the Invention

[0013] According to the present invention, an antigen can be extracted from Mycoplasma pneumoniae contained in a sample, and the antigen can be detected by immunoassay quickly, simply and with high sensitivity. [Brief description of the drawings]

[0014] [Figure 1] This is a schematic cross-sectional view showing an example of an immunochromatography device, in which 1 is a substrate, 2 is a labeled antibody-impregnated member, 3 is a chromatographic development membrane carrier, 4 is an absorption member, 5 is a sample addition member, and 6 is a determination part or capture part where an antibody against an intracellular antigen of a bacteria is fixed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention will now be described in further detail. The present invention provides a method for detecting Mycoplasma pneumoniae contained in a sample, comprising: (a) a step of contacting the sample with a first reagent containing an ionic surfactant to obtain an intermediate composition; (b) a step of contacting the intermediate composition with a second reagent containing a nonionic surfactant to obtain a reaction liquid; and (c) a detection step of subjecting the reaction liquid to an immunoassay using an antibody against an antigen of Mycoplasma pneumoniae to detect the antigen.

[0016] (1) Extraction of antigen: steps (a) and (b) [Detection target bacteria] The present invention is used to detect Mycoplasma pneumoniae.

[0017] 〔antigen〕 In the present invention, an antigen derived from Mycoplasma pneumoniae is detected. The antigen includes various antigens derived from Mycoplasma pneumoniae, such as intracellular ribosomal protein L7 / L12, heat shock protein DnaK, and adhesion organelle-constituting proteins on the cell membrane, P1 protein and P30 protein. Ribosomal protein L7 / L12 is preferred.

[0018] The term "detection" refers to analyzing the presence or absence, or the amount, of the target bacteria or antigen derived therefrom. Therefore, the case where the target bacteria or antigen is not detected as a result of the detection corresponds to the implementation of the method of the present invention, just as in the case where the target bacteria or antigen is detected.

[0019] [Specimen] The specimen in the present invention is not particularly limited as long as it may contain Mycoplasma pneumoniae. Preferably, the specimen includes blood, urine, and the like, as well as pharyngeal swabs, nasal swabs, nasal aspirates, sputum, and the like, which can be collected from the upper respiratory tract such as the pharynx, nose, nasal cavity, and nasopharynx. More preferably, the specimen includes pharyngeal swabs, nasal swabs, nasal aspirates, and sputum, and a particularly preferred example is pharyngeal swabs.

[0020] [First Reagent, Second Reagent] The antigen is extracted in two steps: (a) a sample collected with a cotton swab or the like is contacted with a first reagent containing an ionic surfactant to obtain an intermediate product, and (b) the sample is contacted with a second reagent containing a nonionic surfactant to obtain a reaction solution, which is then subjected to an immunoassay using an antibody against an antigen of Mycoplasma pneumoniae to detect the antigen.

[0021] [Surfactant] The first reagent used in step (a) of the present invention contains an ionic surfactant.

[0022] A surfactant is a substance that acts on an interface to change its properties, and has a structure in which the molecule has a hydrophilic group that shows hydrophilicity and a hydrophobic group that shows lipophilicity. Surfactants that ionize to become ions are called ionic surfactants, and surfactants that do not become ions are called nonionic surfactants. Ionic surfactants are further classified into cationic surfactants, anionic surfactants, and amphoteric surfactants.

[0023] The cationic surfactant that can be used in the first reagent may be any surfactant that, when dissolved in water, ionizes the portion to which the hydrophobic group is attached to a positive ion, and is structurally classified into quaternary ammonium salt type (alkyltrimethylammonium chloride, alkyltrimethylammonium bromide, alkyltrimethylammonium iodide, dialkyldimethylammonium chloride, dialkyldimethylammonium bromide, dialkyldimethylammonium iodide, alkylbenzalkonium chloride, etc.) and alkylamine salt type (monoalkylamine salt, dialkylamine salt, trialkylamine salt, etc.). Either type can be suitably used in the present invention. Preferred examples of the quaternary ammonium salt type are didecyldimethylammonium adipate, didecyldimethylammonium chloride, didecyldimethylammonium bromide, decyltrimethylammonium chloride, decyltrimethylammonium bromide, dodecyltrimethylammonium chloride, trimethyltetradecylammonium chloride, hexadecyltrimethylammonium chloride, trimethylstearylammonium chloride, and benzalkonium chloride, more preferably didecyldimethylammonium adipate.

[0024] When a cationic surfactant is used in the first reagent, its concentration in the first reagent is not particularly limited as long as an effective extraction rate for detection is ensured. For example, the lower limit can be 0.0010 (w / v)% or more, and preferably 0.0025 (w / v)% or more. The upper limit can be 0.50 (w / v)% or less, and preferably 0.25 (w / v)% or less, and more preferably 0.10 (w / v)% or less. The range can be, for example, 0.0010 to 0.50 (w / v)%, and preferably 0.0025 to 0.25 (w / v)%, and more preferably 0.0025 to 0.10 (w / v)%. The concentration of the cationic surfactant in the reaction solution (first reagent + second reagent, the amount of the sample can usually be ignored but may be considered in some cases) is 1 / 2 to 3 / 4 times, for example 2 / 3 times, the concentration described here. Specifically, the lower limit may be 0.00067 (w / v)% or more, preferably 0.0017 (w / v)% or more. The upper limit may be 0.33 (w / v)% or less, preferably 0.17 (w / v)% or less, more preferably 0.067 (w / v)% or less. The range may be, for example, 0.00066 to 0.33 (w / v)%, preferably 0.0017 to 0.17 (w / v)%, more preferably 0.0017 to 0.067 (w / v)%.

[0025] The anionic surfactant that can be used in the first reagent may be any surfactant that dissociates a hydrophobic group into a negative ion when dissolved in water, and is structurally classified into carboxylic acid type (aliphatic monocarboxylate, polyoxyethylene alkyl ether carboxylate, N-acylsarcosine salt, N-acyl glutamate, etc.), sulfonic acid type (dialkyl sulfosuccinate, alkanesulfonate, alpha olefin sulfonate, linear alkyl benzene sulfonate, alkyl (branched) benzene sulfonate, naphthalene sulfonate-formaldehyde condensate, alkyl naphthalene sulfonate, N-methyl-N-acyltaurate, etc.), sulfate type (alkyl sulfate, polyoxyethylene alkyl ether sulfate, fat sulfate, etc.), and phosphate type (alkyl phosphate, polyoxyethylene alkyl ether phosphate, polyoxyethylene alkyl phenyl ether phosphate, etc.). Any of these can be suitably used in the present invention. Preferred examples are linear alkyl benzene sodium sulfonate or sodium dodecyl sulfate.

[0026] When an anionic surfactant is used in the first reagent, its concentration in the first reagent is not particularly limited as long as an effective extraction rate for detection is ensured. For example, the lower limit can be 0.0010 (w / v)% or more, and preferably 0.0025 (w / v)% or more. The upper limit can be 0.50 (w / v)% or less, preferably 0.25 (w / v)% or less, and more preferably 0.10 (w / v)% or less. The range can be, for example, 0.0010 to 0.50 (w / v)%, preferably 0.0025 to 0.25 (w / v)%, and more preferably 0.0025 to 0.10 (w / v)%. The concentration of the anionic surfactant in the reaction solution (first reagent + second reagent, the amount of the sample can usually be ignored but may be considered in some cases) is 1 / 2 to 3 / 4 times, for example 2 / 3 times, the concentration described here. Specifically, the lower limit may be 0.00067 (w / v)% or more, preferably 0.0017 (w / v)% or more. The upper limit may be 0.33 (w / v)% or less, preferably 0.17 (w / v)% or less, more preferably 0.067 (w / v)% or less. The range may be, for example, 0.00066 to 0.33 (w / v)%, preferably 0.0017 to 0.17 (w / v)%, more preferably 0.0017 to 0.067 (w / v)%.

[0027] The amphoteric surfactant that can be used in the first reagent may be any surfactant that, when dissolved in water, exhibits the properties of an anionic surfactant in the alkaline range and the properties of a cationic surfactant in the acidic range, and is structurally classified into carboxybetaine type (alkylbetaine, fatty acid amidopropylbetaine, etc.), 2-alkylimidazoline derivative type (2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine, etc.), glycine type (alkyldiethylenetriamineacetic acid, dialkyldiethylenetriamineacetic acid, etc.), and amine oxide type (alkylamine oxide, etc.). Any of these can be suitably used in the present invention. Preferred examples are 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxypropanesulfonate, and lauryldimethylaminoacetic acid betaine.

[0028] When an amphoteric surfactant is used in the first reagent, its concentration in the first reagent is not particularly limited as long as an effective bacteriolysis rate for detection is ensured, and is, for example, 0.010 to 0.10 (w / v)%. The concentration of the amphoteric surfactant in the reaction solution (first reagent + second reagent, the amount of the sample can usually be ignored but may be considered in some cases) is 1 / 2 to 3 / 4 times, for example 2 / 3 times, the concentration described here. Specifically, the lower limit is more preferably 0.0067 to 0.067 (w / v)%.

[0029] The second reagent used in step (b) of the present invention contains a nonionic surfactant.

[0030] The nonionic surfactant that can be used in the second reagent may be any surfactant having a hydrophilic group that does not ionize when dissolved in water, and is structurally classified into ester type (glycerin fatty acid ester, sorbitan fatty acid ester, sucrose fatty acid ester, etc.), ether type (polyoxyethylene alkyl ether, polyoxyethylene alkylphenyl ether, polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene alkyl ether, etc.), ester ether type (fatty acid polyethylene glycol, polyoxyethylene sorbitan fatty acid ester, etc.), and alkanolamide type (fatty acid alkanolamide, etc.). Any of these types can be suitably used in the present invention. Preferred examples are polyoxyethylene alkyl ether, polyoxyethylene polyoxypropylene alkyl ether, sorbitan fatty acid ester, or polyoxyethylene alkylphenyl ether. More particularly, it is polyoxyethylene (8) oleyl ether, polyoxyethylene (5) alkyl (sec-C11-15) ether, polyoxyethylene (20) polyoxypropylene (8) cetyl ether, polyoxyethylene (9) alkyl (sec-C11-15) ether, p-tert-octylphenoxypolyethyl alcohol, polyoxyethylene (13) oleyl ether, polyoxyethylene sorbitan monostearate, polyoxyethylene (20) stearyl ether, polyoxyethylene sorbitan monopalmitate, polyoxyethylene (20) cetyl ether, or poly(oxyethylene) sorbitan monolaurate. Preferred are polyoxyethylene (20) polyoxypropylene (8) cetyl ether, polyoxyethylene (9) alkyl (sec-C11-15) ether, p-tert-octylphenoxypolyethyl alcohol, polyoxyethylene (13) oleyl ether, polyoxyethylene sorbitan monostearate, polyoxyethylene (20) stearyl ether, polyoxyethylene sorbitan monopalmitate, polyoxyethylene (20) cetyl ether, or polyoxyethylene sorbitan monolaurate, and more preferred is polyoxyethylene sorbitan monolaurate.

[0031] In addition, when selecting a nonionic surfactant to be used in the second reagent, the hydrophile-lipophile balance (HLB) value, which indicates the balance between hydrophilic and hydrophobic groups, can be used as an index. Methods for calculating the HLB value include the Griffin method, the Atlas method, the Davis method, and the Kawakami method. As the nonionic surfactant to be used in the second reagent of the present invention, those having an HLB value based on the Griffin method of 8.0 to 18.5, which is considered to be suitable for the preparation of an oil-in-water type (oil in water O / W) emulsion, can be used, preferably 10.5 to 18.5, and more preferably 12.5 to 16.7. The HLB value based on the Griffin method can be calculated by 20 x the sum of the formula weights of the hydrophilic groups / molecular weight.

[0032] The concentration of the nonionic surfactant in the second reagent is not particularly limited as long as the flow of the developing solution is ensured, for example, when an immunochromatography assay is used. For example, the lower limit can be 0.10 (w / v)% or more, preferably 0.25 (w / v)% or more, and more preferably 0.50 (w / v)% or more. The upper limit of the concentration may be such that it does not significantly inhibit solubilization by the first reagent containing the ionic surfactant or the antigen-antibody reaction in the immune reaction. For example, the upper limit can be 10 (w / v)% or less, preferably 5.0 (w / v)% or less, and more preferably 3.0 (w / v)% or less. The range can be, for example, 0.10 to 10 (w / v)%, preferably 0.25 to 5.0 (w / v)%, and more preferably 0.5 to 3.0 (w / v)%. The concentration of the nonionic surfactant in the reaction solution (the amount of the first reagent + the second reagent and the sample can usually be ignored, but may be considered in some cases) is 1 / 4 to 1 / 2 times, for example, 1 / 3 times, of the concentration described here. Specifically, the lower limit can be 0.033 (w / v)% or more, preferably 0.083 (w / v)% or more, and more preferably 0.17 (w / v)% or more. The upper limit of the concentration may be such that it does not significantly inhibit solubilization by the first reagent containing the ionic surfactant or the antigen-antibody reaction in the immune reaction. For example, the upper limit can be 3.3 (w / v)% or less, preferably 1.7 (w / v)% or less, and more preferably 1.0 (w / v)% or less. The range can be, for example, 0.033 to 3.3 (w / v)%, preferably 0.083 to 1.7 (w / v)%, and more preferably 0.17 to 1.0 (w / v)%.

[0033] [About alkali] The first reagent of the present invention can be configured not to exhibit alkalinity. In the present invention, not exhibiting alkalinity refers to not having an alkaline liquid property (pH exceeding 11). The liquid property of the first reagent that does not exhibit alkalinity may be weakly alkaline (pH exceeding 8.0 and not more than 11.0) or neutral (pH of 6.0 to 8.0). Since the first reagent is not alkaline, there are merits such as safer operation, no need for the trouble of neutralization, no limitations on the material of the container for the first reagent (if the first reagent is not alkaline, a container made of low-density polyethylene, high-density polyethylene, EVA resin, polypropylene, polyvinyl chloride resin, polystyrene, expanded polystyrene, AS resin, ABS resin, polyethylene terephthalate, methacrylic resin, polyvinyl alcohol, vinylidene chloride resin, polycarbonate, polyamide, acetal resin, polybutylene terephthalate, fluororesin, phenolic resin, melamine resin, urea resin, polyurethane, epoxy resin, or unsaturated polyester resin can be used without considering alkali resistance. On the other hand, if the first reagent is alkaline, a container made of a material suitable for alkaline storage must be selected, which may adversely affect the accuracy of the test, such as the occurrence of false positives during long-term storage), and no need to worry about the stability of the antigen extracted in the first reagent (problems such as denaturation can be avoided).

[0034] Both the first and second reagents may contain substances other than the surfactant as long as they exert the intended effect.

[0035] [Two-step operation] The two-step procedure uses a first reagent and a second reagent separately (not mixed) in that order.

[0036] Specifically, the two-stage operation of the present invention can be carried out as follows. When the specimen is a throat swab, the cotton swab with the specimen attached thereto can be immersed in a tube containing a first reagent containing an ionic surfactant, and then a second reagent containing a nonionic surfactant can be added. When an immunochromatography device is used, the second reagent can be prepared in advance by immersing it in a filter provided in the dripping nozzle, a sample addition member (5 in the example of FIG. 1) or a labeled antibody-impregnated member (2 in the example of FIG. 1) of the device, and the first reagent with the specimen attached thereto immersed therein can be directly dripped onto the sample addition member.

[0037] Contact between the first reagent and the specimen can be performed at ambient temperature, if necessary, or under cooled conditions that are less likely to cause deterioration of components such as proteins. The contact can be performed for a period of time that allows sufficient extraction of intracellular antigens, for example, from several seconds to several hours, and stirring or shaking may be performed during the contact, if necessary.

[0038] According to the study by the present inventors, the first and second reagents may be used in any order, and the interval between addition is not particularly limited. The time between the addition of the first and second reagents may be short. In other words, the method of the present invention has the advantage of being able to perform the test quickly.

[0039] The two-step operation allows both efficient bacteriolysis and accurate immunoassay. Since ionic surfactants are charged, they have a high binding affinity with similarly charged proteins. Therefore, it is reasonable to select an ionic surfactant from the viewpoint of bacteriolysis, but in the case of immunoassay, ionic surfactants also have a high binding affinity with the antibodies used in the assay, and as a result, there is a high possibility that they will inhibit the antigen-antibody reaction. The present inventors have found that, in response to this problem, the inhibition of immune reactions by ionic surfactants can be suppressed by adding a specific nonionic surfactant at a fixed ratio. On the other hand, the addition of a nonionic surfactant at a fixed ratio can, in principle, also reduce the bacteriolytic action of the ionic surfactant, so that mixing two types of surfactants in advance can result in insufficient bacteriolysis. Therefore, it is important to use an ionic surfactant alone during bacteriolysis.

[0040] (2) Immunoassay of extracted antigen: step (c) [Type of measurement method] The method for measuring the extracted antigen in the present invention is not particularly limited, but immunoassay using an antigen-antibody reaction utilizing the binding reaction between antigen and antibody molecules is preferable. Immunoassay methods include enzyme immunoassay (EIA), which quantitatively tracks the antigen-antibody reaction using an enzyme to measure the antigen or antibody, and radioimmunoassay (RIA), which quantitatively tracks the antigen-antibody reaction with the aid of a radioisotope to measure the antigen or antibody. The binding reaction between antigens and antibodies is generally highly specific, and they bind relatively easily even at low concentrations, and once bound, they are relatively difficult to dissociate. Therefore, if the antigen-antibody reaction is quantified in some way, it is possible to measure minute amounts of antigens or antibodies.

[0041] The immunoassay in the present invention refers to various immunological assay methods, including, but not limited to, enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, immunoturbidity assay, Western blotting, immunoprecipitation, immunochromatography assay, flow cytometry assay, etc. When carrying out an immunological assay method, a blocking substance for preventing non-specific adsorption or a substance for preventing cross-reaction with bacteria other than the target bacterial species may be used.

[0042] [ELISA method] In this invention, enzyme-linked immunosorbent assay (ELISA) is a method for measuring substances such as proteins using an antigen-antibody reaction. After reacting a sample containing an antigen with a primary antibody that has been previously bound to a solid phase, a secondary antibody labeled with an enzyme is further reacted, and whether or not the antigen binds is measured using the enzyme activity. This method has high detection sensitivity, and there are several types of methods available depending on the purpose of measurement, and it is widely used in biochemical and biological tests.

[0043] An example of a method for detecting an antigen by ELISA is shown below. (1) A saline solution of the primary antibody is added to the wells of a 96-well plastic plate and allowed to stand to allow the primary antibody to adsorb to the surface of the wells; (2) Excess free primary antibody is washed away, and the plastic surface is then treated with an excess of unrelated proteins (such as BSA or casein) to block the surface and prevent nonspecific binding of various proteins; (3) A test sample containing the antigen to be measured is added and reacted with the primary antibody to form an antigen-antibody complex; (4) After removing contaminants by washing, a secondary antibody labeled with peroxidase (HRP) is added and allowed to react; (5) Excess HRP-labeled secondary antibody is removed by washing; (6) TMB, a chromogenic enzyme substrate, is added; (7) This reacts with the HRP bound to the antibody to form a colored final product; (8) The absorbance at 450 nm, which is the specific absorption wavelength of the final product, is measured; and (8) the concentration of the target antigen is calculated from a calibration curve previously prepared from the absorbance of the calibration substance.

[0044] [Immunochromatography] In one preferred embodiment, the present invention uses a device or kit that utilizes an immunochromatography method (sometimes referred to as an "immunochromatography method"). The kit includes an immunochromatography device, and may further include the above-mentioned first reagent and / or second reagent. The kit may further include a tool for collecting a sample, such as a swab and / or a suction catheter. The immunochromatography device has a determination section for determining the presence or absence of bacteria, and an antigen, such as an antibody against ribosomal protein L7 / L12, is immobilized on the determination section. The kit or immunochromatography device may utilize a sandwich assay (sometimes referred to as a "sandwich immunoassay") that uses an antibody that binds to a site different from the immobilized antibody.

[0045] A schematic cross-sectional view of the immunochromatography device is shown in Figure 1. Reference numeral 1 denotes the substrate, 2 denotes a labeled antibody-impregnated member, 3 denotes a membrane carrier for chromatographic development, 4 denotes an absorption member, and 5 denotes a member for adding a sample. Reference numeral 6 denotes a determination portion or a capture portion where an antibody that reacts with an antigen contained in bacteria is fixed.

[0046] The labeled antibody-impregnated member preferably holds a labeled antibody against the antigen to be detected, which binds to a site different from that of the immobilized antibody, or an antigen. When a labeled antibody that binds to a site different from that of the immobilized antibody is held here, the antigen can be detected by a sandwich assay method. When a labeled antigen is held here, a specific substance can be detected by a competitive method. In the present invention, since the sandwich assay method, which has high detection sensitivity and produces a positive antibody detection line, is preferred, it is preferable that the labeled antibody that binds to a site different from that of the immobilized antibody is held here.

[0047] When a labeled antibody that binds to a site different from that of the immobilized antibody is retained, two types of antibodies are used for each antigen: an antibody that is immobilized on the test zone, and a labeled antibody that binds to a site different from that of the immobilized antibody. In order to enable detection of the antigen by a sandwich assay method, it is preferable that the two types of antibodies are antibodies that can simultaneously bind to one antigen, and that the epitope of one antibody is different from the epitope of the antigen recognized by the other antibody.

[0048] Examples of the label include colored particles, enzymes, radioisotopes, etc., but it is preferable to use colored particles that can be detected visually without special equipment. Examples of colored particles include, but are not limited to, fine metal particles such as gold and platinum (also called gold colloid particles or platinum colloid particles), non-metal particles, latex particles, etc. The colored particles may be of any size as long as they can be transported downstream through the voids in the test piece, but a diameter of 1 nm to 10 μm is preferred. More preferably, the diameter is 5 nm to 1 μm, and even more preferably, the diameter is 10 nm to 100 nm.

[0049] The immunochromatography device can be produced by a known method using commercially available materials. As the substrate of the device (1 in the example of FIG. 1), synthetic polymers such as polyethylene, polypropylene, polystyrene, acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, polyurethane, etc., natural polymers themselves such as agarose, cellulose, nitrocellulose, cellulose acetate, chitin, chitosan, alginate, etc., as well as natural polymer derivatives such as crosslinked structures or modified structures thereof, inorganic materials such as hydroxyapatite, glass, alumina, titania, etc., and metals such as stainless steel, titanium, and aluminum can be used in terms of ease of availability, stability, safety, moldability, and sterilization. Among these, synthetic polymers and natural polymer derivatives are preferable. The substrate can be in the form of a flat plate, mesh, woven fabric, nonwoven fabric, sponge-like structure, three-dimensional molded body (block-like), etc.

[0050] The material used for the labeled antibody-impregnated member (2 in the example of Figure 1) is not particularly limited as long as it is capable of performing immunochromatography, but is preferably a fiber matrix such as a cellulose derivative, filter paper, glass fiber, cloth, cotton, etc.

[0051] The material used for the chromatographic development membrane carrier (3 in the example of Figure 1) including the determination part (6 in the example of Figure 1) is not particularly limited as long as it is capable of performing immunochromatography, but is preferably nitrocellulose, mixed nitrocellulose ester, polyvinylidene fluoride, nylon, etc.

[0052] In the present invention, in immobilizing an antibody on the determination part of a membrane carrier for chromatographic development, the antibody molecule may be directly bound to the membrane carrier surface or may be bound via an active group. Any binding state may be used as long as the antibody molecule or active group is immobilized on the membrane carrier surface from the membrane carrier. Examples of the binding state include a single covalent bond, an ionic bond, a van der Waals bond, a hydrogen bond, or a hydrophobic bond, or a combination of a plurality of these bonds. In particular, a physical adsorption method by simple contact between an antibody solution and the membrane carrier surface is simple and particularly suitable for use in the present invention. In addition, a method of immobilizing an antibody on the membrane carrier surface by washing or drying the membrane carrier surface after antibody adsorption, or by applying an antibody solution to the membrane carrier surface and then evaporating the water, can also be used extremely preferably in the present invention.

[0053] [Method of producing antibodies] The antibody used in the present invention may be either a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody.

[0054] The antibody against ribosomal protein L7 / L12 can be prepared by the method described in WO 00 / 06603. The antibody can be prepared by using the full-length protein of ribosomal protein L7 / L12 or a partial peptide thereof as an antigen, but it is preferable to prepare the antibody using the full-length protein as an antigen. The partial peptide or the full-length protein is inoculated into an animal as is, or crosslinked with a carrier protein, and then optionally with an adjuvant, and the serum is collected to obtain an antiserum containing an antibody (polyclonal antibody) that recognizes ribosomal protein L7 / L12. The antibody can also be purified from the antiserum and used. The animal to be inoculated is sheep, horse, goat, rabbit, mouse, rat, etc., and sheep and rabbit are particularly preferable for producing polyclonal antibodies. In addition, it is more preferable to apply a monoclonal antibody obtained by a known method for producing hybridoma cells, and in this case, a mouse is preferable. By screening for a monoclonal antibody that reacts with the ribosomal protein L7 / L12 of a specific bacterium and does not react with the ribosomal protein L7 / L12 of a bacterium belonging to a different species or genus from the specific bacterium, it is possible to diagnose whether or not the patient is infected with an infectious disease caused by the bacterium. The antibody can be similarly produced when an intracellular antigen other than the ribosomal protein L7 / L12 antigen is used as an antigen. Note that, in the following, the present invention may be described by taking an example of detecting the ribosomal protein L7 / L12 antigen, but those skilled in the art can understand the description by appropriately applying it to the case where an intracellular antigen other than the ribosomal protein L7 / L12 is used as an antigen.

[0055] (3) Advantages of the present invention By using the present invention, intracellular antigens can be instantly and efficiently extracted from Mycoplasma pneumoniae, and detected simply, quickly, and with high sensitivity. Specifically, a throat swab is collected from a patient exhibiting symptoms of respiratory infection, the swab with the specimen attached thereto is immersed in a tube containing a first reagent containing an ionic surfactant, and then a second reagent containing a nonionic surfactant is added to prepare a test sample, and the presence or absence of Mycoplasma pneumoniae can be tested.

[0056] Examples of the present invention will be described in detail below, but the present invention is not limited thereto in any way. EXAMPLES

[0057] Example 1: Preparation of bacterial cells Mycoplasma pneumoniae FH strain (ATCC15531) or M129 strain (ATCC29342) was inoculated into PPLO broth (containing horse serum, fresh yeast extract, glucose, thallium acetate, and penicillin G) and aerobically cultured at 37°C to be used as the test bacterial solution.

[0058] Example 2: Preparation of monoclonal antibodies against ribosomal protein L7 / L12 of Mycoplasma pneumoniae Using the method described in Patent No. 6150559, two clones of hybridomas, MPRB-A and MPRB-B, which showed a positive reaction with Ribosomal Protein L7 / L12 of Mycoplasma Pneumoniae, were obtained, and monoclonal antibodies AMMP-A and AMMP-B were produced and recovered according to standard methods.

[0059] The AMMP-A antibody and the AMMP-B antibody are independent antibodies that recognize different antibody sites in Mycoplasma Pneumoniae Ribosomal Protein L7 / L12, and constitute a combination of antibodies that can be detected in the presence of Mycoplasma Pneumoniae Ribosomal Protein L7 / L12 by sandwich ELISA using the antigen.

[0060] Example 3: Preparation of an immunochromatography device (1) Labeled antibody-impregnated member A gold colloid dispersion (60 nm, manufactured by BBI) was mixed with a phosphate buffer (pH 6.2), and AMMP-B adjusted to 100 μg / mL was added and stirred. Then, 100 μg / mL of monoclonal antibody AMMP-B to be labeled with gold colloid was added and left to stand at room temperature for 30 minutes to bind the antibody to the surface of the gold colloid particles. Then, a 10% aqueous solution of bovine serum albumin (BSA) (manufactured by Merck) was added so that the final concentration in the gold colloid solution was 1%, and the remaining surface of the gold colloid particles was blocked with BSA to prepare a solution of monoclonal antibody AMMP-B labeled with gold colloid (hereinafter referred to as "gold colloid-labeled antibody"). This solution was centrifuged (14,000 to 15,000 g, 10 to 60 minutes) to precipitate the gold colloid-labeled antibody, and the supernatant was removed to obtain the gold colloid-labeled antibody. The colloidal gold-labeled antibody was suspended in 20 mM Tris-HCl buffer (pH 8.2) containing 0.25% BSA, 2.5% sucrose, and 35 mM NaCl to obtain a colloidal gold-labeled antibody solution. A 17 mm x 300 mm strip of glass fiber pad was impregnated with 2 mL of the colloidal gold-labeled antibody solution and dried at room temperature to obtain a labeled antibody-impregnated member.

[0061] (2) Chromatographic development membrane carrier A 25mm x 300mm nitrocellulose membrane (Sartorius, product name: UniSart CN140) was prepared as a chromatographic medium for the membrane carrier for chromatographic development. The monoclonal antibody AMMP-A for capturing the complex of gold colloid-labeled antibody and antigen was diluted with 3% trehalose (Fujifilm Wako Pure Chemical Industries, Ltd.) and 0.1M sodium phosphate buffer (pH 8.0) to prepare a concentration of 1.5 mg / mL. The prepared antibody solution was applied in a line shape at 1μL / cm to a position 10mm from the end of the chromatographic development starting point side of the membrane carrier for chromatographic development, and this was dried at 50℃ overnight. After drying, the membrane was washed with Tris buffer (pH 8.0) containing 0.5 (w / v)% sucrose (Fujifilm Wako Pure Chemical Industries, Ltd.) and 0.05 (w / v)% sodium cholate (Fujifilm Wako Pure Chemical Industries, Ltd.), and then dried at 38°C for 3 hours to prepare an immobilized membrane.

[0062] (3) Preparation of immunochromatography device The above-prepared labeled antibody-impregnated member, the above-prepared chromatographic development membrane carrier, a sample addition member (manufactured by Asahi Kasei Corporation, product name: NE107) used for the portion where the sample is added, and an absorption member for absorbing the developed sample and excess gold colloid-labeled antibody were attached to a base material made of a backing sheet (manufactured by Adhesives Research Inc.) with an adhesive layer. The piece was then cut to a width of 4 mm using a cutting machine to prepare an immunochromatography device.

[0063] Example 4: Effect of ionic surfactants on detection of Mycoplasma pneumoniae by immunochromatography To explore the optimal surfactant for the immunochromatographic detection of Mycoplasma pneumoniae ribosomal protein L7 / L12, we compared the performance of ionic surfactants and, as a control, the effect of using a nonionic surfactant as the first reagent.

[0064] [Preparation of the first reagent] The following reagent components were dissolved in purified water to the concentrations shown below to prepare a first reagent. 0.03 (w / v)% of each ionic or nonionic surfactant shown in Table 1

[0065] [Preparation of the second reagent] A second reagent was prepared to have the following composition: 0.15M phosphate buffer (pH 7.5) 2(w / v)% BSA (Merck) 0.05 (w / v)% sodium azide (Nacalai Tesque) 3(w / v)% polyoxyethylene (20) sorbitan monolaurate (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0066] [Preparation of positive sample solution] The Mycoplasma Pneumoniae test bacteria liquid (M129 strain) prepared in Example 1 was diluted 30-fold with physiological saline (Otsuka Pharmaceutical Factory, Ltd.) to prepare a positive specimen liquid.

[0067] [Preparation of measurement samples] The positive sample liquid was treated in any one of steps 1 to 4 below to prepare a measurement sample.

[0068] <<Step 1: Step of treating a specimen with a first reagent and then adding a second reagent>> 20 μL of the positive specimen liquid was added to 300 μL of the first reagent and mixed, and then 150 μL of the second reagent was further added and mixed to prepare the measurement sample for step 1.

[0069] <<Step 2: Treating the specimen with a mixed solution of a first reagent and a second reagent>> A mixed solution was prepared by mixing 300 μL of the first reagent and 150 μL of the second reagent, and 20 μL of the positive specimen solution was added to the mixed solution and mixed to prepare the measurement sample for step 2.

[0070] <Step 3: Treating the specimen with only the first reagent> 20 μL of the positive specimen solution was added to 450 μL of the first reagent and mixed to prepare a measurement sample for step 3.

[0071] <Step 4: Treating the sample with only the second reagent> 20 μL of the positive specimen solution was added to 450 μL of the second reagent and mixed to prepare the measurement sample for step 4.

[0072] [measurement] 120 μL of each measurement sample was dropped onto the sample application member of the immunochromatography device, and visual observation was performed after 15 minutes.

[0073] [result] The results are shown in Table 1. When no signal was observed on the reading zone, it was rated "-", when a weak signal was observed, it was rated "±", and when a signal was observed, it was rated "+".

[0074] A signal was confirmed in the measurement sample processed in step 1, but no signal was observed in the measurement samples processed in steps 2 and 4, suggesting that it is important to add the second reagent containing a nonionic surfactant after the specimen comes into contact with the first reagent containing an ionic surfactant. In addition, in the measurement sample processed in step 3, the liquid did not spread on the immunochromatography device and measurement was impossible, suggesting that it is important to add the second reagent containing a nonionic surfactant in order to spread on the immunochromatography device and promote the antigen-antibody reaction.

[0075] Furthermore, for the measurement samples treated in step 1, signals could be confirmed on the reading zone with all of the ionic surfactants shown in Table 1. As shown in Table 1, when a nonionic surfactant was used in the first reagent, no signal could be confirmed on the reading zone, or the confirmed signal was weak, suggesting that it is important to use an ionic surfactant in the first reagent.

[0076] [Table 1]

[0077] [Example 5: Concentration of ionic surfactant] We investigated the effect of ionic surfactant concentration on the immunochromatographic detection of ribosomal protein L7 / L12 of Mycoplasma pneumoniae.

[0078] [Preparation of the first reagent] Each ionic surfactant shown in Table 2 was dissolved in purified water to a concentration shown in Table 2 to prepare a first reagent.

[0079] [Preparation of the second reagent] A second reagent was prepared to have the same composition as in Example 4.

[0080] [Preparation of positive sample solution] The Mycoplasma Pneumoniae test bacteria liquid (FH strain) prepared in Example 1 was diluted 100-fold with physiological saline (Otsuka Pharmaceutical Factory, Ltd.) to prepare a positive specimen liquid.

[0081] [Preparation of measurement samples] 20 μL of the positive specimen liquid was added to 300 μL of the first reagent and mixed, and then 150 μL of the second reagent was further added and mixed to prepare a measurement sample.

[0082] [measurement] 120 μL of each measurement sample was dropped onto the sample application member of the immunochromatography device, and visual observation was performed after 15 minutes.

[0083] [result] The results are shown in Table 2. When no signal was observed on the test zone, it was marked -, when a weak signal was observed, it was marked ±, when a signal was observed, it was marked +, and when a stronger signal was observed, it was marked ++. For all the ionic surfactants shown in Table 2, signals could be observed on the test zone in the concentration range of 0.0025 to 0.1 (w / v)%.

[0084] [Table 2]

[0085] Example 6: Study on extraction time using ionic surfactants The extraction time required to extract ribosomal protein L7 / L12 from Mycoplasma pneumoniae after adding an ionic detergent was investigated.

[0086] [Preparation of the first reagent] The following reagent components were dissolved in purified water to the concentrations shown below to prepare a first reagent. 0.03 (w / v)% of each ionic surfactant shown in Table 3

[0087] [Preparation of the second reagent] A second reagent was prepared to have the same composition as in Example 4.

[0088] [Preparation of positive sample solution] The Mycoplasma Pneumoniae test bacteria liquid (FH strain) prepared in Example 1 was diluted 50-fold with physiological saline (Otsuka Pharmaceutical Factory, Ltd.) to prepare a positive specimen liquid.

[0089] [Preparation of measurement samples] The measurement samples were prepared by adding 20 μL of positive specimen liquid to 300 μL of the first reagent and mixing, followed immediately by adding an additional 150 μL of the second reagent and mixing, and by adding 20 μL of positive specimen liquid to 300 μL of the first reagent and leaving it for 1 minute, 2 minutes, 5 minutes, and 15 minutes, after which an additional 150 μL of the second reagent was added and mixed.

[0090] [measurement] 120 μL of each measurement sample was dropped onto the sample application member of the immunochromatography device, and visual observation was performed after 15 minutes.

[0091] [Comparative Example: Study on Extraction Time Using Nonionic Surfactants] A reagent was prepared by adding polyoxyethylene cetyl ether (Sigma-Aldrich, product name: Brij58) to 100 mL of Tris·HCl buffer (pH 7.5) containing 0.1 (w / v) % sodium azide (Nacalai Tesque) to make a concentration of 0.5 (w / v) %, and the test was performed in the same manner as in Example 6, except that 20 μL of the positive sample solution was added to 450 μL of the above reagent and mixed.

[0092] [result] The results are shown in Table 3. As shown in Table 3, it was found that the method of the embodiment does not require an extraction time, and extraction is possible immediately after adding the first reagent containing an ionic surfactant. On the other hand, it was found that the signal observed in the method of the comparative example was weak immediately after mixing the sample and the reagent.

[0093] [Table 3]

[0094] [Example 7: Effect of non-ionic surfactant on detection of Mycoplasma pneumoniae by immunochromatography] To find the optimal surfactant for the immunochromatographic detection of ribosomal protein L7 / L12 of Mycoplasma pneumoniae, we compared the performance of nonionic surfactants.

[0095] [Preparation of the first reagent] The following reagent components were dissolved in purified water to the concentrations shown below to prepare a first reagent. 0.03 (w / v)% of each ionic surfactant shown in Table 4

[0096] [Preparation of the second reagent] A second reagent was prepared to have the following composition: 0.15M phosphate buffer (pH 7.5) 2(w / v)% BSA (Merck) 0.05 (w / v)% sodium azide (Nacalai Tesque) 3 (w / v)% of each nonionic surfactant shown in Table 4

[0097] [Preparation of positive sample solution] The Mycoplasma Pneumoniae test bacteria liquid (FH strain) prepared in Example 1 was diluted 100-fold with physiological saline (Otsuka Pharmaceutical Factory, Ltd.) to prepare a positive specimen liquid.

[0098] [Preparation of measurement samples] 20 μL of the positive specimen liquid was added to 300 μL of the first reagent and mixed, and then 150 μL of the second reagent was further added and mixed to prepare a measurement sample.

[0099] [measurement] 120 μL of each measurement sample was dropped onto the sample application member of the immunochromatography device, and visual observation was performed after 15 minutes.

[0100] [result] The results are shown in Table 4. When no signal was observed on the reading zone, it was marked -, when a weak signal was observed, it was marked ±, when a signal was observed, it was marked +, and when a stronger signal was observed, it was marked ++. Table 4 also shows the HLB values ​​of the nonionic surfactants.

[0101] As shown in Table 4, a signal could be confirmed on the reading zone by using any of the nonionic surfactants having an HLB value of 10.5 to 18.5 as the second reagent.

[0102] [Table 4]

[0103] [Example 8: Concentration of nonionic surfactant] The effect of the concentration of a nonionic surfactant on the immunochromatography device for detecting Ribosomal Protein L7 / L12 of Mycoplasma Pneumoniae was investigated. Measurements were performed in the same manner as in Example 7, except that the ionic surfactant and its concentration in the first reagent and the nonionic surfactant and its concentration in the second reagent were changed as shown in Table 5.

[0104] [result] The results are shown in Table 5. When no signal was observed on the reading zone, it was marked -, when a weak signal was observed, it was marked ±, when a signal was observed, it was marked +, and when a stronger signal was observed, it was marked ++. In addition, when Emulgen 705 (Kao Corporation) was used at 10 (w / v)%, the gold colloid-labeled antibody was retained on the chromatographic development membrane carrier, and the reading zone was observed to have a white spot, so it was deemed impossible to measure.

[0105] As is clear from Table 5, for nonionic surfactants with HLB values ​​in the range of 10.5 to 18.5, signals were observed on the test zone at concentrations in the range of 0.25 to 3 (w / v)%. For polyoxyethylene (20) sorbitan monolaurate, strong signals were observed on the test zone at all concentrations observed.

[0106] [Table 5]

[0107] [Example 9: Method for mixing the first and second reagents] We investigated the mixing method of the first and second reagents in an immunochromatographic device for detecting ribosomal protein L7 / L12 of Mycoplasma pneumoniae.

[0108] [Preparation of the first reagent] The following reagent components were dissolved in purified water to the concentrations shown below to prepare a first reagent. 0.03 (w / v)% of each ionic surfactant shown in Table 6

[0109] [Preparation of the second reagent] A second reagent was prepared to have the following composition: 0.15M phosphate buffer (pH 7.5) 2(w / v)% BSA (Merck) 0.05 (w / v)% sodium azide (Nacalai Tesque) 3(w / v)% polyoxyethylene (20) sorbitan monolaurate (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0110] [Preparation of positive sample solution] The Mycoplasma Pneumoniae test bacteria liquid (FH strain) prepared in Example 1 was diluted 100-fold with physiological saline (Otsuka Pharmaceutical Factory, Ltd.) to prepare a positive specimen liquid.

[0111] [Preparation of measurement samples] The positive sample liquid was treated in one of steps 1 to 3 below to prepare a measurement sample.

[0112] <Step 1: Mixing the second reagent as a solution> 20 μL of the positive specimen liquid was added to 300 μL of the first reagent and mixed, and then 150 μL of the second reagent was further added and mixed to prepare the measurement sample for step 1.

[0113] The immunochromatography device used in the measurement was prepared in the same manner as in Example 3.

[0114] <Step 2: Step of immersing the second reagent in the sample addition member of the immunochromatography device> 20 μL of the positive specimen liquid was added to 300 μL of the first reagent and mixed, and then 150 μL of purified water was further added and mixed to prepare a measurement sample.

[0115] The immunochromatography device used in the measurement was prepared by impregnating a strip-shaped glass fiber pad measuring 17 mm x 150 mm with 1.5 mL of the second reagent and drying it under vacuum at room temperature to prepare a sample addition member containing the second reagent. In step 2, the immunochromatography device was prepared in the same manner as in Example 3, except that the sample addition member containing the second reagent was used.

[0116] <<Step 3: Step of immersing the second reagent in the specimen filtration section of the immunochromatography device>> A PVA sponge filter having a diameter of 7 mm attached to a dropping nozzle was impregnated with 0.15 mL of the second reagent and dried in a vacuum at room temperature to prepare a dropping nozzle containing the second reagent.

[0117] 20 μL of the positive specimen liquid was added to 300 μL of the first reagent and mixed, after which 150 μL of purified water was further added and mixed, and the mixture was passed through a dropping nozzle containing the second reagent to prepare a measurement sample.

[0118] The immunochromatography device used in the measurement was prepared in the same manner as in Example 3.

[0119] [measurement] 120 μL of each measurement sample was dropped onto the sample application member of the immunochromatography device, and visual observation was performed after 15 minutes.

[0120] [result] The results are shown in Table 6. A weak signal was observed on the reading zone, and it was marked ±. A signal was observed, and it was marked +. A stronger signal was observed, and it was marked ++.

[0121] As shown in Table 6, a strong signal was confirmed on the reading zone in all cases of step 1, step 2, and step 3.

[0122] [Table 6] [Industrial Applicability]

[0123] The present invention can be used in the diagnosis of respiratory tract infection caused by Mycoplasma pneumoniae. [Explanation of symbols]

[0124] 1 Base material 2. Labeled antibody-impregnated material 3. Chromatographic development membrane carrier 4 Absorption material 5. Sample Addition Parts 6. Detection or capture site

Claims

1. A method for detecting Mycoplasma pneumoniae contained in a specimen, comprising: (a) contacting the sample with a first reagent that contains an ionic surfactant, does not contain a nonionic surfactant, and does not exhibit alkalinity to obtain an intermediate composition; (b) contacting the intermediate composition with a second reagent containing a nonionic surfactant to obtain a reaction solution; and (c) subjecting the reaction solution to an immunoassay using an antibody against an antigen of Mycoplasma pneumoniae to detect the antigen; A method comprising:

2. The method of claim 1 , wherein the ionic surfactant is a cationic surfactant.

3. The method of claim 2 , wherein the cationic surfactant is a quaternary ammonium salt.

4. 4. The method according to claim 3, wherein the quaternary ammonium salt is any one selected from the group consisting of didecyldimethylammonium adipate, didecyldimethylammonium chloride, didecyldimethylammonium bromide, decyltrimethylammonium chloride, decyltrimethylammonium bromide, dodecyltrimethylammonium chloride, trimethyltetradecylammonium chloride, hexadecyltrimethylammonium chloride, trimethylstearylammonium chloride, and benzalkonium chloride.

5. 5. The method according to claim 3 or 4, wherein the quaternary ammonium salt is didecyldimethylammonium adipate.

6. The method of claim 1 , wherein the ionic surfactant is an anionic surfactant.

7. 7. The method of claim 6, wherein the anionic surfactant is sodium linear alkylbenzene sulfonate or sodium dodecyl sulfate.

8. The method according to claim 1, wherein the nonionic surfactant (b) is any one selected from the group consisting of polyoxyethylene alkyl ethers, polyoxyethylene polyoxypropylene alkyl ethers, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, and polyoxyethylene alkyl phenyl ethers.

9. The method according to claim 1, wherein the nonionic surfactant (b) has an HLB of 10.5 to 18.

5.

10. The method according to claim 1, wherein the nonionic surfactant (b) is a nonionic surfactant having an HLB of 12.5 to 16.

7.

11. The method according to any one of claims 1 to 10, wherein the nonionic surfactant (b) is selected from the group consisting of polyoxyethylene (20) polyoxypropylene (8) cetyl ether, polyoxyethylene (9) alkyl (sec-C11-15) ether, p-tert-octylphenoxypolyethyl alcohol, polyoxyethylene (13) oleyl ether, polyoxyethylene sorbitan monostearate, polyoxyethylene (20) stearyl ether, polyoxyethylene sorbitan monopalmitate, polyoxyethylene (20) cetyl ether, and poly(oxyethylene) sorbitan monolaurate.

12. The method of claim 1, wherein the nonionic surfactant (b) is poly(oxyethylene) sorbitan monolaurate.

13. The method according to any one of claims 1 to 12, wherein the intermediate composition is contacted with the second reagent by passing the intermediate composition through a filter provided in a sample addition member or a dropping nozzle impregnated with the second reagent.

14. 14. The method of any one of claims 1 to 13, wherein the antigen is ribosomal protein L7 / L12.

15. The method of claim 1 , wherein the immunoassay is an immunochromatographic assay.

16. The method of any one of claims 1 to 15, wherein the specimen is a throat swab, a nasal swab, a nasal aspirate, or sputum.

17. A kit for carrying out the method of any one of claims 1 to 16, comprising: A first reagent that contains an ionic surfactant, does not contain a nonionic surfactant, and does not exhibit alkalinity A second reagent comprising a non-ionic surfactant

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