JP2022096708A - Method for detecting mycoplasma pneumoniae - Google Patents

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

The present invention relates to a method for detecting Mycoplasma pneumoniae by immunoassay.

Respiratory tract infections are diseases caused by various causative microorganisms, such as viral respiratory tract infections caused by influenza virus and adenovirus, pneumonia bacillus (Streptococcus pneumoniae), pneumonia mycoplasma (Mycoplasma pneumoniae), and pneumonia chlamydia. It is roughly classified into bacterial respiratory tract infections caused by (Chlamydia pneumoniae), Legionella pneumoniae, and Borderella pneumonia.

Mycoplasma pneumoniae is a typical causative agent of atypical pneumonia, and it occurs especially in children, young people, and adults. Mycoplasma pneumonia is characterized by a long-lasting dry and intense cough, and is considered to be a disease that is difficult to distinguish from a common cold at an early stage. Macrolide antibiotics are effective for infection with mycoplasma pneumoniae, but this drug is less effective for other bacterial pneumonia. Conversely, other antibiotics such as cephem that are effective against bacterial pneumonia have no effect on mycoplasma pneumoniae. Therefore, it is important to diagnose mycoplasma pneumonia at an early stage.

Testing for mycoplasma pneumoniae infections includes culture testing, genetic testing, antibody testing, and antigen testing. In the culture test, the mycoplasma pneumoniae itself grows only in a special medium, and the procedure is difficult and requires skill, so the current situation is that it can only be tested in a limited facility, and it takes about 3 weeks to culture. Not suitable for quick judgment. The Polymerase chain reaction (PCR) method and the Loop-mediated isothermal amplification (LAMP) method are known for genetic testing, but generally it takes several hours from sample collection to determination and requires dedicated equipment. It can only be inspected at a limited facility. Furthermore, in the antibody test, the specific antibody against Mycoplasma pneumoniae is measured, but it is necessary to measure the paired sera collected at intervals to see the transition of the antibody titer, which is not suitable for rapid judgment. Rapid antibody titer measurement kits that do not require paired sera are also commercially available, but they are said to be unreliable due to many false positives.

The antigen test is a test method for determining the presence or absence of a microorganism using a target antigen derived from the microorganism and an antibody that specifically detects the target antigen. For example, an enzyme-linked immunosorbent assay (ELISA). ), Immunofluorescence assay, Immunoturbidity assay, Western blotting method, Immunoprecipitation method, Immunochromatography assay, or Flow cytometry assay. Above all, the antigen test by the immunochromatography method does not require any special equipment or technique for the test, and can make a quick judgment.

Antigen tests for pneumonia mycoplasma by immunochromatography include, for example, intracellular ribosomal proteins L7 / L12, heat shock protein DnaK, and adhesive organ constituent proteins on cell membranes P1 protein and P30 protein as target antigens. It is possible to specifically detect pneumonia mycoplasma. On the other hand, according to Non-Patent Document 1, it is reported that when an intracellular protein, particularly ribosomal protein L7 / L12, is used as a target antigen, the sensitivity to viable bacteria is higher than that of killed bacteria.

In order to detect intracellular antigens such as ribosomal proteins L7 / L12, it is essential not only to have high specificity of the antibody against the antigen to be measured, but also to perform pretreatment to disrupt the cells. be. As a method for disrupting cells to detect intracellular antigens, Patent Document 1 describes lysostaffine and an ionic surfactant against staphylococci present in milk, which is one of the causative bacteria of bovine mastitis. Disclosed is that a bacteriostatic agent containing an activator and a nonionic surfactant is mixed and lysed. Further, Patent Document 2 is a method for detecting a bacterium contained in a sample, in which the sample is brought into contact with an alkaline solution, an intracellular antigen of the bacterium contained in the sample is extracted into the alkaline solution, and a sample extract is obtained. The extraction step of obtaining the sample, the neutralization step of contacting the sample extract with the neutralizing solution to obtain the neutralized product, and the immunoassay method using the antibody against the intracellular antigen of the bacterium are applied to the neutralized product in the cells. Disclosed is a method comprising a detection step of detecting an antigen, wherein the neutralizing solution comprises an effective means for suppressing a false positive reaction in the immunoassay. On the other hand, as a method for extracting an intracellular antigen from Mycoplasma pneumoniae, Patent Document 3 describes a method in which a polyoxyethylene alkyl ether or a polyoxyethylene cetyl ether having an HLB value of 12 to 15 is brought into contact with Mycoplasma pneumoniae to extract the antigen. Is disclosed, and it is disclosed that mycoplasma pneumoniae in a sample sample can be detected with high sensitivity and specifically by this method. However, according to Non-Patent Document 2, the conventional ribosomal protein L7 / L12 antigen test for Mycoplasma pneumoniae requires standing for 2 minutes for antigen extraction, leaving room for improvement in operability. There is.

International Publication WO2015 / 093544 (Patent No. 6314156) JP-A-2017-207333 Japanese Unexamined Patent Publication No. 2014-167439 (Patent No. 6150559)

Medical examination vol69 (1), 2020 30-35 Medical Science Digital vol40 (10), 2014 40-44

An object of the present invention is to provide a sample processing method for rapidly and efficiently extracting an antigen from Mycoplasma pneumoniae contained in a sample, and a test method for detecting the extracted antigen by immunoassay.

In order to solve the above-mentioned problems, the present inventors contact the pneumonia mycoplasma in the sample with the first reagent containing an ionic surfactant, and then to a second reagent containing a non-ionic surfactant. We have found that intracellular antigens can be extracted immediately and efficiently from Mycoplasma pneumoniae in a sample by contact with each other, and can be detected easily, quickly and with high sensitivity, and the present invention has been completed. That is, the present invention provides the following.

[1] A method for detecting Mycoplasma pneumoniae contained in a sample.
(A) A step of contacting the sample with a first reagent containing an ionic surfactant that does not exhibit alkalinity to obtain an intermediate composition.
(B) The step of contacting the intermediate composition with a second reagent containing a nonionic surfactant to obtain a reaction solution, and (c) the reaction solution using an antibody against the antigen of the pneumonia mycoplasma. A method comprising a detection step of subjecting to an immunoassay and detecting the antigen.
[2] A method for detecting Mycoplasma pneumoniae contained in a sample.
(A) A step of contacting the sample with a first reagent containing an ionic surfactant to obtain an intermediate composition.
(B) The step of contacting the intermediate composition with a second reagent containing a nonionic surfactant to obtain a reaction solution, and (c) the reaction solution using an antibody against the antigen of the pneumonia mycoplasma. A method comprising a detection step of subjecting to an immunoassay and detecting the antigen.
[3] The method according to [1] or [2], wherein (a), (b), and (c) are performed 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 quaternary ammonium salt is didecyldimethylammonium adipate, didecyldimethylammonium chloride, didecyldimethylammonium bromide, decyltrimethylammonium chloride, decyltrimethylammonium bromide, dodecyltrimethylammonium chloride, trimethyltetradecylammonium chloride, hexa. The method according to [5], which is selected from the group consisting of decyltrimethylammonium chloride, trimethylstearylammonium chloride, and benzalconium 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 amphoteric tenside is 3-[(3-cholamidepropyl) dimethylammonio] propanesulfonate, 3-[(3-colamidepropyl) dimethylammonio] -2-hydroxypropanesulfonate, and lauryldimethyl. The method according to [10], which is betaine aminoacetate.
[12] The nonionic surfactant is selected from the group consisting of polyoxyethylene alkyl ether, polyoxyethylene polyoxypropylene alkyl ether, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, and polyoxyethylene alkylphenyl ether. The method according to any one of [1] to [11], which is any one of the above.
[13] The method according to any one of [1] to [12], wherein the nonionic surfactant is a nonionic surfactant having an HLB of 10.5 to 18.5.
[14] The method according to any one of [1] to [13], wherein the nonionic surfactant is a nonionic surfactant having an HLB of 12.5 to 16.7.
[15] The nonionic surfactant is polyoxyethylene (20) polyoxypropylene (8) cetyl ether, polyoxyethylene (9) alkyl (sec-C11-15) ether, p-tertiary-octylphenoxy. Polyethyl alcohol, polyoxyethylene (13) oleyl ether, polyoxyethylene sorbitan monostearate, polyoxyethylene (20) stearyl ether, polyoxyethylene sorbitan monopalmitate, polyoxyethylene (20) cetyl ether, and poly ( The method according to any one of [1] to [14], which is selected from the group consisting of (oxyethylene) sorbitan monolaurate.
[16] The method according to any one of [1] to [15], wherein the nonionic surfactant is poly (oxyethylene) sorbitan monolaurate.
[17] The 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 0.10. [18] The method according to any one of [1] to [17], which satisfies any of the following, which is 0.067 (w / v)%.
-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. [19] By permeating the intermediate composition into a filter provided in the sample addition member or the dropping nozzle impregnated with the second reagent, which is 17 to 1.0 (w / v)%. The method according to any one of [1] to [18], wherein the intermediate composition is brought into contact 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 immunoassay is an enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, immunoturbidity assay, western brotting method, immunoprecipitation method, immunochromatography assay, or flow cytometry assay. The method according to any one of [1] to [20].
[22] The method according to any one of [1] to [21], wherein the immunoassay is an immunochromatography assay.
[23] The method according to any one of [1] to [22], wherein the sample is pharyngeal swab, nasal swab, nasal suction, or sputum.
[24] A kit for carrying out the method according to any one of [1] to [23], which comprises the following.
-First reagent containing ionic surfactant-Second reagent containing nonionic surfactant

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, easily, and with high sensitivity.

It is sectional drawing which shows an example of the immunochromatography apparatus. 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 unit on which an antibody against the intracellular antigen of a bacterium is immobilized. The capture site is shown.

Hereinafter, the present invention will be described in more detail.
The present invention is a method for detecting pneumonia mycoplasma contained in a sample, wherein (a) the sample is brought into contact with a first reagent containing an ionic surfactant to obtain an intermediate composition, and (b) the intermediate composition. A step of contacting a substance 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, the antigen. Provided is a detection method including a detection step for detecting.

(1) Extraction of antigen: Steps (a) and (b)
[Bacteria to be detected]
The present invention is used to detect Mycoplasma pneumoniae.

〔antigen〕
In the present invention, an antigen derived from Mycoplasma pneumoniae is detected. Examples of the antigen include various proteins derived from mycoplasma pneumoniae, such as intracellular ribosomal protein L7 / L12, heat shock protein DnaK, and P1 protein and P30 protein which are adhesive constituent proteins on the cell membrane. Preferably, it is ribosomal protein L7 / L12.

In addition, detection means to analyze the presence or absence of the detection target bacterium or the antigen derived from it, or the amount thereof. Therefore, even if the detection target bacterium or antigen is not detected as a result of detection, it corresponds to the implementation of the method of the present invention as in the case where it is detected.

[Sample]
The sample in the present invention is not particularly limited as long as it may contain mycoplasma pneumoniae. It preferably contains blood, urine, etc., in addition to pharyngeal swab, nasal swab, nasal suction, sputum, etc., which can be collected from the upper respiratory tract such as pharynx, nose, nasal cavity, and nasal cavity. More preferably, it is a pharyngeal swab, a nasal swab, a nasal aspirate, or sputum, and a particularly preferred example is a pharyngeal swab.

[First reagent, second reagent]
Extraction of the antigen is performed in two steps. That is, (a) a sample collected with a cotton swab or the like is brought into contact with a first reagent containing an ionic surfactant to obtain an intermediate product, and then (b) a second containing a non-ionic surfactant. After contacting with a reagent to obtain a reaction solution, it is subjected to an immunoassay using an antibody against an antigen of pneumonia mycoplasma to detect the antigen.

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

A surfactant is a substance that acts on an interface to change its properties, and has a hydrophilic group showing hydrophilicity and a hydrophobic group showing lipophilicity in the molecule as a structure. Surfactants that ionize into ions are called ionic surfactants, and non-ionic surfactants are called nonionic surfactants. Ionic surfactants are further classified into cationic surfactants, anionic surfactants, and amphoteric surfactants.

The cationic surfactant that can be used as the first reagent may be any surfactant that, when dissolved in water, ionizes positive (positive) ions at the portion having a hydrophobic group, and is structurally structural. Tertiary ammonium salt type (alkyltrimethylammonium chloride, alkyltrimethylammonium bromide, alkyltrimethylammonium chloride, dialkyldimethylammonium chloride, dialkyldimethylammonium bromide, dialkyldimethylammonium cation, alkylbenzalconium chloride, etc.) and alkyl It is classified into amine salt type (monoalkylamine salt, dialkylamine salt, trialkylamine salt, etc.). Any of them can be suitably used in the present invention. Preferred examples are, in the quaternary ammonium salt form, dodecyldimethylammonium adipate, dodecyldimethylammonium chloride, dodecyldimethylammonium bromide, decyltrimethylammonium chloride, decyltrimethylammonium bromide, dodecyltrimethylammonium chloride, trimethyltetradecylammonium chloride. , Hexadecyltrimethylammonium chloride, trimethylstearylammonium chloride, and benzalconium chloride, more preferably didecyldimethylammonium adipate.

When a cationic surfactant is used as the first reagent, the 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, 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 0.0025 to 0.10 (w). / V)% is more preferable. The concentration in the reaction solution of the cationic surfactant (first reagent + second reagent, the amount of the sample is usually negligible but may be considered in some cases) is the concentration described here. It is a value of 1/2 to 3/4 times, for example, 2/3 times. Specifically, the lower limit can be 0.00067 (w / v)% or more, preferably 0.0017 (w / v)% or more. The upper limit can be 0.33 (w / v)% or less, preferably 0.17 (w / v)% or less, and more preferably 0.067 (w / v)% or less. The range can be, for example, 0.00066 to 0.33 (w / v)%, preferably 0.0017 to 0.17 (w / v)%, and 0.0017 to 0.067 (w). / V)% is more preferable.

The anionic surfactant that can be used as the first reagent may be any structurally active agent that ionizes the portion having a hydrophobic group into negative (negative) ions when dissolved in water. Is a carboxylic acid type (aliphatic monocarboxylate, polyoxyethylene alkyl ether carboxylate, N-acylsarcosin salt, N-acylglutamate, etc.), sulfonic acid type (dialkylsulfosulfate, alkanesulfonate, etc.) Alpha olefin sulfonate, linear alkyl benzene sulfonate, alkyl (branched chain) benzene sulfonate, naphthalene sulfonate-formaldehyde condensate, alkyl naphthalene sulfonate, N-methyl-N-acyl taurine salt, etc.), Sulfonic acid ester type (alkyl sulfate, polyoxyethylene alkyl ether sulfate, oil and fat sulfate ester salt, etc.), phosphate ester type (alkyl phosphate, polyoxyethylene alkyl ether phosphate, polyoxyethylene alkyl phenyl ether phosphate, etc.) It is classified into (salt, etc.). Any of them can be suitably used in the present invention. A preferred example is sodium linear alkylbenzene sulfonate, or sodium dodecyl sulphate.

When an anionic surfactant is used as the first reagent, the 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, 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 0.0025 to 0.10 (w). / V)% is more preferable. The concentration of the anionic surfactant in the reaction solution (first reagent + second reagent, the amount of the sample is usually negligible but may be considered in some cases) is the concentration described here. It is a value of 1/2 to 3/4 times, for example, 2/3 times. Specifically, the lower limit can be 0.00067 (w / v)% or more, preferably 0.0017 (w / v)% or more. The upper limit can be 0.33 (w / v)% or less, preferably 0.17 (w / v)% or less, and more preferably 0.067 (w / v)% or less. The range can be, for example, 0.00066 to 0.33 (w / v)%, preferably 0.0017 to 0.17 (w / v)%, and 0.0017 to 0.067 (w). / V)% is more preferable.

The amphoteric surfactant that can be used as the first reagent is a surfactant that, when dissolved in water, exhibits the properties of an anionic surfactant in the alkaline region and the properties of a cationic surfactant in the acidic region. However, structurally, it may be a carboxybetaine type (alkyl betaine, fatty acid amide propyl betaine, etc.) or a 2-alkyl imidazoline-derived type (2-alkyl-N-carboxymethyl-N-hydroxyethyl imidazolinium betaine, etc.). , Glycine type (alkyldiethylenetriamineacetic acid, dialkyldiethylenetriamineacetic acid, etc.), amine oxide type (alkylamineoxide, etc.). Any of them can be suitably used in the present invention. Preferred examples are 3-[(3-Coleamidepropyl) dimethylammonio] propanesulfonate, 3-[(3-Coleamidepropyl) dimethylammonio] -2-hydroxypropanesulfonate, and betaine lauryldimethylaminoacetate. ..

When an amphoteric tenside agent is used as the first reagent, the concentration in the first reagent is not particularly limited as long as an effective lysis rate for detection is secured, and is, for example, 0.010 to 0.10. (W / v)%. The concentration of the amphoteric tenside agent in the reaction solution (first reagent + second reagent, the amount of the sample is usually negligible but may be considered in some cases) is 1 / of the concentration described here. It is a value of 2 to 3/4 times, for example, 2/3 times. Specifically, as the lower limit, 0.0067 to 0.067 (w / v)% is more preferable.

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

The nonionic surfactant that can be used as the second reagent may be any surfactant having a hydrophilic group that does not ionize when dissolved in water, and is structurally an ester type (glycerin fatty acid ester, sorbitan fatty acid). Esters, sucrose fatty acid esters, 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.), alkanolamide type (fatty acid alkanolamide, etc.). Any of them can be suitably used in the present invention. Preferred examples are polyoxyethylene alkyl ethers, polyoxyethylene polyoxypropylene alkyl ethers, sorbitan fatty acid esters, or polyoxyethylene alkyl phenyl ethers. More specifically, 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-tertiary-octylphenoxypolyethyl alcohol, polyoxyethylene (13) oleyl ether, polyoxyethylene sorbitan monostearate, polyoxyethylene (20) stearyl ether, polyoxy Ethylene sorbitan monopalmitate, polyoxyethylene (20) cetyl ether, or poly (oxyethylene) sorbitan monolaurate. Preferably, polyoxyethylene (20) polyoxypropylene (8) cetyl ether, polyoxyethylene (9) alkyl (sec-C11-15) ether, p-tertiary-octylphenoxypolyethyl alcohol, polyoxyethylene ( 13) Oleyl ether, polyoxyethylene sorbitan monostearate, polyoxyethylene (20) stearyl ether, polyoxyethylene sorbitan monopalmitate, polyoxyethylene (20) cetyl ether, or polyoxyethylene sorbitan monolaurate. More preferably, it is polyoxyethylene sorbitan monolaurate.

Further, when selecting the nonionic surfactant to be used as the second reagent, the hydroxyle-lipophilic interface (HLB) value representing the balance between the hydrophilic group and the hydrophobic group can be used as an index. As a method for calculating an HLB value, a Griffin method, an Atlas method, a Davis method, an upstream method and the like are known. As the nonionic surfactant used in the second reagent of the present invention, the HLB value based on the Griffin method is said to be suitable for preparing an oil-in-water emulsion (8.0). Those up to 18.5 can be used, preferably 10.5 to 18.5, more preferably 12.5 to 16.7. The HLB value based on the Griffin method can be obtained by the sum / molecular weight of the formula of 20 × hydrophilic group.

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 when, for example, an immunochromatographic assay is used as the lower limit. 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 the solubilization with the first reagent containing an 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 0.5 to 3.0 (w / v). )% Is more preferable. The concentration of the nonionic surfactant in the reaction solution (first reagent + second reagent, the amount of the sample is usually negligible but may be considered in some cases) is the concentration described here. It is a value set to 1/4 to 1/2 times, for example, 1/3 times. 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. preferable. The upper limit of the concentration may be such that it does not significantly inhibit the solubilization with the first reagent containing an 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 0.17 to 1.0 (w). / V)% is more preferable.

[About alkali]
The first reagent of the present invention can be configured so as not to exhibit alkalinity. In the present invention, not exhibiting alkalinity means preventing the liquid from becoming alkaline (pH exceeds 11). The liquidity of the first reagent that does not exhibit alkalinity is weakly alkaline (pH is more than 8.0 and 11.0 or less) or neutral (pH is 6.0 to 8.0). ) May be. The non-alkaline nature of the first reagent makes the work safer, does not require the hassle of neutralization, and the material of the container for the first reagent is not limited (the first reagent exhibits alkalinity). If not, the materials are low density polyethylene, high density polyethylene, EVA resin, polypropylene, vinyl chloride resin, polystyrene, expanded polystyrene, AS resin, ABS resin, polyethylene terephthalate, methacrylic resin, polyvinyl alcohol, vinylidene chloride resin, polycarbonate, polyamide, Containers made of acetal resin, polybutylene terephthalate, fluororesin, phenol resin, melamine resin, urea resin, polyurethane, epoxy resin, or unsaturated polyester resin can be used without considering alkali resistance, while the first. If the resin of the above is alkaline, if a container made of a material suitable for alkaline storage is not selected, false positives may occur during long-term storage, which may adversely affect the accuracy of the test.) There are merits such as not having to worry about the stability of the extracted antigen (problems such as denaturation can be avoided).

Both the first reagent and the second reagent may contain substances other than the surfactant as long as they exert the desired action.

[Two-step operation]
The two-step operation uses the first and second reagents separately (rather than mixed) in this order.

Specifically, the two-step operation of the present invention can be carried out as follows. If the sample is a throat swab, immerse the swab with the sample in a tube containing the first reagent containing the ionic surfactant, then add the second reagent containing the nonionic surfactant. Can be done. When an immunochromatography device is used, the second reagent includes a filter provided in the dropping nozzle in advance, a sample addition member (5 in the example of FIG. 1) of the device, and a labeled antibody impregnated member (FIG. 1). In the example, the one soaked in 2) may be prepared, and the first reagent in which the cotton swab to which the sample is attached may be directly dropped onto the sample addition member.

If necessary, the contact between the first reagent and the sample can be performed at an environmental temperature, and if necessary, it may be performed under cooling conditions in which deterioration of components such as proteins is less likely to occur. The contact can be carried out for a time during which the intracellular antigen can be sufficiently extracted, for example, from several seconds to several hours, and the contact may be performed with stirring and shaking as necessary.

Further, according to the study by the present inventors, the first reagent and the second reagent may be used in order, and the interval is not particularly limited. The time between the addition of the first reagent and the addition of the second reagent may be short. That is, it is also an advantage of the method of the present invention that it can be inspected quickly.

Highly efficient lysis and accurate immunoassay can be achieved by the two-step operation. Since ionic surfactants are charged, it can be said that they have high binding properties to proteins that are also charged. Therefore, it makes sense to select an ionic detergent from the point of view of lysis, but in the case of immunoassay, the ionic detergent also has high binding properties to the antibody used for the measurement, resulting in an antigen-antibody reaction. Is more likely to interfere. The present inventors have found that the inhibition of the immune reaction by the ionic surfactant can be suppressed by adding a specific nonionic surfactant at a constant ratio to this problem. On the other hand, the operation of adding the nonionic surfactant at a constant ratio can reduce the lytic action of the ionic surfactant in principle, so if the two types of surfactant are mixed in advance, the lysis is insufficient. Can be. Therefore, it is important to act alone with the ionic surfactant during lysis.

(2) Immunoassay of the 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 the molecule of the antigen and the antibody is desirable. Immunoassays include enzyme immunoassay (EIA), which quantitatively tracks antigen-antibody reactions using enzymes and measures antigens or antibodies, and quantifies antigen-antibody reactions with the help of radioisotopes. There is a radioimmunoassay (RIA) that is followed up to measure an antigen or antibody. The binding reaction between an antigen and an antibody is generally highly specific, binding relatively easily even at low concentrations, and once bound, it is relatively difficult to dissociate. Therefore, if the antigen-antibody reaction is quantified by some method, a trace amount of antigen or antibody can be measured.

The immunoassay in the present invention refers to various immunological measurement methods, such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, immunoturbidity assay, western blotting method, and immunity. Precipitation methods, immunochromatographic assays, flow cytometric assays, and the like can be mentioned, but are not limited thereto. In carrying out the immunological measurement method, a substance for blocking to prevent non-specific adsorption or a substance for preventing cross-reactivity with bacteria other than the target bacterial species may be used.

[ELISA method]
The enzyme-linked immunosorbent assay (ELISA) in the present invention is a method for measuring a substance such as a protein using an antigen-antibody reaction, and contains an antigen in a primary antibody previously bound to a solid phase. This is a method in which a secondary antibody labeled with an enzyme is further reacted after reacting the sample, and whether or not the antibody is bound is measured by utilizing the enzyme activity. This method has high detection sensitivity, and there are several types of methods according to the purpose of measurement, and it is widely used for biochemical and biological tests.

An example of the antigen detection method by ELISA is shown below.
(1) Add the physiological saline solution of the primary antibody to the well of the 96-well plastic plate and let it stand to adsorb the primary antibody on the surface of the well. (2) Wash off the excess free primary antibody and further remove the plastic surface from the irrelevant excess. Treat with an amount of protein (BSA or casein) and block to prevent non-specific binding of various proteins thereafter. (3) Add a subject containing the antigen to be measured and react with the primary antibody to react with the primary antibody to form an antigen-antibody complex. (4) After removing impurities by washing, a secondary antibody labeled with peroxidase (HRP) is added and reacted, and (5) excess HRP-labeled secondary antibody is removed by washing. , (6) TMB, which is a color-developing enzyme substrate, is added, and (7) it reacts with HRP bound to the antibody to form a colored final product, and (8) the specific absorption wavelength of the final product, 450 nm. The absorptivity is measured, and (8) the concentration of the target antibody is calculated from the calibration line prepared in advance from the absorbance of the calibration substance.

[Immune Chromatography]
The present invention uses, in one preferred embodiment, an apparatus or kit utilizing an immunochromatography method (sometimes referred to as an "immunochromatography method"). The kit includes an immunochromatographic device, which may also include the first and / or second reagents described above. The kit may further include tools for collecting specimens, such as cotton swabs and / or suction catheters. The immunochromatographic apparatus has a determination unit for determining the presence or absence of bacteria, and an antibody against an antigen such as ribosomal protein L7 / L12 is immobilized on the determination unit. The kit or immunochromatographic apparatus may utilize a sandwich assay (sometimes referred to as a "sandwich immunoassay") using an antibody that binds to a site different from the immobilized antibody.

FIG. 1 shows a schematic cross-sectional view of the immunochromatography apparatus. 1 is a substrate, 2 is a labeled antibody impregnated member, 3 is a chromatographic development membrane carrier, 4 is an absorption member, and 5 is a sample addition member. Reference numeral 6 is a determination unit or a capture site in which an antibody that reacts with an antigen contained in a bacterium is immobilized.

The labeled antibody-impregnated member preferably holds a labeled antibody or an antigen that binds to a site different from the immobilized antibody, which is an antibody against the antigen to be detected. When a labeled antibody that binds to a site different from the immobilized antibody is retained here, the antigen can be detected by the sandwich assay method. Further, when the antigen labeled here is retained, a specific substance can be detected by a competitive method. In the present invention, a sandwich assay method in which the detection sensitivity is high and a positive antibody detection line appears is preferable. Therefore, a labeled antibody that binds to a site different from the immobilized antibody is retained here. It is preferable that it is.

When a labeled antibody that binds to a site different from the immobilized antibody is retained, the antibody immobilized on the determination unit and the site different from the immobilized antibody bind to each antigen for each antigen. Two types of antibodies, labeled antibodies, are used. The two antibodies are antibodies that can bind to one antigen at the same time so that the antigen can be detected by the sandwich assay method, and the epitope of one antibody is the epitope of the antigen recognized by the other antibody. It is preferable that it is different from.

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

The immunochromatographic apparatus can be produced by a known method using commercially available materials. As the base material of the device (1 in the example of FIG. 1), polyethylene, polypropylene, polystyrene, acrylic resin, nylon, polyester, which are excellent in availability, stability, safety, moldability, and sterility, are used. Synthetic polymers such as polycarbonate, polyacrylamide, and polyurethane, natural polymers such as agarose, cellulose, nitrocellulose, cellulose acetate, chitin, chitosan, and arginate, as well as natural polymers such as crosslinked structures and modified structures. Polymer derivatives, hydroxyapatite, glass, alumina, inorganic materials such as titania, and metals such as stainless steel, titanium, and aluminum can be used. Of these, synthetic polymers and natural polymer derivatives are preferable. Further, as the shape of the base material, a flat plate, a mesh, a woven fabric, a non-woven fabric, a sponge-like structure, a three-dimensional molded body (block shape) or the like can be used.

The material used for the labeled antibody-impregnated member (2 in the example of FIG. 1) is not particularly limited as long as it can perform immunochromatography, but is preferably a fiber matrix such as a cellulose derivative, filter paper, glass fiber, cloth, or cotton. And so on.

The material used for the chromatographic development membrane carrier (3 in the example of FIG. 1) including the determination unit (6 in the example of FIG. 1) is not particularly limited as long as it can perform immunochromatography, but nitrocellulose is preferable. , Mixed nitrocellulose ester, Polyvinylidene fluoride, Nylon, etc.

In the present invention, in the immobilization of the antibody on the determination portion of the membrane carrier for chromatographic development, the antibody molecule may be directly bound to the surface of the membrane carrier or may be bound via an active group. Any bound state may be used as long as the antibody molecule or active group is immobilized on the surface of the membrane carrier from the membrane carrier. Examples of the bond state include a covalent bond, an ionic bond, a van der Waals bond, a hydrogen bond, a hydrophobic bond alone, or a plurality of these resultant forces. In particular, a physical adsorption method by simple contact between the antibody solution and the surface of the membrane carrier is simple and is particularly preferably used in the present invention. Further, a method of washing or drying the surface of the membrane carrier after adsorption of the antibody, or applying an antibody solution to the surface of the membrane carrier and then evaporating water to immobilize the antibody on the surface of the membrane carrier is also extremely preferably used in the present invention. be able to.

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

Antibodies to the ribosomal proteins L7 / L12 can be prepared by the method described in International Publication No. 00/06603. The antibody can be prepared by using the full-length protein of the ribosomal protein L7 / L12 or a partial peptide thereof as an antigen, but it is preferable to prepare the antibody by using the full-length protein as an antigen. Anti-antibodies containing an antibody (polyclonal antibody) that recognizes the ribosome protein L7 / L12 by inoculating the animal with this partial peptide or full-length protein as it is or after cross-linking with the carrier protein as needed with an adjuvant and collecting the serum. Serum can be obtained. It is also possible to purify the antibody from antiserum and use it. The animals to be inoculated include sheep, horses, goats, rabbits, mice, rats and the like, and sheep and rabbits are particularly preferable for producing polyclonal antibodies. Further, as the antibody, it is more preferable to apply a monoclonal antibody obtained by a known method for producing hybridoma cells, but in this case, a mouse is preferable. As the monoclonal antibody, a monoclonal antibody that reacts with the ribosomal protein L7 / L12 of a specific bacterium and does not react with the ribosome protein L7 / L12 of a bacterium belonging to a species different from the specific bacterium or a bacterium belonging to a different genus is screened. This makes it possible to use it for diagnosing whether or not an infectious disease caused by the bacterium is present. When an intracellular antigen other than the ribosomal protein L7 / L12 antigen is used as an antigen, an antibody can be prepared in the same manner. In the following, the present invention may be described by exemplifying an embodiment of detecting a ribosomal protein L7 / L12 antigen, but the description thereof will be described by a person skilled in the art as an antigen of an intracellular antigen other than the ribosomal protein L7 / L12. It can be understood by applying it as appropriate even when it is used as.

(3) Advantages of the present invention By using the present invention, intracellular antigens can be extracted immediately and efficiently from Mycoplasma pneumoniae, and can be detected easily, quickly and with high sensitivity. Specifically, a swab of the pharynx is collected from a patient presenting with respiratory infection symptoms with a cotton swab, and the cotton swab with the sample attached is immersed in a tube containing the first reagent containing an ionic surfactant, and then nonionic. A second reagent containing a surfactant can be added to prepare a sample for inspection, and the presence or absence of pneumonia mycoplasma can be inspected.

Examples of the present invention will be described in detail below, but the present invention is not limited thereto.

[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, tallium acetate, penicillin G) and cultured aerobically at 37 ° C. as a test bacterial solution. Used as.

[Example 2: Preparation of monoclonal antibody against Ribosomal Protein L7 / L12 of Mycoplasma pneumoniae]
Two clones of hybridoma MPRB-A cells and hybridoma MPRB-B cells showing a positive reaction with Mycoplasma Pneumoniae's Ribosomal Protein L7 / L12 were obtained by the method described in Patent No. 6150559, and the monoclonal antibody AMMP-A was obtained according to a conventional method. And AMMP-B were produced and recovered.

The AMMP-A antibody and the AMMP-B antibody are independent antibodies having different antibody recognition sites in Mycoplasma Pneumoniae Ribosomal Protein L7 / L12. A group of antibodies in a combination that can be detected if present.

[Example 3: Preparation of immunochromatography apparatus]
(1) Labeled antibody impregnated member A phosphate buffer (pH 6.2) was mixed with a gold colloidal dispersion (manufactured by BBI: 60 nm), AMMP-B adjusted to 100 μg / mL was added, and the mixture was stirred and then gold. A colloid-labeled monoclonal antibody AMMP-B was added at 100 μg / mL and allowed to stand at room temperature for 30 minutes. An aqueous solution of serum albumin (BSA) (manufactured by Merck) was added, and the surface of the residue of the gold colloid particles was blocked with BSA to label the gold colloid-labeled monoclonal antibody AMMP-B (hereinafter referred to as "gold colloid-labeled antibody"). A solution was prepared. This solution was centrifuged (14,000 to 15,000 g, 10 to 60 minutes) to precipitate a gold colloid-labeled antibody, and the supernatant was removed to obtain a gold colloid-labeled antibody. This gold colloid-labeled antibody was suspended in a 20 mM Tris-hydrochloric acid buffer (pH 8.2) containing 0.25% BSA, 2.5% sucrose, and 35 mM NaCl to obtain a gold colloid-labeled antibody solution. A 17 mm × 300 mm strip-shaped glass fiber pad was impregnated with 2 mL of a gold colloid-labeled antibody solution and dried at room temperature to prepare a labeled antibody-impregnated member.

(2) Chromatographic development membrane carrier A 25 mm × 300 mm nitrocellulose membrane (Sartorius, trade name: UniStart CN140) was prepared as a chromatographic development membrane carrier for a chromatographic medium. Monoclonal antibody AMMP-A for capturing the complex of colloidal gold-labeled antibody and antigen was prepared with 3% trehalose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 0.1 M sodium phosphate buffer (pH 8.0). It was diluted to 1.5 mg / mL. The prepared antibody solution was applied in a line at 1 μL / cm at a position 10 mm from the end on the chromatographic development start point side of this chromatographic development membrane carrier, and this was dried at 50 ° C. overnight. After drying, Tris buffer containing 0.5 (w / v)% sucrose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 0.05 (w / v)% sodium cholic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) The mixture was washed at pH 8.0) and dried at 38 ° C. for 3 hours to prepare an immobilized membrane.

(3) Preparation of Immunochromatography Equipment A portion to which a sample is added in addition to the labeled antibody impregnated member prepared above and the chromatographic development membrane carrier on a substrate made of a backing sheet (manufactured by Adhesives Research Inc.) having an adhesive layer. The sample addition member (manufactured by Asahi Kasei Co., Ltd., trade name: NE107), the developed sample, and the absorption member for absorbing the surplus colloid-labeled antibody were bonded together. Then, it was cut to a width of 4 mm using a cutting machine to prepare an immunochromatography apparatus.

[Example 4: Effect of ionic surfactant on detection of mycoplasma pneumoniae by immunochromatography]
The performance of ionic detergents was compared to find the optimal detergent in Mycoplasma Pneumoniae's Ribosomal Protein L7 / L12 detection immunochromatography apparatus. The effects of using a nonionic surfactant as the first reagent as a control were also compared.

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

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

[Preparation of positive sample solution]
The Mycoplasma Pneumoniae test bacterial solution (M129 strain) prepared in Example 1 was diluted 30-fold with physiological saline (manufactured by Otsuka Pharmaceutical Factory) to prepare a positive sample solution.

[Preparation of measurement sample]
The positive sample solution was treated in any of the following steps 1 to 4 to prepare a measurement sample.

<< Step 1: The process of treating the sample with the first reagent and then adding the second reagent >>
20 μL of the positive sample solution was added to 300 μL of the first reagent and mixed, and then 150 μL of the second reagent was further added and mixed, and the sample was used as the measurement sample in step 1.

<< Step 2: Process of treating the sample with a mixed solution of the first reagent and the 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 sample solution was added to the mixed solution and mixed, which was used as the measurement sample in step 2.

<< Step 3: Process of processing the sample with only the first reagent >>
A mixture of 450 μL of the first reagent and 20 μL of the positive sample solution was used as the measurement sample in step 3.

<< Step 4: Process of processing the sample with only the second reagent >>
A mixture of 450 μL of the second reagent added with 20 μL of the positive sample solution was used as the measurement sample in step 4.

[measurement]
120 μL of each measurement sample was dropped onto the sample addition member of the immunochromatography apparatus, and visual judgment was performed 15 minutes later.

[result]
The results are shown in Table 1. Those for which no signal could be confirmed on the judgment unit were marked as-, those for which a weak signal could be confirmed were marked as ±, and those for which a signal could be confirmed were marked as +.

Although the signal could be confirmed in the measurement sample treated in step 1, no signal was observed in the measurement sample treated in steps 2 and 4, so that the first sample containing the sample and the ionic surfactant was contained. It was speculated that it was important to add a second reagent containing the nonionic surfactant after the reagents were in contact. In addition, since the liquid did not develop on the immunochromatography device and measurement was not possible with the measurement sample treated in step 3, in order to develop it on the immunochromatography device and proceed with the antigen-antibody reaction, it is non-ionic. It was speculated that it was important to add a second reagent containing a sex surfactant.

In addition, for the measurement sample treated in step 1, a signal could be confirmed on the determination unit with all the ionic surfactants shown in Table 1. As shown in Table 1, when a nonionic surfactant was used as the first reagent, no signal could be confirmed on the determination unit, or the confirmed signal was weak, so that the first reagent was weak. It was speculated that it is important to use an ionic surfactant.

[Example 5: Concentration of ionic surfactant used]
The effect of the concentration of ionic detergent on the Ribosomal Protein L7 / L12 detection immunochromatography device of Mycoplasma Pneumoniae was investigated.

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

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

[Preparation of positive sample solution]
The Mycoplasma Pneumoniae test bacterial solution (FH strain) prepared in Example 1 was diluted 100-fold with physiological saline (manufactured by Otsuka Pharmaceutical Factory) to prepare a positive sample solution.

[Preparation of measurement sample]
20 μL of the positive sample solution was added to 300 μL of the first reagent and mixed, and then 150 μL of the second reagent was further added and mixed, and the sample was used as a measurement sample.

[measurement]
120 μL of each measurement sample was dropped onto the sample addition member of the immunochromatography apparatus, and visual judgment was performed 15 minutes later.

[result]
The results are shown in Table 2. Those for which no signal could be confirmed on the judgment unit were marked as-, those for which a weak signal could be confirmed were marked as ±, those for which a signal could be confirmed were marked with +, and those with a stronger signal were marked with ++. For all the ionic surfactants shown in Table 2, a signal could be confirmed on the determination unit in the concentration range of 0.0025 to 0.1 (w / v)%.

[Example 6: Examination of extraction time with ionic surfactant]
After the addition of the ionic detergent, the extraction time required to extract the Ribosomal Protein L7 / L12 of Mycoplasma Pneumoniae was examined.

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

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

[Preparation of positive sample solution]
The Mycoplasma Pneumoniae test bacterial solution (FH strain) prepared in Example 1 was diluted 50-fold with physiological saline (manufactured by Otsuka Pharmaceutical Factory) to prepare a positive sample solution.

[Preparation of measurement sample]
20 μL of positive sample solution was added to 300 μL of the first reagent and mixed, and then 150 μL of the second reagent was immediately added and mixed, and 20 μL of positive sample solution was added to 300 μL of the first reagent and mixed for 1 minute and 2 minutes. After allowing to stand for 5 minutes and 15 minutes, 150 μL of the second reagent was further added and mixed, and each was used as a measurement sample.

[measurement]
120 μL of each measurement sample was dropped onto the sample addition member of the immunochromatography apparatus, and visual judgment was performed 15 minutes later.

[Comparative example: Examination of extraction time with nonionic surfactant]
Polyoxyethylene cetyl to be 0.5 (w / v)% in 100 mL of Tris / HCl buffer (pH 7.5) containing 0.1 (w / v)% sodium azide (manufactured by Nacalai Tesque). A reagent containing ether (manufactured by Sigma-Aldrich, trade name: Brij58) was prepared, and the test was carried out 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.

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

[Example 7: Effect of nonionic surfactant on detection of Mycoplasma pneumoniae by immunochromatography]
The performance of nonionic surfactants was compared to find the optimal surfactants in Mycoplasma Pneumoniae's Ribosomal Protein L7 / L12 detection immunochromatography apparatus.

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

[Preparation of second reagent]
A second reagent was prepared to have the following composition.
0.15M phosphate buffer (pH 7.5)
2 (w / v)% BSA (manufactured by Merck & Co., Ltd.)
0.05 (w / v)% sodium azide (manufactured by Nacalai Tesque)
3 (w / v)% Each nonionic surfactant shown in Table 4

[Preparation of positive sample solution]
The Mycoplasma Pneumoniae test bacterial solution (FH strain) prepared in Example 1 was diluted 100-fold with physiological saline (manufactured by Otsuka Pharmaceutical Factory) to prepare a positive sample solution.

[Preparation of measurement sample]
20 μL of the positive sample solution was added to 300 μL of the first reagent and mixed, and then 150 μL of the second reagent was further added and mixed, and the sample was used as a measurement sample.

[measurement]
120 μL of each measurement sample was dropped onto the sample addition member of the immunochromatography apparatus, and visual judgment was performed 15 minutes later.

[result]
The results are shown in Table 4. The signal that could not be confirmed on the judgment unit was defined as-, the signal that could be confirmed as a weak signal was defined as ±, the signal that could be confirmed as +, and the signal that could be confirmed as a stronger signal was defined as ++. Table 4 also shows the HLB values of nonionic surfactants.

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

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

[result]
The results are shown in Table 5. The signal that could not be confirmed on the judgment unit was defined as-, the signal that could be confirmed as a weak signal was defined as ±, the signal that could be confirmed as +, and the signal that could be confirmed as a stronger signal was defined as ++. Further, when Emargen 705 (manufactured by Kao Corporation) was used at 10 (w / v)%, a phenomenon was observed in which the gold colloid-labeled antibody stayed on the membrane carrier for chromatographic development and the determination part was so-called white-out. It was made unmeasurable.

As is clear from Table 5, for nonionic surfactants having an HLB value in the range of 10.5 to 18.5, a signal is signaled on the determination unit in the concentration range of 0.25 to 3 (w / v)%. I was able to confirm. Regarding polyoxyethylene (20) sorbitan monolaurate, a strong signal could be confirmed on the determination unit at any of the confirmed concentrations.

[Example 9: Mixing method of the first reagent and the second reagent]
The method of mixing the first reagent and the second reagent in the Ribosomal Protein L7 / L12 detection immunochromatography apparatus of Mycoplasma Pneumoniae was investigated.

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

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

[Preparation of positive sample solution]
The Mycoplasma Pneumoniae test bacterial solution (FH strain) prepared in Example 1 was diluted 100-fold with physiological saline (manufactured by Otsuka Pharmaceutical Factory) to prepare a positive sample solution.

[Preparation of measurement sample]
The positive sample solution was treated in any of the following steps 1 to 3 to prepare a measurement sample.

≪Step 1: Step of mixing the second reagent as a solution≫
20 μL of the positive sample solution was added to 300 μL of the first reagent and mixed, and then 150 μL of the second reagent was further added and mixed, and the sample was used as the measurement sample in step 1.

As the immunochromatographic apparatus used for the measurement, one prepared by the same method as in Example 3 was used.

<< Step 2: Immersing the second reagent in the sample addition member of the immunochromatography device >>
20 μL of the positive sample solution was added to 300 μL of the first reagent and mixed, and then 150 μL of purified water was further added and mixed, and the sample was used as a measurement sample.

The immunochromatography device used for the measurement is for adding a sample containing the second reagent by impregnating a 17 mm × 150 mm strip-shaped glass fiber pad with 1.5 mL of the second reagent and vacuum-drying the second reagent at room temperature. A member was manufactured. In step 2, an immunochromatography apparatus was produced by the same method as in Example 3 except that the sample addition member containing the second reagent was used.

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

20 μL of the positive sample solution was added to 300 μL of the first reagent and mixed, and then 150 μL of purified water was further added and mixed, and the sample was passed through a dropping nozzle containing the second reagent and used as a measurement sample.

As the immunochromatographic apparatus used for the measurement, one prepared by the same method as in Example 3 was used.

[measurement]
120 μL of each measurement sample was dropped onto the sample addition member of the immunochromatography apparatus, and visual judgment was performed 15 minutes later.

[result]
The results are shown in Table 6. A weak signal was confirmed on the judgment unit as ±, a signal was confirmed as +, and a stronger signal was confirmed as ++.

As shown in Table 6, in any of the cases of step 1, step 2, and step 3, a strong signal could be confirmed on the determination unit.

The present invention can be used in the diagnosis of respiratory infections caused by Mycoplasma pneumoniae.

1 Base material 2 Labeled antibody impregnated member 3 Chromatographic development membrane carrier 4 Absorption member 5 Sample addition member 6 Judgment part or capture part

Claims (18)

A method for detecting Mycoplasma Pneumoniae contained in a sample.
(A) A step of contacting the sample with a first reagent containing an ionic surfactant that does not exhibit alkalinity to obtain an intermediate composition.
(B) The step of contacting the intermediate composition with a second reagent containing a nonionic surfactant to obtain a reaction solution, and (c) immunization of the reaction solution with an antibody against the antigen of mycoplasma pneumoniae. A method comprising a detection step of subjecting to a measurement method and detecting the antigen. A method for detecting Mycoplasma Pneumoniae contained in a sample.
(A) A step of contacting the sample with a first reagent containing an ionic surfactant to obtain an intermediate composition.
(B) The step of contacting the intermediate composition with a second reagent containing a nonionic surfactant to obtain a reaction solution, and (c) immunization of the reaction solution with an antibody against the antigen of mycoplasma pneumoniae. A method comprising a detection step of subjecting to a measurement method and detecting the antigen. The method according to claim 1 or 2, wherein the ionic surfactant is a cationic surfactant. The method of claim 3, wherein the cationic surfactant is a quaternary ammonium salt. The quaternary ammonium salt is didecyldimethylammonium adipate, didecyldimethylammonium chloride, didecyldimethylammonium bromide, decyltrimethylammonium chloride, decyltrimethylammonium bromide, dodecyltrimethylammonium chloride, trimethyltetradecylammonium chloride, hexadecyltrimethylammonium. The method according to claim 4, wherein the method is selected from the group consisting of chloride, trimethylstearyl ammonium chloride, and benzalconium chloride. The method of claim 4 or 5, wherein the quaternary ammonium salt is didecyldimethylammonium adipate. The method according to claim 1 or 2, wherein the ionic surfactant is an anionic surfactant. The method of claim 7, wherein the anionic surfactant is sodium linear alkylbenzene sulfonate or sodium dodecyl sulfate. The nonionic surfactant is 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. The method according to any one of claims 1 to 8. The method according to any one of claims 1 to 9, wherein the nonionic surfactant is a nonionic surfactant having an HLB of 10.5-18.5. The method according to any one of claims 1 to 10, wherein the nonionic surfactant is a nonionic surfactant having an HLB of 12.5 to 16.7. Nonionic surfactants are polyoxyethylene (20) polyoxypropylene (8) cetyl ether, polyoxyethylene (9) alkyl (sec-C11-15) ether, p-tertiary-octylphenoxypolyethyl alcohol. , Polyoxyethylene (13) oleyl ether, polyoxyethylene sorbitan monostearate, polyoxyethylene (20) stearyl ether, polyoxyethylene sorbitan monopalmitate, polyoxyethylene (20) cetyl ether, and poly (oxyethylene). The method according to any one of claims 1 to 11, which is any one selected from the group consisting of sorbitan monolaurates. The method according to any one of claims 1 to 12, wherein the nonionic surfactant is poly (oxyethylene) sorbitan monolaurate. 1 The method according to any one of 13 to 13. The method according to any one of claims 1 to 14, wherein the antigen is ribosomal protein L7 / L12. The method according to any one of claims 1 to 15, wherein the immunoassay is an immunochromatography assay. The method according to any one of claims 1 to 16, wherein the sample is a pharyngeal swab, a nasal swab, a nasal aspirate, or sputum. A kit for carrying out the method according to any one of claims 1 to 17, including the following.
-First reagent containing ionic surfactant-Second reagent containing nonionic surfactant

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