JP7507017B2 - Methods for detecting respiratory infection causative bacteria - Google Patents

Description

The present invention relates to a method for extracting antigens from bacteria that cause respiratory infections contained in a sample and detecting the antigens by immunoassay.

Respiratory infections are diseases caused by various microorganisms and are 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 parapertussis, Bordetella holmesii, etc., which include Bordetella pertussis.

Whooping cough is a bacterial respiratory infection that has been attracting attention in recent years. Whooping cough is a bacterial respiratory infection caused mainly by Bordetella pertussis, whose main symptom is persistent coughing. In infants and young children, typical symptoms such as coughing, paroxysmal coughing, inspiratory whooping, and vomiting after coughing are often present. On the other hand, the clinical picture of adolescent and adult patients is atypical, and they often only have persistent coughing. Bordetella pertussis is transmitted by droplets of the patient's upper respiratory tract secretions or by direct contact, and is transmitted via the respiratory tract. Because it is highly contagious, mass infections have been reported in schools and workplaces. Infection of unvaccinated infants and young children can lead to serious symptoms such as respiratory failure and death. In addition, pertussis is not only caused by Bordetella pertussis, but also by Bordetella parapertussis and Bordetella holmesii, and the symptoms are caused by pertussis toxin (PT), an exotoxin specific to Bordetella pertussis that has ADP-ribosyltransferase activity, and lipopolysaccharide (LPS), an endotoxin present in the outer membrane of the cell wall.

In order to diagnose respiratory infections, it is very important to detect and identify the pathogens in order to provide prompt treatment to patients. Methods for detecting and identifying pathogens include (1) culture and identification tests from pharyngeal swabs of patients, (2) antibody tests using patient serum, (3) genetic tests, and (4) antigen tests. Of these, culture and identification tests are considered essential as the gold standard for diagnosing respiratory infections. However, they are inferior in terms of speed because they take several days to obtain results. Antibody tests are used to diagnose infections based on serum antibody titers, but they basically require measuring paired serum samples from the acute and convalescent phases to observe the progress of antibody titers, and because it takes time for antibody titers to rise after infection, they require several days from infection to diagnosis. Therefore, a diagnostic method that can obtain results quickly and easily is desired. Genetic tests, such as polymerase chain reaction (PCR) tests and loop-mediated isothermal amplification (LAMP) methods, have been put to practical use and are highly regarded for their sensitivity and speed. However, there are still issues with this method, such as the need to extract and purify genes from samples and the need for expensive equipment, meaning it can only be tested at a limited number of facilities.

Well-known antigen tests are methods based on antigen-antibody reactions using antibodies that react specifically with the antigen to be measured (immunoassays), such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, immunoturbidity assay, Western blotting, immunoprecipitation, immunochromatography assay, and flow cytometry assay. In antigen tests, it is important not only that the antibodies have high specificity for the antigen to be measured, but also that the antigen can be extracted efficiently and easily from the target pathogen in the sample. A surfactant may be used to extract the antigen.

For example, Patent Documents 1 and 2 disclose a method for measuring a virus or hepatitis C virus (HCV), which is characterized by measuring a viral antigen by binding to a probe in the presence of a surfactant having an alkyl group with 10 or more carbon atoms and a secondary, tertiary or quaternary amine, or a nonionic surfactant, or both. Patent Document 3 discloses a medium composition for preparing a sample suspension to be used in an immunoassay, which contains an ionic surfactant.

On the other hand, as a measurement target for bacteria, Patent Document 4 discloses that Staphylococcus aureus, which is one of the causative bacteria of bovine mastitis and is present in milk, is lysed by mixing a lysing agent containing lysostaphin, an ionic surfactant, and a nonionic surfactant. Patent Document 5 discloses that Chlamydia major outer membrane protein antigens are extracted by contacting live Chlamydia bacteria with an extraction composition containing a cationic surfactant having a pH of at least 8 and present at at least 1 mg/ml. Patent Document 6 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 the 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 an intracellular antigen of the bacteria to detect the intracellular antigen, and the neutralizing solution includes a means for suppressing false positive reactions in the immunoassay.

JP 2001-215228 A (Patent No. 3623162) International Publication WO2000/007023 (Patent No. 3514729) JP 2005-291783 A International Publication WO2015/093544 (Patent No. 6314156) JP2-211893A (Patent No. 1988072) JP 2017-207333 A

The present invention aims to provide a sample processing method for efficiently extracting antigens from bacteria that cause respiratory infections contained in the sample, and a testing method (diagnostic method) for detecting the extracted antigens by immunoassay.

In order to solve the above problems, the present inventors discovered that respiratory infection-causing bacteria in a specimen can be detected easily, quickly, and with high sensitivity by contacting the respiratory infection-causing bacteria in a specimen with a first reagent containing an ionic surfactant and then with a second reagent containing a nonionic surfactant, and thus completed the present invention. That is, the present invention provides the following.

[1] A method for detecting a causative bacterium of a respiratory infection contained in a specimen, comprising:
(a) contacting the sample with a first reagent comprising an ionic surfactant but not an alkali to obtain an intermediate composition;
(b) contacting the intermediate composition with a second reagent containing a nonionic surfactant to obtain a reaction liquid; and (c) subjecting the reaction liquid to an immunoassay using an antibody against an antigen of the causative bacterium to detect the antigen.
[2] The method according to [1], wherein the causative bacterium is any one selected from the group consisting of Bordetella pertussis, Bordetella parapertussis, Bordetella holmesii, and Legionella pneumophila.
[3] A method for detecting any causative bacterium of a respiratory tract infection selected from the group consisting of Bordetella pertussis, Bordetella parapertussis, Bordetella holmesii, and Legionella pneumophila 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 liquid; and (c) subjecting the reaction liquid to an immunoassay using an antibody against an antigen of the causative bacterium to detect the antigen.
[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, dimethyldioctadecylammonium chloride, 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 any one selected from the group consisting of didecyldimethylammonium adipate, didecyldimethylammonium chloride, didecyldimethylammonium bromide, dimethyldioctadecylammonium chloride, trimethyltetradecylammonium chloride, hexadecyltrimethylammonium chloride, trimethylstearylammonium chloride, and benzalkonium chloride.
[8] The method according to any one of [5] to [7], wherein the quaternary ammonium salt is didecyldimethylammonium adipate.
[9] The method according to any one of [1] to [3], wherein the ionic surfactant is an anionic surfactant.
[10] The method according to [9], wherein the anionic surfactant is sodium linear alkylbenzene sulfonate or sodium dodecyl sulfate.
[11] The method according to any one of [1] to [3], wherein the ionic surfactant is an amphoteric surfactant.
[12] The method according to [11], wherein the amphoteric surfactant is 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxypropanesulfonate, or lauryl dimethylaminoacetic acid betaine.
[13] The method according to any one of [1] to [12], wherein the nonionic surfactant is any one selected from the group consisting of polyoxyethylene alkyl ethers, polyoxyethylene polyoxypropylene alkyl ethers, sorbitan fatty acid esters, and polyoxyethylene alkyl phenyl ethers.
[14] The method according to any one of [1] to [13], wherein the nonionic surfactant has an HLB of 10.0 to 18.5.
[15] The method according to any one of [1] to [14], wherein the nonionic surfactant has an HLB of 12.5 to 16.7.
[16] The method according to any one of [1] to [15], 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.
[17] The method according to any one of [1] to [16], wherein the nonionic surfactant is poly(oxyethylene) sorbitan monolaurate.
[18] The method according to any one of [1] to [17], wherein any one of the following is satisfied:
- the concentration of the ionic surfactant in the first reagent is 0.01 to 0.1 (w/v)%; - the concentration of the ionic surfactant in the reaction solution is 0.0067 to 0.067 (w/v)%. [19] The method according to any one of [1] to [19], which satisfies any one of the following:
- the concentration of the nonionic surfactant in the second reagent is 0.5 to 3 (w/v) %; - the concentration of the nonionic surfactant in the reaction solution is 0.167 to 1 (w/v) %. [20] The method according to any one of [1] to [19], 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 in which the second reagent is immersed.
[21] The method according to any one of [1] to [20], wherein the antigen is ribosomal protein L7/L12.
[22] The method described in any one of [1] to [20], wherein the causative bacterium is a Bordetella bacterium and the antigen is any one selected from the group consisting of ribosomal protein L7/L12, lipopolysaccharide, and Bordetella pertussis toxin.
[23] The method according to [22], wherein the causative bacterium is Bordetella pertussis.
[24] The method according to any one of [1] to [23], wherein the immunoassay is an enzyme-linked immunosorbent assay (ELISA), an immunofluorescence assay, an immunoturbidimetric assay, a Western blotting method, an immunoprecipitation method, an immunochromatography assay, or a flow cytometry assay.
[25] The method according to any one of [1] to [24], wherein the immunoassay is an enzyme-linked immunosorbent assay (ELISA).
[26] The method according to any one of [1] to [24], wherein the immunoassay is an immunochromatographic assay.
[27] The method according to any one of [1] to [26], wherein the sample is a nasal swab, a pharyngeal swab, a nasal aspirate, or sputum.
[28] The method according to any one of [1] to [27] for diagnosing or aiding in the diagnosis of a respiratory infection in humans.
[29] A kit for carrying out the method according to any one of [1] to [28], comprising:
a first reagent comprising an ionic surfactant; and a second reagent comprising a non-ionic surfactant.

The present invention provides a method for extracting antigens from respiratory infection-causing bacteria contained in a sample and detecting the antigens by immunoassay quickly, easily, and with high sensitivity.

Detection of Ribosomal Protein L7/L12 (Process B) Detection of Ribosomal Protein L7/L12 (Treatment C and Treatment D) Detection of lipopolysaccharide (LPS) (Process B) Detection of Lipopolysaccharide (LPS) (Treatment C and Treatment D) Detection of Bordetella pertussis toxin (PT) (Process B) Detection of Pertussis Toxin (PT) (Treatment C and Treatment D) Detection of Bordetella pertussis toxin (PT) (Process B) Detection of Pertussis Toxin (PT) (Treatment C and Treatment D) FIG. 1 is a schematic cross-sectional view showing an example of an immunochromatography device, in which 1 denotes a substrate, 2 denotes a labeled antibody-impregnated member, 3 denotes a chromatographic development membrane carrier, 4 denotes an absorption member, 5 denotes a sample addition member, and 6 denotes a determination portion or capture site where an antibody against an intracellular antigen of bacteria is fixed.

The present invention will now be described in further detail.
The present invention provides a method for detecting bacteria that cause respiratory infections 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 bacteria that cause respiratory infections, and detecting the antigen.

(1) Extraction of antigen: steps (a) and (b)
[Detection target bacteria]
The present invention is used to detect bacteria causing respiratory infections. Respiratory infections are broadly classified into viral respiratory infections caused by viruses and bacterial respiratory infections caused by bacteria, but the respiratory infection in the present invention means bacterial respiratory infections. Examples of bacteria causing bacterial respiratory infections include Streptococcus pneumoniae, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, and Bordetella parapertussis, Bordetella holmesii, including Bordetella pertussis.

Bacteria are broadly divided into gram-positive bacteria, which stain purple when stained with gram stain, and gram-negative bacteria, which do not stain purple and appear red. Streptococcus pneumoniae and Mycoplasma pneumoniae, which cause bacterial respiratory infections, are gram-positive bacteria, but are generally relatively less dangerous. This is because the human body does not have peptidoglycan and therefore has the ability to produce enzymes that damage the peptidoglycan layer of gram-positive bacteria. Gram-positive bacteria are also often highly sensitive to beta-lactam antibiotics such as penicillin. However, Mycoplasma pneumoniae does not have a cell wall. On the other hand, the Bordetella genus, including Chlamydia pneumoniae, Legionella pneumophila, and Bordetella pertussis, is a gram-negative bacterium. Many gram-negative bacteria have an outer membrane covered with a capsule or mucus layer, and although there are exceptions, they generally tend to be relatively pathogenic. This structure acts to hide and camouflage the antigens of the bacterial cell. Since the human immune system recognizes foreign substances by antigens, if the antigens are hidden, it becomes difficult for the body to detect the invader. The presence of a capsule often increases the toxicity of pathogenic bacteria. In addition, gram-negative bacteria have endotoxins, which are lipopolysaccharides, in their outer membranes, which aggravate inflammation and, in severe cases, can cause septic shock.

The present invention can be suitably used when the bacteria to be detected are gram-positive or gram-negative bacteria.

The bacteria to be detected that can be preferably applied to the present invention are Bordetella pertussis, Bordetella parapertussis, Bordetella holmesii, and Legionella pneumophila, and more preferably Bordetella pertussis.

〔antigen〕
In the present invention, an antigen derived from a bacterium to be detected is detected. The antigen may be various substances derived from the bacterium to be detected, such as intracellular substances such as ribosomal protein L7/L12, substances produced by the bacterium to be detected (e.g., toxins), or components constituting the outer membrane of the bacterium to be detected (e.g., lipopolysaccharide (LPS)). The antigen is preferably a proteinaceous substance (e.g., ribosomal protein L7/L12, pertussis toxin (PT)) because it is easy to obtain antibodies for detection. From the viewpoint of identifying the causative bacterium, it is more preferable that the antigen is specific to a species or genus. Here, "specific to a species or genus" means that the antigen possessed by the bacterium to be detected has a structural part such as a unique amino acid sequence that is different from the antigen possessed by bacteria belonging to other genuses other than the genus to which the bacterium belongs, or bacteria of species other than the above-mentioned specific species of the genus to which the bacterium belongs. An example of an antigen specific to a species or genus is ribosomal protein L7/L12.

Note that detection refers to analyzing the presence or absence, or the amount, of the target bacteria or antigens derived therefrom. Therefore, even if the target bacteria or antigens are not detected as a result of detection, this also falls under the implementation of the method of the present invention, just as if they were detected.

[Specimen]
The specimen in the present invention may be any specimen that can be collected from any location of the human body and may contain Bordetella pertussis. Preferably, the specimen includes nasal swabs, pharyngeal swabs, nasal aspirates, sputum, etc., which can be collected from the upper respiratory tract such as the nose, nasal cavity, nasopharynx, and pharynx, as well as blood and urine. More preferably, the specimen is a nasal swabs, pharyngeal swabs, nasal aspirates, or sputum, and a particularly preferred example is a nasal swabs.

[First Reagent, Second Reagent]
The antigen is extracted in two steps: (a) a specimen 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 specimen is contacted with a second reagent containing a nonionic surfactant to obtain a reaction liquid, which is then subjected to an immunoassay using an antibody against an antigen of a bacterium causing a respiratory infection to detect the antigen.

[Surfactant]
The first reagent used in 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 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.

The cationic surfactant that can be used in the first reagent is any surfactant that, when dissolved in water, ionizes the hydrophobic group 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 didecyl dimethyl ammonium adipate, didecyl dimethyl ammonium chloride, didecyl dimethyl ammonium bromide, dimethyl dioctadecyl ammonium chloride, decyl trimethyl ammonium chloride, decyl trimethyl ammonium bromide, dodecyl trimethyl ammonium chloride, trimethyl tetradecyl ammonium chloride, hexadecyl trimethyl ammonium chloride, trimethyl stearyl ammonium chloride, or benzalkonium chloride, preferably didecyl dimethyl ammonium adipate, didecyl dimethyl ammonium chloride, didecyl dimethyl ammonium bromide, dimethyl dioctadecyl ammonium chloride, trimethyl tetradecyl ammonium chloride, hexadecyl trimethyl ammonium chloride, trimethyl stearyl ammonium chloride, or benzalkonium chloride, more preferably didecyl dimethyl ammonium adipate.

It is preferable to use a cationic surfactant as the first reagent. According to the inventors' investigations, a cationic surfactant is suitable for efficiently extracting antigens from the bacteria to be detected in the present invention.

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 0.001 (w/v)% or more, preferably 0.0025 (w/v)% or more, and more preferably 0.01 (w/v)% or more. The upper limit can be 0.5 (w/v)% or less, preferably 0.25 (w/v)% or less, and more preferably 0.1 (w/v)% or less. The range can be, for example, 0.001 to 0.5 (w/v)%, preferably 0.0025 to 0.25 (w/v)%, and more preferably 0.01 to 0.1 (w/v)%.

The anionic surfactant that can be used in the first reagent is a surfactant that, when dissolved in water, dissociates the hydrophobic group into a negative ion. Structurally, they are classified into carboxylic acid type (aliphatic monocarboxylate, polyoxyethylene alkyl ether carboxylate, N-acylsarcosine salt, N-acyl glutamate, etc.), sulfonic acid type (dialkyl sulfosuccinate, alkane sulfonate, 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 types can be suitably used in the present invention. Preferred examples are sodium linear alkyl benzene sulfonate or sodium dodecyl sulfate.

It may be preferable to use an anionic surfactant as the first reagent. According to the inventors' investigations, anionic surfactants are suitable for efficiently extracting antigens from the bacteria to be detected in the present invention.

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 bacteriolysis rate for detection is ensured, but is, for example, 0.01 to 0.1 (w/v)%.

The amphoteric surfactant that can be used in the first reagent may be any surfactant that, when dissolved in water, exhibits the properties of a negative surfactant in the alkaline region and the properties of a positive surfactant in the acidic region. Structurally, they are classified into carboxybetaine type (alkylbetaine, fatty acid amidopropyl betaine, 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 types 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.

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, but is, for example, 0.01 to 0.1 (w/v)%.

The second reagent used in step (b) of the present invention contains a nonionic surfactant.
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, etc.), ester ether type (fatty acid polyethylene glycol, fatty acid polyoxyethylene sorbitan, 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 (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. Preferably, it is poly(oxyethylene) sorbitan monolaurate.

When selecting a nonionic surfactant for use 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 for use 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 preparing an oil-in-water (O/W) emulsion, can be used, with those having an HLB value of 10.0 to 18.5 being preferred, and those having an HLB value of 12.5 to 16.7 being more preferred. 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.

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 0.1 (w/v)% or more, preferably 0.25 (w/v)% or more, and more preferably 0.5 (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 (w/v)% or less, and more preferably 3 (w/v)% or less. The range can be, for example, 0.1 to 10 (w/v)%, preferably 0.25 to 5 (w/v)%, more preferably 0.5 to 3 (w/v)%, or more preferably 0.25 to 1.5 (w/v)%.

[About alkali]
The first reagent of the present invention may be configured to be free of alkali. Free of alkali in the present invention refers to a liquid that is not alkaline (pH is not greater than 11). The liquid property of the first reagent that does not contain alkali may be weakly alkaline (pH is greater than 8.0 and not greater than 11.0) or neutral (pH is 6.0 to 8.0). The absence of alkali in the first reagent provides the following advantages: safer operation, no need for the trouble of neutralization, no restrictions on the material of the container for the first reagent (no need for alkali resistance), no need to worry about the stability of the antigen extracted in the first reagent (problems such as denaturation can be avoided).

Both the first and second reagents may contain substances other than surfactants as long as they exert the intended effect.

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

The two-step operation of the present invention can be specifically carried out as follows. When the specimen is a nasal 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. In addition, when an immunochromatography device is used, the second reagent can be prepared by pre-immersing it in a filter provided in the drip nozzle, a sample addition member (5 in the example of FIG. 9) of the device, or a labeled antibody-impregnated member (2 in the example of FIG. 9) of the device, and the first reagent with the specimen attached thereto soaked in the cotton swab can be dripped directly onto the sample addition member. By using the second reagent pre-immersed in a filter provided in the drip nozzle or a sample addition member, there are advantages such as improved operability (the effort of dripping the second reagent can be omitted, and operational errors can be reduced) and concentration of the antigen amount (the amount of reaction liquid can be reduced).

If necessary, the first reagent can be contacted with the specimen at ambient temperature, or, if necessary, under cooled conditions that are less likely to cause deterioration of components such as proteins. The contact can be carried out for a period of time that allows sufficient extraction of intracellular antigens, for example, from a few seconds to a few hours, and, if necessary, stirring or shaking can be carried out during the contact.

Furthermore, according to the inventors' investigations, the first and second reagents may be used in any order, and the interval between additions is not particularly limited. The time between the addition of the first and second reagents may be short. In other words, one of the advantages of the method of the present invention is that it allows for rapid testing.

The two-step operation allows for both highly efficient bacteriolysis and accurate immunoassay. Since ionic surfactants are electrically charged, they have a high binding affinity with similarly charged proteins. Therefore, from the viewpoint of bacteriolysis, it makes sense to select an ionic surfactant. However, in the case of immunoassay, ionic surfactants also have a high binding affinity with the antibodies used in the assay, which results in a high possibility of inhibiting the antigen-antibody reaction. The 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 the ionic surfactant alone during bacteriolysis.

(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 trace amounts of antigens or antibodies.

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, and flow cytometry assay. When carrying out an immunological assay method, a blocking substance to prevent non-specific adsorption or a substance to prevent cross-reaction with bacteria other than the target bacterial species may be used.

[ELISA method]
The enzyme-linked immunosorbent assay (ELISA) in this invention is a method for measuring substances such as proteins using an antigen-antibody reaction, in which a sample containing an antigen is reacted with a primary antibody that has been previously bound to a solid phase, and then an enzyme-labeled secondary antibody is reacted with the sample, and whether or not the antigen is bound 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.

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 be adsorbed onto 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.

[Immunochromatography]
In one preferred embodiment, the present invention uses an apparatus or kit that utilizes an immunochromatography method (sometimes referred to as an "immunochromatography method"). The kit includes an immunochromatography apparatus, and may further include the first reagent and/or the second reagent described above. 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, for example, an antibody against ribosomal protein L7/L12, is immobilized on the determination section. The kit or immunochromatography device may utilize a sandwich assay (sometimes called a "sandwich immunoassay") that uses an antibody that binds to a site different from the immobilized antibody.

Figure 9 shows a schematic cross-sectional view of an immunochromatography device. 1 is the substrate, 2 is a member impregnated with a labeled antibody, 3 is a membrane carrier for chromatographic development, 4 is an absorption member, and 5 is a member for adding a sample. 6 is a determination part or capture part where an antibody that reacts with an antigen contained in bacteria is fixed.

The labeled antibody-impregnated member preferably holds a labeled antibody against the antigen to be detected that 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.

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 determination section, 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 capable of simultaneously binding to one antigen, and that the epitope of one antibody is different from the epitope of the antigen recognized by the other antibody.

Labels include colored particles, enzymes, radioisotopes, etc., but it is preferable to use colored particles that can be detected visually without the need for special equipment. 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-metallic particles, latex particles, etc. 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.

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. 9), synthetic polymers such as polyethylene, polypropylene, polystyrene, acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, polyurethane, etc., natural polymers such as agarose, cellulose, nitrocellulose, cellulose acetate, chitin, chitosan, alginate, etc., as well as structures crosslinked 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 view of their ease of availability, stability, safety, moldability, and sterilizability. Among these, synthetic polymers and derivatives of natural polymers 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.
The material used for the labeled antibody-impregnated member (2 in the example of Figure 9) 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.
The material used for the chromatographic development membrane carrier (3 in the example of Figure 9) including the determination part (6 in the example of Figure 9) is not particularly limited as long as it is capable of performing immunochromatography, but is preferably nitrocellulose, mixed nitrocellulose ester, polyvinylidene fluoride, nylon, etc.

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 is acceptable as long as the antibody molecule or active group is immobilized from the membrane carrier to the membrane carrier surface. 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. 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.

[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.

Antibodies against ribosomal protein L7/L12 can be produced by the method described in International Publication No. 00/06603. The antibodies can be produced using the full-length protein of ribosomal protein L7/L12 or a partial peptide thereof as an antigen, but it is preferable to produce the antibodies 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. Antibodies can also be purified from the antiserum and used. Examples of animals to be inoculated include sheep, horses, goats, rabbits, mice, and rats, and sheep and rabbits are particularly preferable for producing polyclonal antibodies. It is more preferable to apply monoclonal antibodies obtained by a known method for producing hybridoma cells, but in this case, mice are 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 a patient is infected with an infectious disease caused by the bacterium. Antibodies can be produced in a similar manner when an intracellular antigen other than the ribosomal protein L7/L12 antigen is used as an antigen. Note that, although the present invention will be described below using an example of detecting the ribosomal protein L7/L12 antigen as an example, those skilled in the art can understand the description by appropriately applying it to cases where an intracellular antigen other than the ribosomal protein L7/L12 is used as an antigen.

(3) Advantages of the present invention By using the present invention, it is possible to identify multiple types of respiratory infection causative bacteria by collecting a specimen from a subject in one go. Specifically, a nasal swab is collected from a patient exhibiting symptoms of respiratory infection in one go with a cotton swab, the cotton 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 sample for testing. For example, the presence or absence of multiple types of respiratory infection causative bacteria can be detected at once using immunodetection reagents for Streptococcus pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, and Bordetella pertussis. In addition, for example, if whooping cough is suspected, a similarly prepared test sample can be measured using immunodetection reagents for Bordetella pertussis, Bordetella parapertussis, Bordetella holmesii, pertussis toxin (PT), and lipopolysaccharide (LPS), an endotoxin specific to Bordetella pertussis, to determine in more detail whether or not there is whooping cough infection.

Examples of the present invention are described in detail below, but the present invention is not limited thereto in any way.

Example 1: Preparation of bacterial cells
[Method for preparing Bordetella pertussis, Bordetella parapertussis, and Bordetella holmesii bacterial liquid]
Bordetella pertussis (ATCC No. BAA-589), Bordetella parapertussis (ATCC No. BAA-587), and Bordetella holmesii (ATCC No. 700053) were prepared as test bacterial solutions by the following method: Bordetella CFDN agar medium (Nikken Biomedical Research Institute) was inoculated and cultured at 37°C for 3 days under aerobic conditions, and the colonies on the medium were suspended in physiological saline (Japanese Pharmacopoeia physiological saline) to be used as the test bacterial solution.

[Legionella pneumophila bacterial liquid preparation method]
Legionella pneumophila (ATCC No. 33152) was cultured on BCYE agar medium at 37°C for 4 days, after which a single colony was added to 3 ml of BYE medium and cultured overnight at 37°C with stirring. The bacteria was then added to 3 ml of fresh BYE medium and cultured at 37°C with stirring to be used as the test bacteria solution.

Example 2: Preparation of monoclonal antibodies against Bordetella pertussis Ribosomal Protein L7/L12
(1) Cloning of Ribosomal Protein L7/L12 Genes from Bordetella pertussis PCR (polymerase chain reaction) was performed using 50 ng of purified Bordetella pertussis genomic DNA. PCR was performed using Taq polymerase (Takara Shuzo Co., Ltd., code R001A). 5 μL of the buffer provided with the enzyme, 5 μL of the dNTP mixture provided with the enzyme, and 260 pmol each of synthetic oligonucleotide A (catggatccatggcacttagcaaagctgaa SEQ ID NO: 1) and sequence oligonucleotide B (gtagaattcttattacttgacttcgaccttgg SEQ ID NO: 2) were added to a final volume of 50 μL.

This mixture was subjected to one cycle of 94°C for 2 minutes using a TaKaRa PCR Thermal Cycler 480, followed by 30 cycles of 98°C for 10 seconds, 55°C for 30 seconds, and 68°C for 1 minute. A portion of this PCR product was electrophoresed in a 1.5% agarose gel, stained with ethidium bromide (manufactured by Nippon Gene Co., Ltd.), and observed under ultraviolet light to confirm that approximately 400 bp of cDNA had been amplified. The resulting PCR product was inserted into the plasmid pCR-BluntII-TOPO according to the procedure of the Zero blunt (registered trademark) TOPO (registered trademark) PCR Cloning Kit (Invitrogen). After further digestion with the restriction enzymes BamHI and EcoRI, electrophoresis was performed in a 1.5% agarose gel, and after staining with ethidium bromide, a band of approximately 370 bp was excised from the gel and purified with Suprec01 (Takara Shuzo Co., Ltd.), and then incorporated into the commercially available vector pGEX-6P-1 (Pharmacia Co., Ltd.). This vector can function as an expression vector for the target molecule that can express a fusion protein with the GST protein by incorporating the target gene fragment into an appropriate restriction enzyme site.

Specifically, the vector pGEX-6P-1 and the above DNA were mixed at a molar ratio of 1:3, and the DNA was incorporated into the vector using T4 DNA ligase (Invitrogen). The vector pGEX-6P-1 with the DNA incorporated was transfected into E. coli One Shot Competent Cells (Invitrogen), plated on a plate of semi-solid medium LLB-Broth (Takara Shuzo) containing 50 μg/mL ampicillin (Sigma), and left at 37°C for about 12 hours. Colonies that appeared were randomly selected and planted in 2 mL of LLB-Broth liquid medium containing the same concentration of ampicillin, and cultured with shaking at 37°C for about 8 hours. The bacterial cells were then collected and subjected to QIAprep Spin Miniprep. The plasmid was isolated using a kit (QIAGEN) according to the attached instructions, and the plasmid was digested with the restriction enzymes BamHI/EcoRI to confirm that the PCR product had been incorporated by excising approximately 370 bp of DNA. The base sequence of the incorporated DNA was then determined for the confirmed clones.

The clone obtained had sequence homology with the probe used in PCR, and the DNA sequence clearly matched the ribosomal protein L7/L12 gene sequence. The complete base sequence of the structural gene portion and the corresponding amino acid sequence were as follows. This gene fragment clearly codes for the ribosomal protein L7/L12 gene of Bordetella pertussis.

atggcacttagcaaagctgaaatccttgacgccatcgctggcatgtccgtgctcgagctgtccgagctgatcaaggaaatggaagaaaagtttggcgtgtcggctgctgccgccgccgtggccgtggccgccccggccgctggtggcgctggcgccgctgctgctgctgaagagcagaccgagttcaccgttgtgctgctggaagccggcgcgaacaaggtcagcgtcatcaaggccgtgcgcgagctgaccggtctgggtctgaaggaagccaaggacctggttgacggcgctccgaagcccgtcaaggaagcgctgcccaaggctgacgccgaagccgccaagaagaagctggaagaagctggcgccaaggtcgaagtaa (SEQ ID NO: 3)

MALSKAEILDAIAGMSVLELSELIKEMEEKFGVSAAAAAVAVAAPAAGGAGAAAAEEQTEFTVVLLEAGANKVSVIKAVRELTGLGLKEAKDLVDGAPKPVKEALPKADAEAAKKKLEEAGAKVEVK (SEQ ID NO: 4)

(2) Large-scale expression and purification of ribosomal protein L7/L12 genes from Bordetella pertussis in Escherichia coli Escherichia coli incorporating an expression vector was cultured overnight at 37°C in 50 mL of LB medium. 50 mL of the overnight cultured Escherichia coli solution was added to 500 mL of TB medium. After 1 hour, 550 μL of 100 mM isopropyl β-D(-)-thiogalactopyranoside (IPTG) was added and cultured for 4 hours, after which the medium was recovered, 1/100th the amount of BugBuster (Merck) was added, and the mixture was shaken at room temperature for 20 minutes, centrifuged at 10,000 rpm for 30 minutes, and the supernatant was recovered.

Next, the above-mentioned supernatant was adsorbed onto a glutathione agarose column conditioned with phosphate-buffered saline (PBS). The column was then washed with 2 bed volumes of a washing solution containing 20 mM Tris buffer pH 7.4, 4.2 mM MgCl2, and 1 mM dithiothreitol (DTT). The column was then eluted with 50 mM Tris buffer pH 9.6 containing 5 mM glutathione, and the protein content in the fractionated fraction was detected by a dye-binding method (Bradford method; Biorad) to obtain the main fraction. The purity of the obtained purified GST fusion ribosomal protein L7/L12 was confirmed by electrophoresis to be about 75%, which was sufficient purity for use as an immunogen.

(3) Preparation of Monoclonal Antibody Against Ribosomal Protein L7/L12 of Bordetella pertussis First, for immunization of mice, 100 μg of GST fusion ribosomal protein L7/L12 antigen of Bordetella pertussis was dissolved in 200 μL of PBS, and then 200 μL of Freund's complete adjuvant was added and mixed to form an emulsion, and 200 μL of the emulsion was injected intraperitoneally.

The same emulsion antigen was injected intraperitoneally after 2, 4, and 6 weeks, and then a double-concentration antigen emulsion was injected intraperitoneally after 10 and 14 weeks. Three days after the final immunization, the spleen was removed and subjected to cell fusion.

10 ⁸ mouse spleen cells were aseptically removed, and 2×10 ⁷ myeloma cells were placed in a glass tube, mixed thoroughly, and centrifuged at 1,500 rpm for 5 minutes. The supernatant was discarded, and the cells were then mixed thoroughly.

The myeloma cells used for cell fusion were NS-1 cell line, cultured in RPMI1640 medium containing 10% fetal calf serum (FCS), and then cultured for one week in RPMI1640 medium containing 0.13 mM azaguanine, 0.5 μg/mL MC-210, and 10% FCS from two weeks before cell fusion, and then cultured for another week in RPMI1640 medium containing 10% FCS. 30 mL of 50 mL RPMI1640 medium kept at 37°C was added to the mixed cell sample, centrifuged at 1,500 rpm, the supernatant was removed, 1 mL of 50% polyethylene glycol kept at 37°C was added, and the mixture was treated with vigorously stirring for 2 minutes, after which 10 mL of RPMI1640 medium kept at 37°C was added, and the liquid was vigorously stirred and mixed for about 5 minutes while aspirating and discharging with a sterile pipette.

After centrifugation at 1,000 rpm for 5 minutes and removal of the supernatant, an additional 30 mL of HAT medium was added to adjust the cell concentration to 5 x ¹⁰ cells/mL and homogenized. After stirring, 0.1 mL was dispensed into a 96-well culture plate and cultured at 37°C under 7% CO2 conditions. 0.1 mL of HAT medium was added on the 1st day, 1 week, and 2 weeks.

Next, an ELISA evaluation was performed to screen for cells producing the desired antibody. Bordetella pertussis GST fusion ribosomal protein L7/L12 and GST protein dissolved in PBS containing 0.05% sodium azide were diluted to a concentration of 10 μg/mL, and 100 μL of each solution was dispensed into a 96-well plate and allowed to adsorb overnight at 4°C. After removing the supernatant, 200 μL of 1% bovine serum albumin (BSA) solution (in PBS) was added and reacted at room temperature for 1 hour for blocking. After removing the supernatant, the cells were washed with a washing solution (Tween 20 0.02%, PBS), and 100 μL of the culture solution of the fused cells was added on top and reacted at room temperature for 2 hours, after which the supernatant was removed and the cells were washed with a washing solution. 100 μL of 500 ng/mL peroxidase-labeled goat anti-mouse IgG antibody solution was added to this, and the reaction was carried out at room temperature for 1 hour. The supernatant was removed and the plate was washed with a washing solution, after which 100 μL of TMB solution (KPL) was added and the plate was reacted at room temperature for 20 minutes. After color development, 100 μL of 1N sulfuric acid was added to stop the reaction, and the absorbance at 450 nm was measured.

As a result, positive wells were found that reacted only with GST fusion ribosomal protein L7/L12 and not with GST protein, revealing that they contained antibodies against ribosomal protein L7/L12.

The cells in the positive wells were then collected and cultured in HAT medium in a 24-well plastic plate. The cultured fusion medium was diluted with HT medium to a cell count of approximately 20 cells/mL, and 50 μL of the diluted medium was mixed with 10 ⁶ thymocytes from 6-week-old mice suspended in HT medium in a 96-well culture plate, and then cultured at 37° C. under 7% CO ₂ conditions for 2 weeks. The antibody activity in the culture supernatant was assayed in the same manner as above using the ELISA method, and cells that reacted positively with ribosomal protein L7/L12 were collected.

Furthermore, the same dilution assay and cloning procedures were repeated to obtain a total of seven hybridoma strains: BPRB-5, -7, -10, -11, -12, -13, and -14.

(4) Selection of Monoclonal Antibodies Reacting with Ribosomal Protein L7/L12 of Bordetella pertussis Monoclonal antibodies were produced and collected according to a standard method using the positive hybridoma cells obtained as described above.

Specifically, cells that had been subcultured using RPMI1640 medium (containing 10% FCS) were injected intraperitoneally at 5 x ¹⁰ cells (in PBS) into a Balb/C mouse that had been intraperitoneally injected with 0.5 mL of pristane two weeks prior, and the ascites was collected three weeks later, and the supernatant was obtained by centrifugation.

The antibody-containing solution obtained was adsorbed onto a Protein A column (5 mL bed, Pharmacia), washed with 3 bed volumes of PBS, and eluted with citrate buffer at pH 3. The antibody fraction was collected to obtain the monoclonal antibodies produced by each hybridoma.

The monoclonal antibodies derived from these seven hybridoma strains were used as the primary or secondary antibodies to evaluate the detection performance of 42 combinations of Bordetella pertussis by ELISA. The antibodies were evaluated using a sandwich assay method, and the prepared monoclonal antibodies were used as the secondary antibodies in the ELISA method by chemically binding them to peroxidase to prepare enzyme-labeled antibodies. That is, enzyme labeling was performed using horseradish peroxidase (Sigma grade VI) and the reagent S-acetylthioacetate N-hydroxysuccinimide for binding, according to the method described in Analytical Bio-chemistry 132 (1983), 68-73. In the ELISA reaction, the monoclonal antibody dissolved in PBS containing 0.05% sodium azide was used as the primary antibody, and the solution diluted to a concentration of 10 μg/mL was dispensed in 100 μL portions into a 96-well plate and allowed to adsorb overnight at 4°C.

After removing the supernatant, 200 μL of 1% BSA solution (in PBS) is added and reacted at room temperature for 1 hour for blocking. After removing the supernatant, the plate was washed with a washing solution (Tween 20 0.02%, PBS), and 100 μL of an antigen solution (6× ¹⁰⁵ cells/mL) extracted from a Bordetella pertussis culture solution with 0.3% Triton X-100 at room temperature for 5 minutes was added thereon, and the plate was incubated at room temperature for 2 hours. The supernatant was removed, and the plate was further washed with a washing solution. After that, 100 μL of 5 μg/mL peroxidase-labeled anti-ribosomal protein L7/L12 antibody solution was added and incubated at room temperature for 1 hour. The supernatant was removed and the plate was further washed with a washing solution. Then, 100 μL of TMB solution (KPL) was added and incubated at room temperature for 20 minutes. After color development, 100 μL of 1N sulfuric acid was added to stop the reaction, and the absorbance at 450 nm was measured. The Bordetella pertussis antigen was identified based on the difference with the negative control signal. The combination of Bordetella pertussis ribosomal protein L7/L12 antibodies capable of detecting Bordetella pertussis was evaluated. In addition, the reactivity of each antibody combination with 31 major bacterial species other than Bordetella pertussis was evaluated. The ELISA conditions were the same as those described above, and the test was performed by providing samples prepared in 1 x 10 ⁸ cells/mL, 0.5% Triton X-100, and PBS for the other major bacteria instead of Bordetella pertussis as the antigen.

As a result, 10 antibody combinations were obtained that could detect Bordetella pertussis and did not react with other major bacteria.

Table 1 shows ELISA absorbance data confirming reaction with Bordetella pertussis for 10 monoclonal antibody combinations derived from hybridomas BPRB-5, -7, -10, -11, -12, -13, and -14 that can detect these 10 pairs of Bordetella pertussis and do not react with 31 types of bacteria other than Bordetella pertussis. Table 2 also shows a list of other bacteria that were confirmed to not cross-react by ELISA. The values in Table 1 indicate the values obtained by subtracting the measurement value of the negative control from the measurement value of the positive sample for each antibody combination.

Example 3: Preparation of monoclonal antibodies against Legionella pneumophila ribosomal protein L7/L12
Similar to the method disclosed in JP 2004-201605 A, two clones of hybridoma LEG-1 cells and hybridoma LEG-2 cells that showed a positive reaction with Legionella pneumophila Ribosomal Protein L7/L12 were obtained, and monoclonal antibodies LEG-1 and LEG-2 were produced and collected according to a standard method. The LEG-1 antibody and the LEG-2 antibody are independent antibodies that recognize different antibody sites in Legionella pneumophila Ribosomal Protein L7/L12, and are a combination of antibodies that can detect the presence of the antigen by sandwich ELISA using Legionella pneumophila Ribosomal Protein L7/L12 as the antigen.

Example 4: Preparation of an immunochromatography device
As an example, the procedure for preparing an immunochromatography device for Bordetella pertussis is described below. It is also possible to prepare immunochromatography devices for other bacteria using similar procedures.

(1) Labeled antibody-impregnated member 0.9 mL of gold colloid solution (particle size 60 nm, Tanaka Kikinzoku Co., Ltd.) was mixed with 0.05 M TAPS buffer pH 8.0, 100 μg/mL of monoclonal antibody BPRB-7 to be labeled with gold colloid was added, and the mixture was left at room temperature for 10 minutes to bind the antibody to the surface of the gold colloid particles. After that, 10% bovine serum albumin (BSA) (Merck) aqueous solution 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 BPRB-7 labeled with gold colloid (hereinafter referred to as "gold colloid-labeled antibody"). This solution was centrifuged (15,000 g, 10 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.

(2) Membrane carrier for chromatographic development
A 25 mm x 300 mm nitrocellulose membrane (Sartorius, product name: UniSart CN140) was prepared as a chromatographic medium for the membrane carrier. The monoclonal antibody BPRB-5 for capturing the complex of gold colloid-labeled antibody and antigen was diluted with 3% trehalose (Fujifilm Wako Pure Chemical Industries, Ltd.) and 0.01 M TAPS buffer (pH 9.0) to prepare a concentration of 2.0 mg/mL. The prepared antibody solution was applied in a line shape at 1 μL/cm to a position 10 mm from the end of the chromatographic development start point of the membrane carrier for the chromatographic development, and this was dried at 55 °C overnight.

(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 in the portion where the sample is added, and an absorption member (manufactured by Nippon Paper Crecia Co., Ltd., product name: CR Cushion) for absorbing the developed sample and excess gold colloid-labeled antibody were attached to a base material made of a backing sheet having an adhesive layer (manufactured by Adhesives Research Inc.). The sheet was then cut into a width of 5 mm using a cutting machine to prepare an immunochromatography device.

Example 5: Effect of ionic surfactants on detection of Bordetella pertussis by immunochromatography
To explore the optimal surfactant for the immunochromatographic detection of ribosomal protein L7/L12 in Bordetella pertussis, we compared the performance of ionic surfactants and, as a control, the effect of using a nonionic surfactant as the first reagent.

[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)% each of the ionic surfactants or nonionic surfactants shown in Table 3

[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.)

[Preparation of positive sample solution]
5 μL of the Bordetella pertussis test bacteria solution prepared in Example 1 was diluted with PBS (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to a final volume of 1 mL to prepare a positive specimen solution.

[Preparation of measurement sample]
The positive sample liquid was treated by any one of steps 1 to 4 described below to prepare a measurement sample.
<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 after mixing, 150 μL of the second reagent was further added and mixed to prepare the measurement sample for step 1.
<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.
<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.
<Step 4: Treating the specimen 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 a measurement sample for step 4.

[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.

[result]
The results are shown in Table 3. When no signal was observed on the reading zone, it was rated "-", when a weak signal was observed, it was rated "±", when a signal was observed, it was rated "+", and when a stronger signal was observed, it was rated "++".

The measurement sample processed in step 1 showed a high signal intensity, whereas no signal was observed in the measurement samples processed in steps 2 and 4. This suggests 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, the measurement sample processed in step 3 was not able to be measured because the liquid did not spread on the immunochromatography device. This suggests 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.

In addition, for the measurement samples processed in step 1, signals could be confirmed on the test zone with all of the ionic surfactants shown in Table 3. In particular, it was found that high signal strength was observed in the sample processing method using didecyl dimethyl ammonium adipate, didecyl dimethyl ammonium chloride, didecyl dimethyl ammonium bromide, hexadecyl trimethyl ammonium chloride, trimethyl stearyl ammonium chloride, and benzalkonium chloride. As shown in Table 3, no signal could be confirmed on the test zone when a nonionic surfactant was used in the first reagent, suggesting that it is important to use an ionic surfactant in the first reagent.

[Example 6: Concentration of ionic surfactant]
The effect of the concentration of an ionic surfactant on the immunochromatography device for detecting ribosomal protein L7/L12 of Bordetella pertussis was examined. Measurement was performed in the same manner as in Example 5, except that the concentration of the ionic surfactant in the first reagent was changed to the concentration shown in Table 4.

[result]
The results are shown in Table 4. A signal that was not observed on the reading zone was marked as -, a weak signal that was observed was marked as ±, a signal that was observed was marked as +, and a stronger signal was marked as ++. For all the ionic surfactants shown in Table 4, a signal could be observed on the reading zone in the concentration range of 0.01 to 0.1 (w/v)%.

Example 7: Study on extraction time using ionic surfactants
After the addition of an ionic surfactant, the extraction time required for extracting ribosomal protein L7/L12 from Bordetella pertussis was examined.

[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)% each of the ionic surfactants shown in Table 5

[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 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)

[Preparation of positive sample solution]
5 μL of the Bordetella pertussis test bacteria solution prepared in Example 1 was diluted with PBS to a final volume of 1 mL to prepare a positive specimen solution.

[Preparation of measurement sample]
The measurement samples were prepared by adding 20 μL of the positive specimen liquid to 300 μL of the first reagent and mixing, and then immediately adding and mixing 150 μL of the second reagent, or by leaving the mixture for 1 minute, 2 minutes, 5 minutes, and 15 minutes, and then adding and mixing 150 μL of the second reagent.

[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.

[result]
The results are shown in Table 5. As shown in Table 5, it was found that the method of the present invention does not require an extraction time, and extraction is possible immediately after the addition of the first reagent containing an ionic surfactant.

Example 8: Effect of non-ionic surfactants on detection of Bordetella pertussis by immunochromatography
To explore the optimal surfactant for an immunochromatographic device for detecting ribosomal protein L7/L12 of Bordetella pertussis, we compared the performance of nonionic surfactants.

[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)% each of the ionic surfactants shown in Table 6

[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 6

[Preparation of positive sample solution]
20 μL of the Bordetella pertussis test bacteria solution prepared in Example 1 was diluted with PBS to a final volume of 1 mL to prepare a positive specimen solution.

[Preparation of measurement sample]
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.

[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.

[result]
The results are shown in Table 6. A weak signal was observed on the reading zone, and a signal was observed, which was rated as ±. As the signal became stronger, it was rated as ++ and +++. Table 6 also shows the HLB values of the nonionic surfactants.

As shown in Table 6, a signal could be confirmed on the test zone by using any of the nonionic surfactants with HLB values between 10 and 18.5 as the second reagent.

[Example 9: Concentration of nonionic surfactant]
The effect of the concentration of a nonionic surfactant on the immunochromatography device for detecting ribosomal protein L7/L12 of Bordetella pertussis was investigated. Measurements were performed in the same manner as in Example 8, except that the concentration of the nonionic surfactant contained in the second reagent was changed as shown in Table 7.

[result]
The results are shown in Table 7. When a weak signal was confirmed on the reading zone, it was rated ±, when a signal was confirmed, it was rated +, and as the signal became stronger, it was rated ++ and +++.

As is clear from Table 7, for nonionic surfactants with HLB values in the range of 10 to 18.5, signals could be confirmed on the test zone at concentrations in the range of 0.5 to 3 (w/v)%. In addition, for polyoxyethylene (20) sorbitan monolaurate, signals could be confirmed on the test zone at all concentrations confirmed, but a particularly strong signal was confirmed in the range of 0.5 to 10 (w/v)%.

Example 10: Detection of respiratory infection-causing bacteria by immunochromatography
We investigated whether the antigens of Bordetella pertussis, Bordetella parapertussis, Bordetella holmesii, and Legionella pneumophila, which are causative bacteria of respiratory infections, could be detected using the optimal combination of ionic and nonionic surfactants using each Ribosomal Protein L7/L12 detection immunochromatography device.

[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)% each of the ionic surfactants shown in Table 8

[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.)

[Preparation of positive sample solution]
(Preparation of positive specimens for Bordetella pertussis, Bordetella parapertussis, and Bordetella holmesii)
5 μL of the test bacteria solution of Bordetella pertussis, Bordetella parapertussis, or Bordetella holmesii prepared in Example 1 was diluted with PBS (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to a final volume of 1 mL to prepare a positive sample solution for each bacteria.

(Preparation of positive specimen for Legionella pneumophila)
A positive specimen solution was prepared by diluting 10 μL of the test bacteria solution of Legionella pneumophila prepared in Example 1 with physiological saline (Japanese Pharmacopoeia physiological saline) to a final volume of 1 mL.

[Preparation of measurement sample]
(Preparation of samples for measuring Bordetella pertussis, Bordetella parapertussis, and Bordetella holmesii)
The positive specimen liquid was treated by either step 1 or 2 described below to prepare a measurement sample.
<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 after mixing, 150 μL of the second reagent was further added and mixed to prepare the measurement sample for step 1.
<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.

(Preparation of samples for measuring Legionella pneumophila)
Except for using 3 μL of positive sample liquid, 100 μL of the first reagent, and 50 μL of the second reagent, the measurement samples were prepared in the same manner as in the preparation of the measurement samples for Bordetella pertussis, Bordetella parapertussis, and Bordetella holmesii described above, by processing in either step 1 or 2.

[measurement]
120 μL of each measurement sample was dropped onto the sample application member of the immunochromatography device for each bacterium, and visual judgment was performed after 15 minutes. If no signal was observed on the judgment part, it was rated as -, if a signal was observed, it was rated as +, and if the signal was stronger, it was rated as ++.

[result]
The results are shown in Table 8. As is clear from Table 8, all respiratory infection causative bacteria could be detected by the method of the present invention. As for Bordetella pertussis, ATCC Nos. 8467, 9797, and 9340 strains, which were prepared from test bacterial solutions in the same manner as for Bordetella pertussis (ATCC No. BAA-589) in Example 1, could also be detected.

Example 11: Diagnosis of pertussis infection by immunochromatography using specimens collected from patients with respiratory infection symptoms
[Specimen collection]
Specimens were collected from patients with respiratory infection symptoms. Nasal swabs were obtained by inserting a sterile cotton swab into the patient's nasal cavity and rotating the swab once it reached the back of the cavity.

[Preparation of measurement sample]
300μL of the first reagent containing 0.03(w/v)% Osmolin DA-50 (Sanyo Chemical Industries, Ltd.) was placed in a polyethylene tube, and the cotton swab containing the sample was inserted into the tube and rubbed well from the outside 5 to 20 times, after which the cotton swab was removed. 150μL of the second reagent containing 0.15M phosphate buffer (pH7.5), 2(w/v)% BSA (Merck, Inc.), 0.05(w/v)% sodium azide (Nacalai Tesque, Inc.), and 3(w/v)% polyoxyethylene (20) sorbitan monolaurate (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the tube. The resulting solution was used as the measurement sample.

[Measurement using an immunochromatography device]
A dropping nozzle equipped with a PVA filter was attached to the polyethylene tube containing the measurement sample, and four drops of the measurement sample (volume approximately 120 μL) were dropped onto the sample addition member of the immunochromatography device. After 15 minutes, the result was visually judged.

[PCR using residual liquid of measurement sample]
DNA was extracted from the test samples, and PCR was performed using primers targeting IS481 of Bordetella pertussis to quantify the copy number of IS481. The procedure is shown below.
A QIAamp DNA Micro kit (QIAGEN) was used to extract DNA. Extraction and purification procedures were performed according to the protocol included with the kit. PCR was performed using the resulting DNA extract as a template. PCR conditions were based on the National Institute of Infectious Diseases, Pathogen Testing Manual, Whooping Cough, October 2011.

[result]
The results of visual judgment using the immunochromatography device and the results of PCR are shown in Tables 9 and 10. As shown in Table 9, when the PCR method using the test sample was used as the standard, the results of visual judgment using the immunochromatography device showed a sensitivity of 85.7% (12/14) and a specificity of 92.8% (141/152). Even when nasal swabs collected from patients with respiratory infection symptoms were used, patients with Bordetella pertussis infection could be detected by using this processing method. Table 10 shows the copy numbers of IS481 contained in the test samples that were positive by the PCR method and the results of visual judgment. As is clear from Table 10, all test samples containing IS481 copies of 8.9 x ¹⁰⁵ (copies/swab) or more were judged to be positive by visual judgment.

Example 12: Detection of various antigens of Bordetella pertussis by enzyme-linked immunosorbent assay (ELISA)
We investigated whether this sample processing method can detect ribosomal protein L7/L12, lipopolysaccharide (LPS), and pertussis toxin (PT) contained in Bordetella pertussis.

[Peroxidase labeling process for detection antibodies]
The peroxidase-labeled antibodies used in the detection described below were prepared using Peroxidase Labeling Kit-NH2 (Dojindo Chemical Industries, Ltd.). The antibodies used for peroxidase labeling were BPRB-7 antibody or monoclonal antibody (Bordetella pertussis LOS-A Monoclonal Antibody (D26E), Thermo Fisher Scientific Co., Ltd.), and the labeling procedure was performed according to the protocol attached to the kit.

[Detection process]
(1) Detection of Ribosomal Protein L7/L12 2.5 μg/mL BPRB-5 antibody prepared in Example 2 and 50 μL of PBS solution were dispensed into a 96-well ELISA plate (Nunc MaxiSorp ELISA plate) and adsorbed overnight at 4 ° C. After removing the supernatant, 200 μL of 1% BSA solution (in PBS) was added and reacted at room temperature for 2 hours for blocking. After removing the supernatant, the plate was washed several times with a washing solution (0.05% Tween 20, PBS). 50 μL of PBS (for control) or Bordetella pertussis test bacteria solution (for this test) prepared in Example 1 treated with the solution shown in Table 11 was added and reacted at room temperature for 1 hour. After further removing the supernatant, peroxidase-labeled BPRB-7 antibody was diluted with 0.05% Tween 20 and PBS to a final concentration of 2 μg/mL, and 50 μL of each was added and reacted at room temperature for 1 hour. After removing the supernatant and washing several times with washing solution, 100 μL of TMB solution (KPL) was added to each well and reacted at room temperature for 10 minutes, and then 100 μL of 1N hydrochloric acid was added to stop the reaction, and the absorbance at 450 nm was measured. The results of treatment B compared to no treatment and treatment A are shown in Figure 1, and the results of treatment C and treatment D compared to no treatment and treatment A are shown in Figure 2.

(2) Detection of lipopolysaccharide (LPS) Measurements were performed in the same manner as in (1) above, except that a monoclonal antibody (Bordetella pertussis LOS-A Monoclonal Antibody (D26E), Thermo Fisher Scientific) at 30 μg/mL and a peroxidase-labeled monoclonal antibody (Bordetella pertussis LOS-A Monoclonal Antibody (D26E), Thermo Fisher Scientific) at a final concentration of 5 μg/mL were used as the antibody, and blocking was performed with a 5% skim milk solution (in PBS). The results of treatment B compared to no treatment and treatment A are shown in Figure 3, and the results of treatment C and treatment D compared to no treatment and treatment A are shown in Figure 4.

(3) Detection of pertussis toxin (PT)
50μL of a 2500-fold diluted polyclonal antibody (Bordetella pertussis Toxin antibody, manufactured by GeneTex) and PBS solution were dispensed into a 96-well ELISA plate (Nunc MaxiSorp ELISA plate) and adsorbed overnight at 4°C. After removing the supernatant, 200μL of a 1% BSA solution (in PBS) was added and reacted at room temperature for 2 hours for blocking. After removing the supernatant, the plate was washed several times with a washing solution (0.05% Tween 20, PBS). 50μL of PBS (for control) or the Bordetella pertussis test bacteria solution (for this test) prepared in Example 1 treated with the solution shown in Table 11 was added and reacted at room temperature for 1 hour. After removing the supernatant, 50 μL of monoclonal antibody (Bordetella Pertussis Toxin antibody [1280/204], manufactured by GeneTex) was diluted with 0.05% Tween 20 and PBS to a final concentration of 2 μg/mL, and then added and reacted at room temperature for 1 hour. After removing the supernatant, 50 μL of peroxidase-labeled monoclonal antibody (Anti mouse IgG (Fc specific) Peroxidase, antibody (goat), manufactured by SIGMA-ALDRICH) was diluted 4000-fold with 0.05% Tween 20 and PBS, and then added and reacted at room temperature for 1 hour. After removing the supernatant, the plate was washed several times with a washing solution, and then 100 μL of TMB solution (manufactured by KPL) was added and reacted at room temperature for 10 minutes. After that, 100 μL of 1N hydrochloric acid was added to stop the reaction, and the absorbance at 450 nm was measured. The results for treatment B compared to no treatment and treatment A are shown in FIG. 5, and the results for treatment C compared to no treatment and treatment A, and treatment D are shown in FIG.

[result]
By using this sample treatment method using ionic and non-ionic surfactants (treatments B, C, and D), it was possible to detect all antigens derived from Bordetella pertussis. In particular, in the system for detecting antigens contained inside the bacteria, such as ribosomal protein L7/L12 and Bordetella pertussis toxin (PT), this treatment method showed a higher treatment effect than the control treatment method (treatment A) and no treatment.

Example 13: Detection of Bordetella pertussis pertussis toxin (PT) by enzyme-linked immunosorbent assay (ELISA)
This sample treatment method was used to examine whether or not pertussis toxin (PT) contained inside the Bordetella pertussis bacteria can be detected. The Bordetella pertussis test bacteria solution (for this test) used was prepared by centrifuging 500 μL of the Bordetella pertussis test bacteria solution prepared in Example 1 at 10° C. and 10,000 rpm for 10 minutes, removing all the supernatant, and suspending the precipitate in 500 μL of PBS (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). The results of treatment B compared to no treatment and treatment A are shown in FIG. 7, and the results of treatment C and treatment D compared to no treatment and treatment A are shown in FIG. 8.

[result]
By using the sample treatment methods (treatments B, C, and D) that used ionic and nonionic surfactants, it was possible to detect pertussis toxins (PT) contained inside Bordetella pertussis cells. These treatment methods showed a higher treatment effect than the control treatment method (treatment A) and no treatment.

[Example 14: Method of 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 Bordetella pertussis.

[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)% each of the ionic surfactants shown in Table 12

[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 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)

[Preparation of positive sample solution]
5 μL of the Bordetella pertussis test bacteria solution prepared in Example 1 was diluted with PBS (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to a final volume of 1 mL to prepare a positive specimen solution.

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

<Step 1: Step of 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.
The immunochromatography device used in the measurement was prepared in the same manner as in Example 4.

<Step 2: Step of immersing the second reagent in the sample addition member of the immunochromatography device>
20 μL of the positive specimen solution 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 for step 2.
The sample addition member containing the second reagent was prepared by immersing a 17 mm x 150 mm strip of glass fiber pad in 1.5 mL of the second reagent and drying it under vacuum at room temperature. In step 2, an immunochromatography device was prepared in the same manner as in Example 4, except that this sample addition member containing the second reagent was used.

<Step 3: Step of immersing the second reagent in the specimen filtration section of the immunochromatography device>
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 the measurement sample for step 3.

The dropping nozzle containing the second reagent was prepared by impregnating a PVA sponge filter having a diameter of 7 mm attached to the dropping nozzle with 0.15 mL of the second reagent.
The immunochromatography device used in the measurement was prepared in the same manner as in Example 4.

[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.

[result]
The results are shown in Table 12. A weak signal was observed on the reading zone, and the result was indicated as ±. A signal was observed, and the result was indicated as +. A stronger signal was observed, and the result was indicated as ++.
As shown in Table 12, a signal was confirmed on the reading zone in all cases of step 1, step 2, and step 3.

The present invention can be used to diagnose respiratory infections caused by Bordetella pertussis and other bacteria.

1 Substrate 2 Labeled antibody-impregnated member 3 Chromatographic development membrane carrier 4 Absorption member 5 Sample addition member 6 Judgment portion or capture portion

Claims (21)

A method for detecting a pathogenic bacterium of a respiratory tract infection contained in a specimen, comprising:
(a) contacting the sample with a first reagent comprising an ionic surfactant but not an alkaline or non-ionic surfactant to obtain an intermediate composition;
(b) contacting the intermediate composition with a second reagent containing a nonionic surfactant to obtain a reaction liquid; and (c) subjecting the reaction liquid to an immunoassay using an antibody against an antigen of the causative bacterium to detect the antigen. The method according to claim 1, wherein the causative bacterium is any one selected from the group consisting of Bordetella pertussis, Bordetella parapertussis, Bordetella holmesii, and Legionella pneumophila. A method for detecting any one of respiratory tract infection causative bacteria selected from the group consisting of Bordetella pertussis, Bordetella parapertussis, Bordetella holmesii, and Legionella pneumophila in a sample, comprising:
(a) contacting the sample with a first reagent comprising an ionic surfactant but no non-ionic surfactant to obtain an intermediate composition;
(b) contacting the intermediate composition with a second reagent containing a nonionic surfactant to obtain a reaction liquid; and (c) subjecting the reaction liquid to an immunoassay using an antibody against an antigen of the causative bacterium to detect the antigen. The method according to any one of claims 1 to 3, wherein the ionic surfactant is a cationic surfactant. The method according to claim 4, wherein the cationic surfactant is a quaternary ammonium salt. The method according to claim 5, wherein the quaternary ammonium salt is any one selected from the group consisting of didecyldimethylammonium adipate, didecyldimethylammonium chloride, didecyldimethylammonium bromide, dimethyldioctadecylammonium chloride, trimethyltetradecylammonium chloride, hexadecyltrimethylammonium chloride, trimethylstearylammonium chloride, and benzalkonium chloride. The method according to claim 5 or 6, wherein the quaternary ammonium salt is didecyldimethylammonium adipate. The method according to any one of claims 1 to 3, wherein the ionic surfactant is an anionic surfactant. The method according to claim 8, wherein the anionic surfactant is sodium linear alkylbenzene sulfonate or sodium dodecyl sulfate. 10. The method according to claim 1, wherein the nonionic surfactant contained in the second reagent is any one selected from the group consisting of polyoxyethylene alkyl ethers, polyoxyethylene polyoxypropylene alkyl ethers, sorbitan fatty acid esters, and polyoxyethylene alkyl phenyl ethers. The method according to any one of claims 1 to 10, wherein the nonionic surfactant contained in the second reagent is a nonionic surfactant whose HLB is 10.0 to 18.5. The method according to any one of claims 1 to 11, wherein the nonionic surfactant contained in the second reagent is a nonionic surfactant whose HLB is 12.5 to 16.7. 13. The method according to any one of claims 1 to 12, wherein the nonionic surfactant contained in the second reagent 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. 14. The method according to any one of claims 1 to 13, wherein the nonionic surfactant contained in the second reagent is poly(oxyethylene) sorbitan monolaurate. The method according to any one of claims 1 to 14, wherein the intermediate composition is brought into contact with the second reagent by passing the intermediate composition through a filter provided on a sample addition member or a dropping nozzle in which the second reagent is immersed. The method according to any one of claims 1 to 15, wherein the antigen is ribosomal protein L7/L12. The method according to any one of claims 1 to 16, wherein the causative bacterium is a Bordetella bacterium, and the antigen is any one selected from the group consisting of ribosomal protein L7/L12, lipopolysaccharide, and Bordetella pertussis toxin. The method of any one of claims 1 to 17, wherein the immunoassay is an immunochromatographic assay. The method according to any one of claims 1 to 18, wherein the sample is a nasal swab, a throat swab, a nasal aspirate, or sputum. A method according to any one of claims 1 to 19 for diagnosing or aiding in the diagnosis of a respiratory infection in a human. 21. A kit for carrying out the method of any one of claims 1 to 20 comprising:
a first reagent that contains an ionic surfactant but does not contain a non-ionic surfactant ; and a second reagent that contains a non-ionic surfactant.

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