JP6676464B2 - Method and kit for detecting Chlamydia pneumoniae - Google Patents

Description

  The present invention relates to a method and a kit for detecting Chlamydia pneumoniae contained in a specimen using an intracellular antigen.

  Bacterial infections are usually diagnosed based on the detection of causative bacteria at the site of infection or the like, or the detection of antibodies to causative bacteria in blood or body fluids. In particular, for this diagnosis, detection of the causative bacteria is important in terms of enabling rapid treatment of a patient.

  Infectious disease-causing bacteria can be detected by a culture method that identifies the causative microorganism based on its biochemical properties through isolation and culture of the causative microorganism, and a PCR method based on the gene specific to the causative microorganism. It can be broadly classified into a gene method for amplification and detection, and an immunization method for detecting the causative bacterium using a specific reaction between the antibody and the surface antigen marker of the causative bacterium. However, since the culture method and the gene method require a long time to obtain a detection result, an immunization method that can detect the causative bacteria in a short time and with high sensitivity is of value in that it leads to prompt and appropriate treatment of patients. high. Conventionally, various combinations of marker antigens and antibodies are used to detect infectious disease-causing bacteria by the immunization method depending on the bacterial species.

  Patent Document 1 distinguishes a bacterium belonging to Chlamydia pneumoniae from a bacterium of another genus by using an antibody that reacts with ribosomal protein L7 / L12 of a bacterium belonging to Chlamydia pneumoniae, which is one of the causative bacteria of respiratory tract infection. Provide a way to Non-Patent Document 1 describes the growth process of Chlamydia pneumoniae and its bacterial cell structure. According to this, Chlamydia pneumoniae forms an elementary body (EB) having an outer membrane during the growth process, and the EB outer membrane is stable to physical shocks such as osmotic pressure and ultrasonic waves and has a material permeability. Disclose that it is scarce. Further, in Non-Patent Document 2, using Chlamydia trachomatis, which is the same genus of Chlamydia as Chlamydia pneumoniae, compared the efficiency of solubilizing a 39500-molecular-weight protein constituting the outer membrane of EB for multiple types of surfactants. ing. According to this, sodium dodecyl sulfate (SDS), which is a strong anionic surfactant, solubilizes the EB outer membrane with high efficiency, but Triton X-100 which is a nonionic surfactant and amphoteric surfactant It discloses that Zweittergent TM-314 as an agent and sodium deoxycholate as an anionic surfactant have low solubilization efficiency. Similarly, Patent Document 2 discloses the detection of Chlamydia trachomatis. Here, as a method of reducing false positive and false negative results in enzyme immunoassay for chlamydia antigen, it is proposed to extract a sample into an aqueous solution containing a strong base. As a method of extracting a protein to be detected from a cell sample, Patent Document 3 discloses (a) contacting fixed cells or unfixed cells with an extraction reagent to produce an intermediate composition having a pH of at least about 10.0. And (b) contacting the intermediate composition with a neutralizing reagent to neutralize the pH of the intermediate composition and produce a protein extract. A method for producing an extract is proposed, wherein one or both of the extraction reagent and the neutralization reagent comprises a polyoxyethylene alkyl ether. It also states that proteins extracted by this method are applicable for detection by immunochromatographic assays.

WO 01/057089 (Patent No. 5331284) Japanese Unexamined Patent Publication No. Hei 3-502967 (Patent No. 2782256) JP 2011-504308 A

Chlamydia pneumoniae infectious disease for practitioners Basic and clinical (Pharmaceutical Journal), first edition published on February 25, 2002, I-3 (p26-34) Infection and Immunity, Mar. 1981, p. 1161-1176

  An object of the present invention is to detect Chlamydia pneumoniae contained in a specimen with high accuracy.

  The present inventors have found that in order to detect Chlamydia pneumoniae contained in a sample with high precision, the present inventors have found that the intracellular antigen of Chlamydia pneumoniae contained in a sample is efficiently extracted and subjected to an immunoassay, and It was concluded that it was important to suppress non-specific reactions (false positive reactions) originating from the sample in the measurement method. The present inventors have also found that a false positive reaction occurs when a sample comes into contact with an alkaline solution. Therefore, as a result of conducting various experiments and conducting intensive studies, the specimen was contacted with an alkaline solution to extract intracellular antigens, which were neutralized with a neutralizing solution containing a specific buffer and a nonionic surfactant. The present inventors have found that it is possible to detect Chlamydia pneumoniae with high accuracy while suppressing non-specific reactions derived from a specimen by detecting the same by immunochromatography, and completed the present invention.

The present invention provides the following.
[1] A method for detecting Chlamydia pneumoniae contained in a sample,
Contacting the specimen with an alkaline solution, extracting the intracellular antigens of Chlamydia pneumoniae contained in the specimen into the alkaline solution, an extraction step of obtaining a specimen extract,
Contacting the sample extract with a neutralizing solution to obtain a neutralized product;
Subjecting the neutralized product to immunochromatography to detect the intracellular antigen;
Including
The neutralization solution contains a nonionic surfactant and a buffer solution effective for suppressing false positive reactions,
Method.
[2] The method according to 1, wherein the intracellular antigen is ribosomal protein L7 / L12.
[3] The method according to 1 or 2, wherein the alkaline solution contains an anionic surfactant, and the anionic surfactant is sodium deoxycholate and / or sodium cholate.
[4] The nonionic surfactant is poly (oxyethylene) nonylphenyl ether,
Poly (oxyethylene) poly (oxypropylene) alkyl ether,
Poly (oxyethylene) alkyl ether,
The method according to any one of claims 1 to 3, which is at least one member selected from the group consisting of poly (oxyethylene) octylphenyl ether and poly (oxyethylene) sorbitan monolaurate.
[5] The method according to any one of items 1 to 4, wherein the pH of the neutralized product is 10.0 or less.
[6] The method according to any one of 1 to 5, wherein the sample is an upper respiratory sample.
[7] The method according to 6, wherein the upper respiratory tract sample is any of a pharyngeal swab, a nasopharyngeal swab, a nasal aspirate, saliva, and sputum.
[8] The method according to any one of 1 to 7, wherein the buffer effective for suppressing the false positive reaction is a tris (hydroxymethyl) aminomethane buffer and / or a phosphate buffer.
[9] A kit for detecting Chlamydia pneumoniae contained in a sample,
An alkali solution for contacting with the sample and extracting a Chlamydia pneumoniae intracellular antigen contained in the sample and obtaining a sample extract, and a neutralizing method for contacting the sample extract to obtain a neutralized product Liquid, a neutralized substance is provided, including an immunochromatography apparatus for detecting intracellular antigens,
The neutralization solution contains a nonionic surfactant and a buffer solution effective for suppressing false positive reactions,
Detection kit.
[10] The detection kit according to 9, wherein the immunochromatography apparatus contains an antibody that binds to ribosomal protein L7 / L12 of Chlamydia pneumoniae.
[11] The detection kit according to 9 or 10, wherein the alkaline solution contains an anionic surfactant, and the anionic surfactant is sodium deoxycholate and / or sodium cholate.
[12] The nonionic surfactant is poly (oxyethylene) nonylphenyl ether,
Poly (oxyethylene) poly (oxypropylene) alkyl ether,
Poly (oxyethylene) alkyl ether,
12. The detection kit according to any one of 9 to 11, which is at least one selected from the group consisting of poly (oxyethylene) octylphenyl ether and poly (oxyethylene) sorbitan monolaurate.
[13] The detection kit according to any one of items 9 to 12, wherein the neutralizing solution makes the pH of the neutralized product 10.0 or less.
[14] The detection kit according to any one of items 9 to 13, wherein the sample is an upper respiratory tract sample, and further includes a tool for collecting the sample.
[15] The detection kit according to any one of 9 to 14, wherein the buffer effective for suppressing the false positive reaction is a tris (hydroxymethyl) aminomethane buffer and / or a phosphate buffer.

  According to the method of the present invention, Chlamydia pneumoniae contained in a specimen can be accurately detected.

It is a cross section showing an example of an immunochromatography device. 1 is a substrate, 2 is a member impregnated with a labeled antibody, 3 is a membrane carrier for developing a chromatograph, 4 is a member for absorption, 5 is a member for adding a sample, and 6 is an antibody immobilized with a ribosomal protein L7 / L12 of Chlamydia pneumoniae. The captured part (determination part) is shown.

Hereinafter, the present invention will be described in more detail.
The present invention is a method for detecting Chlamydia pneumoniae contained in a sample, comprising contacting the sample with an alkaline solution, extracting intracellular antigens of Chlamydia pneumoniae contained in the sample into the alkaline solution, and extracting the sample. An extraction step of obtaining a product, a neutralization step of bringing the sample extract into contact with a neutralizing solution to obtain a neutralized product, and a detection step of subjecting the neutralized product to immunochromatography to detect the intracellular antigen. Wherein the neutralizing solution comprises a nonionic surfactant and a buffer effective for suppressing false positive reactions. In addition, "detecting" means analyzing the presence or absence or the amount of Chlamydia pneumoniae. Therefore, the case where Chlamydia pneumoniae is not detected as a result of the detection also corresponds to the implementation of the method of the present invention, as in the case where Chlamydia pneumoniae is detected.

[About Chlamydia pneumoniae]
Chlamydia pneumoniae is the causative bacterium of respiratory infections. It is considered to be one of the major causative bacteria of community-acquired pneumonia, and causes respiratory infections such as acute bronchitis, acute upper respiratory tract inflammation, sinusitis, and otitis media in addition to community-acquired pneumonia. Other fungi belonging to the genus Chlamydia include Chlamydia trachomatis and Chlamydia shittasi.
Chlamydia pneumoniae is an obligate intracellular parasitic bacterium that forms inclusion bodies in the cytoplasm of host cells and adopts three different forms of the cell structure common to the genus. One elementary body (EB) that is infectious and resistant to external stimuli, one is a reticulative body (RB) that has the ability to proliferate, and the other is their translocation. It is an intermediate form (IF) which is a bacterial body. Of these, EB is stable against physical stimuli such as osmotic pressure and ultrasonic waves.

[About samples]
The specimen used in the present invention may be any substance that can be collected from any place in a human body and may contain Chlamydia pneumoniae. It is preferably a sample collected from the upper respiratory tract, more preferably a pharyngeal swab, a nasopharyngeal swab, a nasal aspirate, saliva, sputum and the like. The method for collecting these samples is not particularly limited, and a known method can be adopted. For example, a pharyngeal swab, a nasopharyngeal swab, saliva, and sputum can be collected using a cotton swab, and a nasal aspirate can be collected using a suction catheter.
The specimen contains various substances other than Chlamydia pneumoniae to be measured. For example, the pharyngeal swab may contain tissues such as Chlamydia pneumoniae and other bacteria in addition to human-derived tissue fragments and secretions. Some of them exist in a suspended or dissolved state in a solution obtained by treating a specimen.
When subjected to measurement by immunochromatography in a state containing these, false positives may occur due to non-specific reactions.

[Extraction of intracellular components]
When Chlamydia pneumoniae is detected by immunochromatography, it is desirable to measure an antigen of Chlamydia pneumoniae, preferably an intracellular antigen. As the intracellular antigen, ribosomal protein L7 / L12 is preferable.
When the ribosomal protein L7 / L12 is to be detected, it is necessary to expose the protein by destroying the outer membrane covering the cell since the protein is present in the cell.
The inventors of the present invention have found that, by treating Chlamydia pneumoniae with an alkaline solution, the outer membrane of the basic body (EB) of Chlamydia pneumoniae can be destroyed and ribosomal protein L7 / L12, which is an intracellular component, can be extracted. Further, it has been found that, by treating with an alkaline solution containing an anionic surfactant, the extraction efficiency is improved and the detection sensitivity in an immunochromatography apparatus is improved.

[Alkaline solution and anionic surfactant]
As the alkaline solution that can be used in the present invention, for example, an aqueous solution of a hydroxide of a Group 1 element or a Group 2 element such as sodium hydroxide or potassium hydroxide can be used. The pH of the alkaline solution is preferably from 11.0 to 13.5. The concentration of the alkali metal can be 5 to 1000 mM, preferably 10 to 500 mM.

  In the present invention, an anionic surfactant can be used simultaneously with the alkaline solution. Anionic surfactants are preferred because they have the function of solubilizing cell membranes. The anionic surfactant that can be used in the present invention is not particularly limited as long as it can destroy the outer membrane of bacteria. Preferred examples include sodium N-lauroyl sarcosine, sodium cholate, and sodium deoxycholate. Can be. Among them, sodium cholate and sodium deoxycholate are more preferred.

  The anionic surfactant of the present invention is preferably added in an amount of 0.1% to 2.0%, more preferably 0.3% to 1.0%, based on the alkali solution. If the concentration is low, the expected effect may not be obtained sufficiently. If the concentration is high, the specificity (reaction between the antigen and the antibody) may be inhibited, or the nonspecific reaction may be induced or promoted. Or may be.

  The contact between the analyte and the alkaline solution containing an anionic surfactant can be performed at ambient temperature, if necessary, and, if necessary, even under cooling conditions in which deterioration of components such as proteins is less likely to occur. Good. The contact can be carried out for a time during which the intracellular antigen can be sufficiently extracted, for example, several seconds to several hours. At the time of the contact, stirring and shaking may be performed as necessary.

[Neutralizing solution, nonionic surfactant and buffer solution]
In the present invention, it was described above that ribosomal protein L7 / L12, which is an intracellular component of Chlamydia pneumoniae, contained in a specimen can be extracted using an alkaline solution containing an anionic surfactant. If this extract is used directly for immunochromatography, the antibody used for immunochromatography will be denatured and cannot be detected. Therefore, it is preferable to neutralize the pH of the extract before measurement by the immunochromatography method.

  The pH after the neutralization is not particularly limited as long as the anti-ribosomal protein L7 / L12 antibody properly functions, but according to the study of the present inventors, the pH can be set to 10.0 or less. , 9.8 or less. If the pH value exceeds 10, the antibody may be denatured. The lower limit of the pH is determined mainly by the capacity of the buffer contained in the neutralizing solution. The pH after neutralization can be, for example, 6.9 or higher, pH 7.0 or higher, or pH 7.2 or higher.

  Preferably, the neutralizing solution is also a mixture of at least a nonionic surfactant and a buffer. This is because in the immunochromatography method described later, it is effective to use a nonionic surfactant in order to improve the spreadability of the solution. On the other hand, as described above, in the present invention, a specimen is brought into contact with a neutralizing solution after the specimen is brought into contact with an alkaline solution, and the specimen analyte obtained is detected by immunochromatography. In the present invention, when a nonionic surfactant is added to an alkaline solution for the purpose of improving the solution expandability by immunochromatography, the nonionic surfactant is hydrolyzed over a long period of time. It is difficult to ensure the desired effect (improving the solution expandability). Therefore, in the present invention, it is considered that a nonionic surfactant is added to the neutralized solution, and as a result, it is possible to improve the spreadability of the solution by immunochromatography and maintain the effect over a long period of time. It has become possible.

  The nonionic surfactant that can be used by being added to the neutralization solution in the present invention may be any as long as it can improve the solution expandability by immunochromatography, and specifically, for example, poly (oxyethylene) ) Nonylphenyl ether, poly (oxyethylene) poly (oxypropylene) alkyl ether, poly (oxyethylene) alkyl ether, poly (oxyethylene) octylphenyl ether, poly (oxyethylene) sorbitan monolaurate, etc. can be used. Among them, poly (oxyethylene) poly (oxypropylene) alkyl ether is preferable.

  By the way, in the study of the present inventors, a false positive reaction occurs when a sample is brought into contact with an alkaline solution and then neutralized under a specific condition and the sample analyte is measured by immunochromatography. Was newly found. It is presumed that treatment of the specimen with an alkaline solution resulted in the generation of a component that causes a non-specific reaction with the anti-Chlamydia pneumoniae ribosomal protein L7 / L12 antibody. Such a false-positive reaction also occurred when the specimen was other than a pharyngeal swab, such as a nasopharyngeal swab, a nasal aspirate, saliva, and sputum.

  The present inventors have intensively studied a method for suppressing the false positive reaction and found that it is effective to use a neutralizing solution containing a buffer effective for suppressing the false positive reaction. By using such a neutralizing solution, components generated when the sample comes into contact with an alkaline solution containing an anionic surfactant cause a non-specific reaction with the anti-Chlamydia pneumoniae ribosomal protein L7 / L12 mouse monoclonal antibody This has made it possible to achieve high detection accuracy. "Effective in suppressing false positive reactions" means that there is an effect of preventing or reducing false positive reactions generated when a sample is treated with alkali. Those skilled in the art can select a buffer effective for suppressing false-positive reactions by performing preliminary experiments and appropriately selecting from existing buffers without trial and error. Although the act itself of selecting from existing ones is general, it is the first time confirmed by the present inventors that the specimen used in the present invention shows false positives when subjected to alkali treatment, and that false positive reaction It has also been discovered for the first time by the inventors that suppression can be achieved with a particular buffer. An example of a buffer that is effective in suppressing false positive reactions is one that contains tris (hydroxymethyl) aminomethane and / or phosphoric acid as a buffer component.

  The contact between the sample extract and the neutralizing solution with the alkali solution can be performed at ambient temperature, and if necessary, may be performed under cooling conditions in which components such as proteins are less likely to deteriorate. The contact can be carried out for a time required for neutralization, for example, several seconds to several hours. At the time of the contact, stirring and shaking may be performed as necessary.

[About immunochromatography apparatus and kit]
The method of the present invention uses an apparatus or a kit utilizing an immunochromatography method (sometimes referred to as “immunochromatography method”). The kit includes an immunochromatography apparatus, and in addition, an alkali solution for contacting with a sample, extracting a Chlamydia pneumoniae intracellular antigen contained in the sample, and obtaining a sample extract, and / or a sample extract And a neutralizing solution for obtaining a neutralized product. The alkaline solution and the neutralizing solution have been described above. The kit may further include tools for collecting the specimen, such as a swab, and / or a suction catheter.

  The immunochromatography apparatus has a judgment unit for judging the presence or absence of bacteria, on which an intracellular antigen of Chlamydia pneumoniae, preferably an antibody against ribosomal protein L7 / L12, is immobilized. . The kit or the immunochromatography apparatus may utilize a sandwich assay using an antibody that binds to a site different from the immobilized antibody (sometimes referred to as a “sandwich immunoassay”).

  More specifically, the ribosomal protein L7 / L12 of Chlamydia pneumoniae, which is an antigen, is sandwiched between an antibody that binds to a site different from the antibody immobilized on the determination part and an antibody immobilized on the determination part. It can be detected by an assay. Alternatively, detection may be performed by a competition method using an antibody that binds to a site different from the labeled antigen and the antibody immobilized on the determination unit. However, in the present invention, a sandwich assay in which the detection sensitivity is high and an antibody detection line appears positively is preferable.

  FIG. 1 shows a schematic cross-sectional view of an example of an immunochromatography apparatus. Reference numeral 1 denotes a substrate, 2 denotes a labeled antibody-impregnated member, 3 denotes a chromatographic development membrane carrier, 4 denotes an absorption member, and 5 denotes a sample addition member. Reference numeral 6 denotes a determination unit on which an antibody that reacts with the ribosomal protein L7 / L12 of Chlamydia pneumoniae is fixed. Since the antigen, which is the ribosomal protein L7 / L12 of Chlamydia pneumoniae, is captured, it is also referred to as a capture site. is there.

  The immunochromatography apparatus can be manufactured using a commercially available material by a known method. The following is a specific example thereof.

[About the substrate]
The device base material is a solid capable of forming an interface with an aqueous solution, and a solid obtained by modifying the surface thereof. As the material of the base material, the synthesis of polyethylene, polypropylene, polystyrene, acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, polyurethane, etc. in that it is easily available, stable, safe, moldable and sterilizable Polymers, natural polymers such as agarose, cellulose, nitrocellulose, cellulose acetate, chitin, chitosan, and alginate, as well as cross-linked and modified structures thereof, and inorganic materials such as hydroxyapatite, glass, alumina, and titania Materials, metals such as stainless steel, titanium, and aluminum can be used. Among them, synthetic polymers and natural polymer derivatives are preferred. As the shape of the substrate, a flat plate, a mesh, a woven fabric, a nonwoven fabric, a sponge-like structure, a three-dimensional molded body (block shape), or the like can be used, and a flat plate is particularly preferable.

[About labeled antibodies and labeled antibody-impregnated members]
The labeled antibody-impregnated member holds the labeled antibody. Examples of the label include colored particles, enzymes, radioisotopes, etc., and it is preferable to use colored particles that can be visually detected without special equipment. Examples of the colored particles include fine metal particles such as gold and platinum (sometimes referred to as colloidal gold particles and colloidal platinum particles), nonmetal particles, latex particles, and the like. It is not something to be done. The colored particles may be of any size as long as they can be transported downstream through the voids of the test piece, but preferably have a diameter of 1 nm to 10 μm. More preferably, the thickness is 5 nm to 1 μm, and still more preferably, 10 nm to 100 nm.
The material used for the labeled antibody-impregnated member is not particularly limited as long as it can perform immunochromatography, but is preferably a fiber matrix such as a cellulose derivative, filter paper, glass fiber, cloth, cotton, and more preferably glass. Fiber.

[About chromatographic carrier]
The material used for the carrier for chromatographic development is not particularly limited as long as it can perform immunochromatography, and is preferably nitrocellulose, mixed nitrocellulose ester, polyvinylidene fluoride, nylon, or the like.

[About the judgment unit]
The above-mentioned carrier for chromatograph development is provided with a judgment part, on which an antibody that binds to the ribosomal protein L7 / L12 of Chlamydia pneumoniae is immobilized. In the immobilization of the antibody on the determination portion (that is, the carrier for chromatographic development), the antibody molecule may be directly bonded to the surface of the substrate, or may be bonded via an active group. Any binding state may be used as long as the antibody molecule or active group is immobilized on the substrate surface from the substrate. Examples of the bonding state include a single bond of a covalent bond, an ionic bond, a Van der Waals bond, a hydrogen bond or a hydrophobic bond, or a combined force of a plurality of these bonds. In particular, a physical adsorption method by simple contact between the antibody solution and the substrate surface is simple and is particularly preferably used in the present invention. Further, a method of immobilizing the antibody on the substrate surface by washing or drying the substrate surface after the adsorption of the antibody, or evaporating water after applying the antibody solution to the substrate surface, is also extremely suitably used in the present invention. be able to.

After the Chlamydia pneumoniae ribosomal protein L7 / L12 contained in the sample is developed on the chromatographic carrier while being bound to the above-mentioned labeled antibody, it is bound at a site different from the labeled antibody in the determination part. By forming a sandwich between an antibody and an antigen, the ribosomal proteins L7 / L12 of Chlamydia pneumoniae can be detected (sandwich assay).
The antibody immobilized on the determination part and the labeled antibody are both antibodies that react with the ribosomal protein L7 / L12 of Chlamydia pneumoniae, and are antibodies that can simultaneously bind to one such protein. . Therefore, it is preferable that the epitope to which one antibody binds is different from the epitope to which the other antibody binds.
When a labeled antigen is retained in the determination unit, a specific substance can be detected by a competition method (competition assay method). In the present invention, a sandwich assay in which the detection sensitivity is high and an antibody detection line appears positively is more preferable.

[Method for producing antibody]
The antibody used in the present invention may be either a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody.
An antibody against the ribosomal protein L7 / L12 antigen can be prepared by the method described in WO 00/06603 (Patent Document 1). The antibody can be prepared using a full-length protein of ribosomal protein L7 / L12 or a partial peptide thereof as an antigen, but is preferably prepared using the full-length protein as an antigen. The partial peptide or full-length protein can be inoculated to an animal as it is or after cross-linking with a carrier protein, if necessary, together with an adjuvant, and its serum can be collected to obtain an antibody containing an antibody (polyclonal antibody) that recognizes the L7 / L12 ribosomal protein. Serum can be obtained. Further, the antibody can be purified from the antiserum and used. The animals to be inoculated are sheep, horses, goats, rabbits, mice, rats, and the like. Particularly, sheep, rabbits, and the like are preferable for preparing polyclonal antibodies. As the antibody, it is more preferable to apply a monoclonal antibody obtained by a known method for producing hybridoma cells, but in this case, a mouse is preferable. As the monoclonal antibody, a monoclonal antibody that reacts with ribosomal protein L7 / L12 of a specific bacterium and does not react with a bacterium belonging to a different species from the specific bacterium or a ribosome protein L7 / L12 of a bacterium belonging to a different genus is screened. This makes it possible to use the diagnosis to determine whether or not the bacterium is infected by the bacterium.

In the present invention, Chlamydia pneumoniae is detected. The antibody used herein is an antibody that reacts with the Chlamydia pneumoniae ribosomal protein L7 / L12 antigen in a species-specific or genus-selective manner. Here, “species-specific” means that it reacts only with a certain kind of bacterium belonging to the genus Chlamydia (eg, Chlamydia pneumoniae) and belongs to a bacterium belonging to another genus other than the genus Chlamydia, It means that it does not react with bacteria of a species other than the specific species. The term "selective for the genus" means that it reacts only with some or all bacteria belonging to the genus Chlamydia and does not react with bacteria belonging to other genera other than the genus Chlamydia.
The antibody used in this embodiment can be prepared using the full length of Chlamydia pneumoniae ribosomal protein L7 / L12 or a partial peptide thereof as an antigen. The amino acid sequence of the Chlamydia pneumoniae ribosomal protein L7 / L12 is known and is described, for example, in SEQ ID NO: 2 of WO 01/057089.

The antibody against the Chlamydia pneumoniae ribosomal protein L7 / L12 antigen used in this embodiment can be obtained by the method described in a) or b) below, or other similar methods, but is not limited to these methods. Not something.
a) Since the gene sequence encoding the amino acid sequence of the chlamydia pneumoniae ribosomal protein L7 / L12 is known, the amino acid sequence of the protein in a microorganism whose amino acid sequence encoding the amino acid sequence of another ribosomal protein L7 / L12 is known A target antibody can be obtained by synthesizing a peptide fragment of about 5 to 30 amino acids in a region having little similarity to the above and preparing a polyclonal antibody or a monoclonal antibody using the peptide fragment as an immunogen.

b) The full-length sequence of the gene can be obtained by using a conventional gene manipulation technique such as gene amplification by PCR using a DNA sequence at both ends of the gene as a probe and hybridization using a homologous partial sequence as a template probe. Can be obtained. After that, a fusion gene with another protein gene is constructed, and the relevant fusion gene is inserted into a host by a known gene transfer method using Escherichia coli or the like as a host, expressed in a large amount, and then antibody affinity to the protein used as the fusion protein is obtained. The target protein antigen can be obtained by purifying the expressed protein by a column method or the like.
In this case, since the full-length protein of Chlamydia pneumoniae ribosomal protein L7 / L12 serves as an antigen, obtaining an antibody against an amino acid portion conserved between different microorganisms does not meet the purpose of the present invention. Therefore, for the antigen obtained by this method, a hybridoma producing a monoclonal antibody is obtained by a known method, and a clone producing an antibody that reacts only with the ribosomal protein L7 / L12 of Chlamydia pneumoniae is selected. Can be obtained.

  When the degree of purification of the ribosomal protein L7 / L12 serving as an antigen is insufficient, the protein is purified by a known purification technique such as ion exchange chromatography, hydrophobic chromatography, gel filtration and the like, and then described in WO 01/057089. The elution fraction of Chlamydia pneumoniae ribosomal protein L7 / L12 can be identified by a method such as Western blotting using an antibody that has already been prepared, and a protein with a higher degree of purification can be obtained. A hybridoma or polyclonal antibody is obtained by a known method based on the obtained purified Chlamydia pneumoniae ribosomal protein L7 / L12 antigen, and similarly, a hybridoma or polyclonal antibody specifically reacting with the target microorganism is selected. The desired antibody can be obtained.

[Example 1: Production of immunochromatography apparatus]
(Preparation of antibody)
An antibody against the chlamydia pneumoniae ribosomal protein L7 / L12 can be prepared by the method described in WO 01/057089. Specifically, for example, Escherichia coli transformed with an expression vector incorporating a DNA encoding the entire or a part of the amino acid sequence of ribosomal protein L7 / L12 of Chlamydia pneumoniae is cultured in an LB medium or the like, and the affinity column is used. To purify it as a fusion protein using the tag sequence derived from the expression vector. Animals are inoculated with this partial peptide or full-length protein together with an adjuvant as needed. The animals to be inoculated are sheep, horses, goats, rabbits, mice, rats, and the like. In particular, when monoclonal antibodies are obtained, mice are preferable. Mouse spleen cells are removed and fused with myeloma cells to produce antibody-producing hybridomas. Monoclonal antibodies produced from the hybridomas are selected by ELISA or the like to obtain monoclonal antibodies that specifically react with Chlamydia pneumoniae.

(Preparation of chromatographic membrane support)
Antibodies to the Chlamydia pneumoniae ribosomal protein L7 / L12 and trehalose were diluted in 50 mM sodium phosphate buffer to 2.0 mg / ml and 3% (v / v), respectively. This was applied to a commercially available nitrocellulose membrane cut to a width of 2.5 cm and a length of 30 cm at a liquid volume of 1 μl per cm, and then dried to obtain a chromatographic development membrane carrier.

(Preparation of colloidal gold labeled antibody and labeled antibody impregnated member)
A 1/10 volume of 0.1 M sensitized Buffer and an antibody against Chlamydia pneumoniae ribosomal protein L7 / L12 were added to a gold colloid solution (particle size: 60 nm), mixed, and allowed to stand at room temperature for 30 minutes to remove the antibody. After binding to the particle surface, 1% bovine serum albumin aqueous solution was added so that the final concentration in the colloidal gold solution was 0.1%, and blocking was performed to prepare a colloidal gold-labeled anti-ribosomal protein L7 / L12 antibody solution.
This liquid was impregnated into a commercially available glass fiber sheet, and then dried overnight in a drying oven at a humidity of 0 to 60% to obtain a labeled antibody infiltrating member.

(Assembly of immunochromatography equipment)
In addition to the chromatographic development membrane carrier and the gold colloid-labeled antibody-impregnated member prepared by the above-described procedure, a cotton cloth as a sample addition member and a filter paper as an absorption member were further prepared. Then, after bonding these members to the base material, the members were cut into a width of 6 mm, and an immunochromatography apparatus similar to that of FIG. 1 was produced.

[Example 2: Preparation of test bacterial solution of Chlamydia pneumoniae]
HEp-2 cells were cultivated in a monolayer on a 24-well plate. Chlamydia pneumoniae TW183 strain was inoculated into the culture, a culture solution was added thereto, and the cells were cultured at 36 ° C. in a 5% CO ₂ atmosphere for 3 days. Each cell was peeled off using a rubber policeman, a culture solution containing Chlamydia pneumoniae cells was recovered, and Chlamydia pneumoniae cells were purified by density gradient centrifugation using urografin injection 76% (Bayer Yakuhin).

[Example 3: Preparation of alkaline solution]
Sodium cholate (Wako Pure Chemical), sodium deoxycholate (Wako Pure Chemical), and sodium N-lauroyl sarcosine (Nacalai Tesque) as anionic surfactants were added to an aqueous sodium hydroxide solution (Wako Pure Chemical) at a final concentration of 0.1. It was added so as to be 5% to prepare the following six kinds of alkaline solutions.
Alkaline solution (1): 50 mM sodium hydroxide (pH 12.5)
Alkaline solution (2): 100 mM sodium hydroxide (pH 13.0)
Alkaline solution (3): 50 mM sodium hydroxide, 0.5% sodium cholate (pH 12.5)
Alkaline solution (4): 100 mM sodium hydroxide, 0.5% sodium cholate (pH 13.0)
Alkaline solution (5): 50 mM sodium hydroxide, 0.5% sodium deoxycholate (pH 12.5)
Alkaline solution (6): 50 mM sodium hydroxide, 0.5% N-lauroyl sarcosine sodium (pH 12.5)

[Example 4: Preparation 1 of neutralization solution]
34.84 g of dipotassium hydrogen phosphate (Wako Pure Chemical Industries) was dissolved in distilled water, and the volume was increased to 400 ml to obtain a 0.5 M dipotassium hydrogen phosphate aqueous solution. Further, 17.01 g of potassium dihydrogen phosphate (Wako Pure Chemical) was dissolved in distilled water, and the volume was increased to 250 ml to obtain a 0.5 M aqueous solution of potassium dihydrogen phosphate. These were mixed to prepare a 0.5 M potassium phosphate buffer (pH 7.0).
In a beaker, 0.58 g of sodium chloride (Wako Pure Chemical), 0.25 g of bovine serum albumin (SIGMA), and 0.025 g of sodium azide (Wako Pure Chemical) are each weighed, and the above 0.5 M potassium phosphate buffer ( (pH 7.0) and made up to 50 ml (neutralization solution (1)).
In addition, Pelletex-1224 (Nikko Chemicals), PBC-44 (Nikko Chemicals), BrijS100 (SIGMA), Brij35 (SIGMA), TritonX-405 (SIGMA), TritonX- as nonionic surfactants in the neutralizing solution (1). 100 (SIGMA), Triton X-114 (SIGMA), and Tween 20 (BioRad) were added to a final concentration of 0.75% (neutralization solutions (2) to (9)).

The final composition is as follows:
Neutralizing solution (1): 0.2 M NaCl, 0.5% BSA, 0.05% NaN3, 0.5 M phosphoric acid K / pH 6.8)
Neutralizing solution (2): 0.2M NaCl, 0.5% BSA, 0.75% Pelletex-1224, 0.05% NaN3, 0.5M phosphoric acid K / pH 6.8
Neutralizing solution (3): 0.2M NaCl, 0.5% BSA, 0.75% PBC-44, 0.05% NaN3, 0.5M phosphoric acid K / pH 6.8
Neutralizing solution (4): 0.2M NaCl, 0.5% BSA, 0.75% BrijS100, 0.05% NaN3, 0.5M phosphoric acid K / pH 6.8
Neutralizing solution (5): 0.2 M NaCl, 0.5% BSA, 0.75% Brij35, 0.05% NaN3, 0.5 M phosphoric acid K / pH 6.8
Neutralizing solution (6): 0.2M NaCl, 0.5% BSA, 0.75% Triton X-405, 0.05% NaN3, 0.5M phosphoric acid K / pH 6.8
Neutralizing solution (7): 0.2M NaCl, 0.5% BSA, 0.75% Triton X-100, 0.05% NaN3, 0.5M phosphoric acid K / pH 6.8
Neutralizing solution (8): 0.2M NaCl, 0.5% BSA, 0.75% Triton X-114, 0.05% NaN3, 0.5M phosphoric acid K / pH 6.8
Neutralizing solution (9): 0.2 M NaCl, 0.5% BSA, 0.75% Tween 20, 0.05% NaN3, 0.5 M phosphoric acid K / pH 6.8

[Example 5: Preparation 2 of neutralization solution]
In a beaker, weigh 0.58 g of sodium chloride (Wako Pure Chemical), 0.25 g of bovine serum albumin (SIGMA), and 0.025 g of sodium azide (Wako Pure Chemical), and further 3.03 g of tris (hydroxymethyl) aminomethane Was added, and PBC-44 (Nikko Chemicals) was added to a final concentration of 1.0%. Hydrochloric acid (Wako Pure Chemical) was added dropwise to adjust the pH to 6.6, and then distilled water was added to make up to 50 ml. (Neutralizing solution (10)).
PBC-44 (Nikko Chemicals) was not added in the neutralization solution (10), and phosphoric acid (Tokyo Kasei) was added dropwise instead of hydrochloric acid to adjust the pH to 6.6, and then the volume was made up to 50 ml with distilled water. (Neutralizing solution (11)). Further, PBC-44 (Nikko Chemicals) and Triton X-405 (SIGMA) were added to the neutralized solution (11) so that the final concentration was 1.0% (neutralized solutions (12) and (13)).

The final composition is as follows:
Neutralizing solution (10): 0.2M NaCl, 0.5% BSA, 0.05% NaN3, 0.3M Tris (hydroxymethyl) aminomethane-hydrochloric acid / pH 6.6
Neutralizing solution (11): 0.2 M NaCl, 0.5% BSA, 0.05% NaN3, 0.3 M Tris (hydroxymethyl) aminomethane-phosphoric acid / pH 6.6
Neutralizing solution (12): 0.2M NaCl, 0.5% BSA, 0.05% NaN3, 1.0% PBC-44, 0.3M Tris (hydroxymethyl) aminomethane-phosphoric acid / pH 6.6
Neutralizing solution (13): 0.2 M NaCl, 0.5% BSA, 0.05% NaN3, 1.0% Triton X-405, 0.3 M tris (hydroxymethyl) aminomethane-phosphate / pH 6.6

[Example 6: Preparation 3 of neutralization solution]
In a beaker, 0.58 g of sodium chloride (Wako Pure Chemical), 0.25 g of bovine serum albumin (SIGMA), 0.025 g of sodium azide (Wako Pure Chemical), and 0.5 g of PBC-44 (Nikko Chemicals) were weighed. . Here, as buffer components, 2.38 g of HEPES (Dojindo), 1.55 g of boric acid (Wako Pure Chemical), 2.04 g of Bicine (Dojindo), 2.67 g of BES (Dojindo), 2.87 g of TES (Dojindo), Any of 3.24 g of TAPSO (Dojindo) was added, and the volume was made up to 50 ml with distilled water (neutralization solutions (14) to (19)).

The final composition of each neutralizing solution is as follows.
Neutralizing solution (14): 0.2M NaCl, 0.5% BSA, 1.0% PBC-44, 0.05% NaN3, 0.2M HEPES
Neutralizing solution (15): 0.2M NaCl, 0.5% BSA, 1.0% PBC-44, 0.05% NaN3, 0.25M boric acid neutralizing solution (16): 0.2M NaCl, 0 0.5% BSA, 1.0% PBC-44, 0.05% NaN3, 0.25 MBine
Neutralizing solution (17): 0.2M NaCl, 0.5% BSA, 1.0% PBC-44, 0.05% NaN3, 0.25M BES
Neutralizing solution (18): 0.2M NaCl, 0.5% BSA, 1.0% PBC-44, 0.05% NaN3, 0.25M TES
Neutralizing solution (19): 0.2M NaCl, 0.5% BSA, 1.0% PBC-44, 0.05% NaN3, 0.25M TAPSO

[Example 7: Preparation of neutralization solution 4]
13.93 g of dipotassium hydrogen phosphate (Wako Pure Chemical Industries, Ltd.) was dissolved in distilled water and made up to 400 ml to obtain a 0.2 M dipotassium hydrogen phosphate aqueous solution. Further, 6.80 g of potassium dihydrogen phosphate (Wako Pure Chemical Industries, Ltd.) was dissolved in distilled water, and the volume was increased to 250 ml to obtain a 0.2 M potassium dihydrogen phosphate aqueous solution. These were mixed to prepare a 0.2 M potassium phosphate buffer (pH 7.0).
In a beaker, 0.58 g of sodium chloride (Wako Pure Chemical), 0.25 g of bovine serum albumin (SIGMA), and 0.025 g of sodium azide (Wako Pure Chemical) are each weighed, and the above 0.2 M potassium phosphate buffer ( (neutralized solution (20)).

The final composition is as follows:
Neutralizing solution (20): 0.2M NaCl, 0.5% BSA, 0.05% NaN3, 0.75% BrijS100, 0.2M phosphoric acid K / pH 7.0

[Example 8: Effect of anionic surfactant]
10 μl of the Chlamydia pneumoniae cell suspension prepared in Example 2 was added to 300 μl of the alkaline solutions (1) to (6) prepared in Example 3, followed by 200 μl of the neutralized solution (12) prepared in Example 5. As a positive sample. A negative sample using PBS (Wako Pure Chemical) instead of the Chlamydia pneumoniae body fluid was used as a negative sample.
140 μl of the positive sample and the negative sample were dropped on the immunochromatography apparatus prepared in Example 1, and visually judged 15 minutes later. The results are shown below.

  Compared with sodium hydroxide alone (alkali solutions (1) and (2)), those containing sodium cholate or sodium deoxycholate as anionic surfactants (alkali solutions (3), (4), ( In 5)), a positive sample was visually determined to be strongly positive, and a negative sample was visually determined to be negative. That is, the detection sensitivity in the immunochromatography apparatus was improved.

[Example 9: Effect of adding nonionic surfactant to buffer]
10 μl of the Chlamydia pneumoniae cell suspension prepared in Example 2 was added to 300 μl of the alkaline solution (3) prepared in Example 3, followed by neutralization solutions (1) to (9) prepared in Examples 4 and 5. ) And 200 μl of any of (11) to (13) were added to obtain a positive sample. A negative sample using PBS (Wako Pure Chemical) instead of the Chlamydia pneumoniae body fluid was used as a negative sample.
140 μl of the positive sample and the negative sample were dropped on the immunochromatography apparatus prepared in Example 1, and visually judged 15 minutes later. The results are shown below.

When a neutralizing solution containing no nonionic surfactant (neutralizing solutions (1) and (11)) was used, the spreadability of the solution in the immunochromatography apparatus was extremely poor, and as a result, a positive sample was measured. In any case, it was visually judged negative.
On the other hand, when a neutralizing solution containing a nonionic surfactant (neutralizing solution (2) to (9), (12), (13)) is used, the spreadability of the solution in the immunochromatography apparatus is good. Yes, a positive sample was visually determined to be positive, and a negative sample was visually determined to be negative.

[Example 10: Generation of false positives by treating a sample with an alkaline solution and a neutralizing solution, and effect of suppressing false positives by a buffer constituting the neutralizing solution]
(Sample collection)
The posterior wall of the human pharynx was rubbed with a sterile cotton swab (Heiwa Medic, for pharynx) and used as a specimen.

(Preparation of positive and negative samples for measurement)
10 μl of the Chlamydia pneumoniae cell suspension prepared in Example 2 was added to 300 μl of the alkaline solution (3) prepared in Example 3, followed by neutralization (10%) prepared in Examples 4, 5 and 6. ), (12), (3), (4), (14), (15), (16), (17), (18), and (19) were added to 200 µl to prepare positive samples. A negative sample using PBS (Wako Pure Chemical) instead of the Chlamydia pneumoniae body fluid was used as a negative sample.

(Preparation of sample containing sample)
{Step 1: Step of neutralizing the sample after treating it with an alkaline solution}
300 μl of the alkaline solution (3) (50 mM sodium hydroxide, 0.5% sodium cholate (pH 12.5)) prepared in Example 3 was placed in a low-density polyethylene tube, and a specimen was collected therein. A cotton swab was inserted and rubbed well from the outside of the tube 5 to 20 times. Here, neutralized solutions (10), (12), (3), (4), (14), (15), (16) and (17) prepared in Examples 4, 5 and 6 were prepared. After adding 200 μl of any of (18) and (19), the swab was taken out. The obtained solution was used as a sample of Step 1.

{Step 2: Step of treating the specimen with a mixed solution of an alkaline solution and a neutralizing solution}
300 μl of the alkaline solution (3) (50 mM sodium hydroxide, 0.5% sodium cholate (pH 12.5)) prepared in Example 3 was placed in a tube made of low-density polyethylene. Neutralized solutions (10), (12), (3), (4), (14), (15), (16), (17), (18), (19) prepared in Examples 5 and 6 ) Was added in an amount of 200 μl. The swab from which the specimen was collected was inserted into the tube, and the swab was taken out from the outside of the tube after rubbing well 5 to 20 times. The obtained solution was used as a sample of Step 2.

(Measurement with immunochromatography equipment)
140 μl of the sample was dropped on the immunochromatography apparatus, and the judgment was visually made after 15 minutes. The results are shown below.

  In all of the neutralizing solutions (10), (12), (3), (4), (14), (15), (16), (17), (18), and (19), positive samples were visually observed. Was determined to be positive, and the negative sample was visually determined to be negative.

When neutralizing solutions (14) to (19) (neutralizing solutions containing any of HEPES, boric acid, Bicine, BES, TES, and TAPSO as buffer components) were used, the sample was subjected to {step 1}. When treated, all neutralization solutions generated false positives with high frequency. On the other hand, when the sample was treated in {Step 2}, no false positives occurred in any of the neutralized solutions. From this, it is presumed that a component which causes a non-specific reaction with the anti-Chlamydia pneumoniae ribosomal protein L7 / L12 mouse monoclonal antibody is generated by first treating the sample with an alkaline solution (so-called {Step 1}). You.
On the other hand, when a neutralizing solution (10), (12), (3), (4) (a neutralizing solution containing tris (hydroxymethyl) aminomethane and / or phosphoric acid as a buffer component) is used, When the sample was treated in {Step 2}, no false positives occurred in any of the neutralized solutions, which is the same result as in the neutralized solutions (14) to (19) described above. , No false positives occurred in any of the neutralized solutions, and results different from those of the neutralized solutions (14) to (19) were obtained.
That is, when the neutralizing solutions (10), (12), (3) and (4) were used, the effects of components causing a non-specific reaction with the anti-Chlamydia pneumoniae ribosomal protein L7 / L12 mouse monoclonal antibody were suppressed. it can.

Example 11: False Positives Occurred by Treating a Sample with an Alkaline Solution and Neutralizing Solution, and Suppressing Effect of False Positives by Buffer Constituting Neutralizing Solution No. 2
(Sample collection)
The posterior wall of the human pharynx was rubbed with a sterile cotton swab (Heiwa Medic, for pharynx) and used as a specimen.

(Preparation of sample containing sample)
{Step 1: Step of neutralizing the sample after treating it with an alkaline solution}
300 μl of the alkaline solution (1) (50 mM sodium hydroxide (pH 12.5)) prepared in Example 3 was put into a low-density polyethylene tube, and a cotton swab from which a sample was collected was inserted into the tube. Kneaded well 5-20 times. After adding 200 μl of any of the neutralizing solutions (10), (12), (3), (4) and (14) prepared in Examples 4, 5 and 6, the cotton swab was taken out. Was.

  After treating the sample with the alkaline solution (1), neutralization solutions (10), (12), (3), and (4) (tris (hydroxymethyl) aminomethane and / or phosphoric acid In the case of neutralization with a neutralizing solution containing no false positives, no false positives occurred. On the other hand, when neutralized with the neutralizing solution (14), false positives occurred.

Looking at the case where the neutralizing solution (14) was used as the neutralizing solution in step 1 of Example 11 and Example 10, false positives occurred in both cases. The difference between the two is that the specimen comes in contact with the alkaline solution (1) (50 mM NaOH (pH 12.5) containing no anionic surfactant) or the alkaline solution (3) (anionic surfactant Or 50 mM NaOH (pH 12.5) containing sodium cholate as From these results, a component that causes a non-specific reaction with the anti-Chlamydia pneumoniae ribosomal protein L7 / L12 mouse monoclonal antibody when the sample first comes in contact with the alkaline solution is generated. This phenomenon is related to the presence or absence of an anionic surfactant. It is presumed that this would occur without.
When neutralized with neutralizing solution (10), (12), (3), (4) (neutralizing solution containing tris (hydroxymethyl) aminomethane and / or phosphoric acid as a buffer component) The effect of a component that causes a non-specific reaction with an antibody can be suppressed.

[Example 12: pH after neutralization]
(Preparation of positive and negative samples for measurement)
10 μl of the Chlamydia pneumoniae cell suspension prepared in Example 2 was added to 300 μl of the alkaline solution (3) prepared in Example 3, followed by the neutralized solution (4) prepared in Examples 5, 6 and 7. ), (12), (13), and (20) were added as 200 μl to obtain a positive sample. A negative sample using PBS (Wako Pure Chemical) instead of the Chlamydia pneumoniae body fluid was used as a negative sample.

(Measurement of pH after addition of neutralizing solution and measurement with immunochromatography apparatus)
The pH of the negative sample prepared by the above method was measured, and the measured value was taken as the pH after neutralization.
In addition, 140 μl of the positive sample and the negative sample were dropped on the immunochromatography apparatus, and the judgment was visually made after 15 minutes. The results are shown below.

  When the pH after neutralization was pH 10.8, a positive sample was judged visually negative. This is considered to be because the antigen-antibody reaction was inhibited due to the high pH condition. On the other hand, when the pH after the neutralization was lower than 9.8, the positive sample was visually determined to be positive under any conditions.

[Example 13: Evaluation using a sample collected from a patient suspected of having Chlamydia pneumoniae infection]
(Sample collection)
Specimens were collected from patients suspected of having Chlamydia pneumoniae infection. Pharyngeal swabs were obtained by rubbing the patient's posterior pharyngeal wall with a sterile cotton swab. The nasopharyngeal swab was obtained by inserting a sterile cotton swab from the nasal cavity of the patient and rotating the swab when reaching the interior of the nasal cavity. The nasal aspirate was collected by inserting a tracheal catheter with a trap through the nasal cavity of the patient and aspirating when it reached the interior of the nasal cavity. The aspirate collected in the trap was wiped off with a sterile cotton swab, and this was used as a nasal aspirate sample.

(Preparation of sample for measurement)
300 μl of the alkali solution (3) prepared in Example 3 was put into a low-density polyethylene tube, and a cotton swab from which a sample was collected was inserted into the tube, and the tube was thoroughly rubbed 5 to 20 times from the outside of the tube. After 200 µl of the neutralizing solution (12) prepared in Example 5 was added thereto, the cotton swab was taken out. The obtained solution was used as a sample.

(Measurement with immunochromatography equipment)
A dropping nozzle equipped with a filter paper filter was attached to the low-density polyethylene tube containing the sample, and 4 drops (approximately 140 μl in volume) were dropped into the immunochromatography apparatus. The judgment was made visually after 15 minutes.

(PCR using residual solution of sample)
DNA was extracted from the residual solution of the sample, and PCR was performed using primers for 16S rRNA of Chlamydia pneumoniae. The procedure is shown below.
A commercially available kit (QIAGEN: DNAminiKIT) was used for DNA extraction. 100 μl of the sample was mixed with protease K and bufferAL included in the kit, heated at 56 ° C. for 10 minutes, and then ethanol was further added. This was applied to a spin column included in the kit, and after centrifugal adsorption, washed with BufferAW1 and BufferAW2, 100 μl of BufferEL was applied and centrifuged to obtain a DNA extract.
PCR was performed using the obtained DNA extract as a template. PCR conditions are described in Journal of Molecular Diagnostics, Vol. 6, No. 2, May 2004.

(Measurement result)
The results of the visual judgment using the immunochromatography apparatus and the results of the PCR are shown below.

  Regarding the pharyngeal swabs, nasopharyngeal swabs, and nasal aspirates collected from patients suspected of having Chlamydia pneumoniae infection, any of the kits that tested positive by PCR were also positive in the kit. In addition, the kit determination was negative for the sample determined negative by PCR.

  INDUSTRIAL APPLICABILITY The present invention can be used for diagnosis of bacterial infections caused by Chlamydia pneumoniae.

Claims (15)

A method for detecting Chlamydia pneumoniae contained in a sample,
Contacting the specimen with an alkaline solution, extracting the intracellular antigens of Chlamydia pneumoniae contained in the specimen into the alkaline solution, an extraction step of obtaining a specimen extract,
Contacting the sample extract with a neutralizing solution to obtain a neutralized product;
Subjecting the neutralized product to immunochromatography to detect the intracellular antigen;
Including
The neutralization solution contains a nonionic surfactant and a buffer solution effective for suppressing false positive reactions,
Method. The method according to claim 1, wherein the intracellular antigen is a ribosomal protein L7 / L12. The method according to claim 1 or 2, wherein the alkaline solution comprises an anionic surfactant, wherein the anionic surfactant is sodium deoxycholate and / or sodium cholate. The nonionic surfactant is poly (oxyethylene) nonylphenyl ether;
Poly (oxyethylene) poly (oxypropylene) alkyl ether,
Poly (oxyethylene) alkyl ether,
The method according to any one of claims 1 to 3, wherein the method is at least one selected from the group consisting of poly (oxyethylene) octylphenyl ether and poly (oxyethylene) sorbitan monolaurate. The method according to any one of claims 1 to 4, wherein the pH of the neutralized product is 10.0 or less. The method according to claim 1, wherein the specimen is an upper respiratory tract specimen. The method according to claim 6, wherein the upper respiratory tract specimen is any of a pharyngeal swab, a nasopharyngeal swab, a nasal aspirate, saliva, and sputum. The method according to any one of claims 1 to 7, wherein the buffer effective for suppressing the false positive reaction is a tris (hydroxymethyl) aminomethane buffer and / or a phosphate buffer. A kit for detecting Chlamydia pneumoniae contained in a sample,
An alkali solution for contacting with the sample and extracting a Chlamydia pneumoniae intracellular antigen contained in the sample and obtaining a sample extract, and a neutralizing method for contacting the sample extract to obtain a neutralized product Liquid, a neutralized substance is provided, including an immunochromatography apparatus for detecting intracellular antigens,
The neutralization solution contains a nonionic surfactant and a buffer solution effective for suppressing false positive reactions,
Detection kit. 10. The detection kit according to claim 9, wherein the immunochromatography apparatus comprises an antibody that binds to Chlamydia pneumoniae ribosomal protein L7 / L12. The detection kit according to claim 9 or 10, wherein the alkaline solution contains an anionic surfactant, and the anionic surfactant is sodium deoxycholate and / or sodium cholate. The nonionic surfactant is poly (oxyethylene) nonylphenyl ether;
Poly (oxyethylene) poly (oxypropylene) alkyl ether,
Poly (oxyethylene) alkyl ether,
The detection kit according to any one of claims 9 to 11, wherein the detection kit is at least one selected from the group consisting of poly (oxyethylene) octylphenyl ether and poly (oxyethylene) sorbitan monolaurate.   The detection kit according to any one of claims 9 to 12, wherein the neutralizing solution adjusts the pH of the neutralized product to 10.0 or less. The detection kit according to any one of claims 9 to 13, wherein the specimen is an upper respiratory tract specimen, and further includes a tool for collecting the specimen. The detection kit according to any one of claims 9 to 14, wherein the buffer effective in suppressing the false positive reaction is a tris (hydroxymethyl) aminomethane buffer and / or a phosphate buffer.

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