WO2016194986A1

WO2016194986A1 - Immunochromatographic analyzer for mycoplasma pneumoniae detection

Info

Publication number: WO2016194986A1
Application number: PCT/JP2016/066310
Authority: WO
Inventors: 鈴木　啓太; 久彦岩本
Original assignee: 田中貴金属工業株式会社
Priority date: 2015-06-01
Filing date: 2016-06-01
Publication date: 2016-12-08

Abstract

The purpose of the present invention is to provide an immunochromatographic analyzer whereby Mycoplasma pneumoniae can be quickly and easily detected at a high sensitivity so that mycoplasma pneumonia can be more surely and quickly diagnosed. The present invention relates to an immunochromatographic analyzer for detecting Mycoplasma pneumoniae, said immunochromatographic analyzer comprising a sample addition part, a marker substance-holding part, a chromatography medium part including a detection section, and an absorption part, wherein the marker substance-holding part and the detection section contain an antibody that strongly recognizes domain III of Mycoplasma pneumoniae protein P30 comprising the amino acid sequence represented by SEQ ID NO: 2.

Description

Immunochromatographic analyzer for detection of Mycoplasma pneumoniae

The present invention relates to an immunochromatography analyzer for detecting Mycoplasma pneumoniae, an immunochromatography kit, and a detection method thereof.

Mycoplasma pneumonia is a respiratory infection mainly caused by Mycoplasma pneumoniae. Mycoplasma pneumoniae is a typical causative agent of atypical pneumonia. The age of mycoplasma pneumonia patients is mainly in early childhood, schoolchildhood, and adolescence (5 to 35 years). The initial symptoms are cold syndrome-like symptoms, so-called cold-like symptoms, but cough over time. May continue and may continue for about a month after the fever.

Various methods have been used for diagnosis of mycoplasma pneumonia. For example, a detection method using an antibody against Mycoplasma pneumoniae is used. In mycoplasma pneumonia, there is a high need in the medical field to determine the presence or absence of Mycoplasma pneumoniae infection at the early stage of infection for the selection of antibiotics, but by applying it to an immunochromatographic analysis method that is easy to operate, mycoplasma can be quickly and easily performed.・ Pneumoniae can be detected and mycoplasma pneumonia can be diagnosed.

The simplest structure of an immunochromatographic analyzer used for immunochromatographic analysis is a structure in which a sample addition part, a labeling substance holding part, a chromatographic medium part having a detection part, and an absorption part are connected to each other.

For example, Patent Document 1 discloses an immunochromatographic analyzer using a monoclonal antibody specific for the P1 protein of Mycoplasma pneumoniae.

Further, Patent Document 2 discloses an immunochromatographic analyzer using an antibody specific for Mycoplasma pneumoniae ribosomal protein Ribosomal Protein L7 / L12.

Japanese Unexamined Patent Publication No. 2013-72663 Japanese Unexamined Patent Publication No. 2014-167439

However, the conventional diagnostic method using an immunochromatography analyzer using an antibody against Mycoplasma pneumoniae is not sufficiently sensitive to Mycoplasma pneumoniae and there are many non-specific reactions. As it was not enough.

Therefore, as a result of intensive studies, the present inventors have focused on the P30 protein of Mycoplasma pneumoniae as a target protein that can be detected with higher sensitivity than the P1 protein of Mycoplasma pneumoniae, and in particular, recognized a specific site of the P30 protein. It was found that the antibody to detect Mycoplasma pneumoniae with high sensitivity.

Further, the present inventors have found that mycoplasma pneumoniae infection can be detected quickly and easily with high sensitivity according to an immunochromatography analyzer using an antibody that binds to a specific site of the P30 protein of Mycoplasma pneumoniae, and the present invention has been achieved.

Therefore, the present invention is as follows.
1. An immunochromatography analyzer for detecting Mycoplasma pneumoniae, comprising a sample addition part, a labeling substance holding part, a chromatographic medium part having a detection part and an absorption part,
The immunochromatographic analyzer, wherein the labeling substance holding part and the detection part contain an antibody that strongly recognizes the domain III of the P30 protein of Mycoplasma pneumoniae consisting of the amino acid sequence of SEQ ID NO: 2.
2. 2. The immunochromatographic analyzer according to 1 above, wherein the antibody recognizes the amino acid sequence of SEQ ID NO: 3 in the domain III.
3. 3. The immunochromatographic analysis according to 1 or 2 above, wherein the labeling substance contained in the labeling substance holding part is gold particles, and the labeling substance holding part contains 0.25 to 0.7 μg / cm ^{2 of the} gold particles. apparatus.
4). An immunochromatography analysis kit comprising the immunochromatography analyzer according to any one of 1 to 3 above and a sample diluent for diluting and developing the sample.
5. 5. The immunochromatographic analysis kit according to 4 above, wherein the specimen diluent contains at least one nonionic surfactant.
6). 6. The immunochromatographic analysis kit according to 5 above, wherein 50% or more of the nonionic surfactant contained in the specimen diluent is a nonionic surfactant having an HLB value of 13 to 18.
7). A method for detecting Mycoplasma pneumoniae in a specimen using an immunochromatographic analyzer including a sample addition unit, a labeled substance holding unit, a chromatographic medium unit having a detection unit, and an absorption unit, the following step (1) An immunochromatographic analysis method comprising (4).
(1) A step of adding a sample-containing solution obtained by diluting a sample with a sample dilution solution to a sample addition portion (2) P30 protein of Mycoplasma pneumoniae having the amino acid sequence of SEQ ID NO: 2 held in the labeling substance holding portion A step of recognizing Mycoplasma pneumoniae by an antibody that strongly recognizes domain III (hereinafter referred to as antibody P30 (A)) (3) a step of developing the specimen and antibody P30 (A) as a mobile phase in a chromatographic medium part (4) A step of detecting Mycoplasma pneumoniae in the developed mobile phase with the antibody P30 (A) contained in the detection unit.

According to the present invention, Mycoplasma pneumoniae can be detected quickly and easily with high sensitivity by using an immunochromatographic analyzer using an antibody that binds to a specific site of the P30 protein of Mycoplasma pneumoniae. That is, the diagnosis of pneumonia mycoplasma can be performed more reliably and quickly.

FIG. 1 is a cross-sectional view for explaining the structure of an immunochromatographic analyzer according to an embodiment of the present invention. FIG. 2 is a graph showing the results of detection of Mycoplasma pneumoniae using the immunochromatography analyzer of the present invention and measuring the color intensity. FIG. 3 is a graph showing the results of detection of Mycoplasma pneumoniae using the immunochromatography analyzer of the present invention and measuring the color intensity. FIG. 4 is a graph showing the results of the detection of Mycoplasma pneumoniae P30 protein using the immunochromatography analyzer of the present invention and the measurement of its color intensity. FIG. 5 is a graph showing the results of performing a competitive inhibition ELISA test using peptide 1 or peptide 2 as an antigen and measuring the color intensity in Test Example 4 using antibody P30 (a). FIG. 6 is a graph showing the results of a competition inhibition ELISA test using peptide 1 or peptide 2 as an antigen and the color intensity measured in test example 4 using antibody P30 (b).

Hereinafter, modes for carrying out the present invention will be described.

The immunochromatography analyzer for detecting Mycoplasma pneumoniae of the present invention is a chromatograph having a sample addition part for adding a specimen, a labeling substance holding part for holding a labeling substance, and a detection part for detecting Mycoplasma pneumoniae. A medium part and an absorption part that absorbs the liquid that has passed through the detection part, wherein the labeling substance holding part and the detection part contain an antibody that strongly recognizes the domain III of the P30 protein of Mycoplasma pneumoniae .

The antibody used in the immunochromatography analyzer of the present invention is an antibody that strongly recognizes domain III of the P30 protein of Mycoplasma pneumoniae (hereinafter also referred to as antibody P30 (A)).

Mycoplasma pneumoniae P30 protein is an adhesion protein consisting of 274 amino acids shown in SEQ ID NO: 1, rich in proline (20.7%), and having a molecular weight of 29,743.

Moreover, domain III of the P30 protein shown in SEQ ID NO: 2 is a region corresponding to the 177th to 274th amino acid sequence of the P30 protein (SEQ ID NO: 1), and is a domain rich in a specific amino acid repetitive sequence. . There are three types of amino acid repetitive sequences in domain III, 7 amino acid sequences shown in SEQ ID NO: 3 (PGMAPR), 3 amino acid sequences shown in SEQ ID NO: 4 (PGMPPH), and shown in SEQ ID NO: 5 (PGGFPPQ). There are three amino acid sequences.

The antibody P30 (A) used in the present invention recognizes domain III rich in this amino acid repetitive sequence. Therefore, by performing analysis using the immunochromatography analyzer of the present invention, the sandwich structure as shown below is obtained. It is estimated that the detection sensitivity is remarkably improved and the effect is remarkably exhibited.

That is, when antibody P30 (A) is contained in the labeling substance holding part and the detection part of the immunochromatography analyzer of the present invention, first, antibody P30 (A) held in the labeling substance holding part is Mycoplasma pneumoniae. It binds to part of the repeat sequence of domain III in the P30 protein.

Subsequently, the antibody P30 (A) immobilized on the detection unit binds to the same repetitive sequence present at a location different from the location where the antibody P30 (A) held in the labeling substance holding unit is bound, A sandwich structure is formed by sandwiching the P30 protein with the antibody P30 (A), and Mycoplasma pneumoniae is detected.

Thus, since antibody P30 (A) recognizes domain III having the above-mentioned repetitive sequence, it can be estimated that it can recognize a plurality of locations of P30 protein and can increase the detection sensitivity of Mycoplasma pneumoniae. Is done.

The antibody P30 (A) used in the present invention preferably recognizes at least the amino acid sequence of SEQ ID NO: 3 (PGMAPR) among the repeated sequences of amino acids in the domain III. As shown in the results of Examples below, the antibody recognizing the above sequence more strongly recognizes the domain III of the P30 protein of Mycoplasma pneumoniae, so that Mycoplasma pneumoniae can be detected with high sensitivity.

The antibody P30 (A) in the present invention is an antibody that "recognizes strongly the domain III of the P30 protein of Mycoplasma pneumoniae" as an antibody having an absorbance at 450 nm of 0.2 Abs or more when performing an ELISA test. Can be defined.

Specifically, an ELISA test is performed according to the following protocol. First, Mycoplasma pneumonia P30 ((amino acids 96-274) was expressed and purified in a conventional manner using Escherichia coli in a 96-well plate for Nunc Immuno modules (Thermo Fisher Scientific, code 469949) ELISA 4Pg / n4 Add 100 μL of mL in 50 mM carbonate buffer pH 9.5 and incubate at 4 ° C. for 16 hours. After 16 hours, the P30 solution is removed, and the wells are washed 3 times with 300 μL PBST (0.05% Tween 20 in PBS). Remove the liquid remaining in the well by tapping on a paper towel.

As a blocking solution, add 300 μL of 5% BSA in PBST (BSA: manufactured by Oriental Yeast), and incubate at 37 ° C. for 1 hour. Then, the BSA solution is removed, and the wells are washed 3 times with 300 μL PBST (0.05% Tween20 in PBS). Remove the liquid remaining in the well by tapping on a paper towel.

As a primary antibody, anti-Mycoplasma pneumoniae P30 antibody (antibody P30 (A)) 5 μg / mL in 50% blocking solution 100 μL is added to the well and incubated at 37 ° C. for 1 hour. Thereafter, the primary antibody solution is removed, and the wells are washed 3 times with 300 μL PBST (0.05% Tween 20 in PBS).

As a secondary antibody, add 1 mg / mL of Anti Mouse IgG (H + L), Rabbit, IgG Whole, Peroxidase Cojugated (manufactured by Wako Pure Chemical Industries, Ltd., code 014-17611) to the well, and incubate at 37 ° C. for 1.5 hours. . Then, the BSA solution is removed, and the wells are washed 3 times with 300 μL PBST (0.05% Tween20 in PBS). Remove the liquid remaining in the well by tapping on a paper towel.

100 μL of Sure Blue Reserve TMB Microwell Peroxidase Substrate (1-Component) (manufactured by KPL, code 53-00-01) is added to the well as a chromogenic substrate, reacted for 15 minutes, and 100 μL of 2N sulfuric acid is added to stop the reaction.
Absorbance at 450 nm is measured with a microplate reader (BIORAD).

An antibody having a value obtained by subtracting the absorbance of a blank (secondary antibody without adding a primary antibody, well in which color development reaction was performed) by the above method to be 0.2 Abs or more was designated as “Mycoplasma pneumoniae P30 protein domain III. Antibody ".

Further, the antibody P30 (A) in the present invention is an antibody that "recognizes strongly the domain III of the P30 protein of Mycoplasma pneumoniae", specifically, the full-length Mycoplasma pneumonia P30 protein (amino acids 1-274: SEQ ID NO: 1) or aminoplasm 96-274 fragment of Mycoplasma pneumonia P30 protein (these proteins are referred to as protein A) and one or more of three types of repeated sequences of amino acids in domain III or domain III (SEQ ID NOs: 3 to 5) When a competitive inhibition test (competitive inhibition ELISA test) by the ELISA method with a domain III fragment containing these proteins (referred to as protein B) was performed, the antibody reaction against protein A was Both the antibody in the case where the reaction is inhibited by the presence of click quality B can be defined. The competitive inhibition ELISA test can be defined by either a direct competitive method or an indirect competitive method.

To illustrate the test by the indirect competition method, Nunc Immuno modules (Thermo Fisher Scientific, code 469949) ELISA 96-well plate Mycoplasma pneumonia P30 (amino acid 1-274: SEQ ID NO: 1) 4 ngm 50 mL Add 100 μL of carbonate buffer pH 9.5 and incubate at 4 ° C. for 16 hours. After 16 hours, the P30 solution is removed, and the wells are washed 3 times with 300 μL PBST (0.05% Tween 20 in PBS). Remove the liquid remaining in the well by tapping on a paper towel.

As a blocking solution, add 300 μL of 5% BSA in PBST (BSA: manufactured by Oriental Yeast), and incubate at 37 ° C. for 1 hour. Then, the BSA solution is removed, and the wells are washed 3 times with 300 μL PBST (0.05% Tween20 in PBS). The liquid remaining in the well is removed by tapping on a paper towel to create a full-length protein-fixed well.

Next, 100 μL of anti-Mycoplasma pneumoniae P30 antibody (antibody P30 (A)) 5 μg / mL in 50% blocking solution is added to the well as a primary antibody in the full-length protein-fixed well, and incubated at 37 ° C. for 1 hour. Thereafter, the primary antibody solution is removed, and the wells are washed 3 times with 300 μL PBST (0.05% Tween 20 in PBS).

In addition to the addition of the primary antibody alone, the primary antibody anti-Mycoplasma pneumoniae P30 antibody (antibody P30 (A)) 5 μg / mL, and the primary antibody amount 40 times or 80 times the domain III or 100 μL of a 50% blocking solution containing a domain III fragment containing one or more of the three repeating sequences of amino acids in domain III is added to the wells and incubated at 37 ° C. for 1 hour. Thereafter, the primary antibody solution is removed, and the wells are washed 3 times with 300 μL PBST (0.05% Tween 20 in PBS).

As a secondary antibody, Anti Mouse IgG (H + L), Rabbit, IgG Whole, Peroxidase Cojugated (manufactured by Wako Pure Chemicals Co., Ltd., code 04-117611) 1 mg / mL, 100 μL, was added to each of the two types of wells prepared above, 37 Incubate at C for 1.5 hours. Then, the BSA solution is removed, and the wells are washed 3 times with 300 μL PBST (0.05% Tween20 in PBS). Remove the liquid remaining in the well by tapping on a paper towel.

Using the above method, it is confirmed that the absorbance of the well added with the primary antibody and the domain III fragment containing one or more of the three types of repeating sequences of amino acids in domain III or domain III decreases from the absorbance of the well added with only the primary antibody. It is possible to confirm that “domain III of P30 protein of Mycoplasma pneumoniae is strongly recognized”.

In the present invention, the absorbance is reduced by 30% or more when the domain III or a fragment of domain III containing one or more of three types of repeating sequences of amino acids in domain III is used 40 times the amount of the primary antibody, or 80 times that of the primary antibody. It can be confirmed that the domain III of the P30 protein of Mycoplasma pneumoniae is more strongly recognized if the absorbance when used in an amount decreases by 50% or more.

Examples of the antibody P30 (A) used in the present invention include polyclonal antibodies and monoclonal antibodies. A monoclonal antibody is preferable from the viewpoint of sensitivity. The antibody P30 (A) can be obtained by purchasing, for example, trade name: Mp-P30-3-AB from Fucal Research.

Next, an embodiment of the immunochromatographic analyzer of the present invention will be described with reference to the drawings. In this specification, “fixed” means that the antibody is arranged on a carrier such as a membrane so that it does not move, and “hold” means that the inside or the surface of the carrier such as a membrane can move. It means to be placed.

As an embodiment of the immunochromatography analyzer of the present invention, as shown in FIG. 1, a sample addition part (also called a sample pad) (1), a labeling substance holding part (also called a conjugate pad) (2), a chromatograph It is comprised from the graph medium part (3), the detection part (4), the absorption part (5), and the backing sheet (6).

The sample addition part (1) is a part to which a sample containing a specimen is added in the immunochromatography analyzer. In the sample addition part (1), the sample is rapidly absorbed, but it can be constituted by a porous sheet having such a property that the sample moves quickly. Examples of the porous sheet include cellulose filter paper, glass fiber, polyurethane, polyacetate, cellulose acetate, nylon, and cotton cloth.

The labeling substance holding part (2) contains a labeling substance (marker substance) described later, and the labeling substance is held as a labeled antibody (hereinafter also simply referred to as a labeled antibody) bound to the antibody P30 (A). Held in the part (2). When the specimen moves in the labeling substance holding part, the labeled antibody and Mycoplasma pneumoniae in the specimen are combined. For the labeling substance holding part (2), a film of glass fiber, cellulose or the like is usually used.

The content of the labeled antibody in the labeling substance holding part is usually 0.06 to 0.25 μg / device, preferably 0.1 to 0.2 μg / device, more preferably 0.1 to 0.15 μg. / Device. The content of the labeled antibody per unit area of the labeling substance holding part is usually 0.1 to 0.42 μg / cm ² , preferably 0.17 to 0.33 μg / cm ² , more preferably 0.17 to 0.25 μg / cm ² .

An enzyme or the like is generally used for labeling a detection reagent in immunochromatographic analysis, but it is preferable to use an insoluble carrier as the labeling substance because it is suitable for visually determining the presence of the substance to be detected. A labeled detection reagent can be prepared by sensitizing the antibody P30 (A) to an insoluble carrier. The means for sensitizing the antibody P30 (A) to the insoluble carrier may be a known method.

As the insoluble carrier as a labeling substance, metal particles such as gold, silver or platinum, metal oxide particles such as iron oxide, non-metallic particles such as sulfur and latex particles made of synthetic polymer, or other insoluble carriers are used. be able to. As described above, the insoluble carrier is a labeling substance suitable for visually determining the presence of the substance to be detected, and is preferably colored to facilitate visual determination. The metal particles and the metal oxide particles themselves exhibit a specific natural color corresponding to the particle diameter, and the color can be used as a label.

In particular, gold particles are preferable because they are easy to detect and are difficult to aggregate and non-specific color development is unlikely to occur. The average particle diameter of the gold particles is, for example, 10 nm to 250 nm, preferably 35 nm to 120 nm. The average particle diameter was measured with a transmission electron microscope (TEM: manufactured by JEOL Ltd., JEM-2010), and the projected area circle equivalent diameter of 100 particles was measured randomly using a photograph taken. It can be calculated from the average value.

The gold particles contained in the labeling substance holding part are usually 0.25 to 0.7 μg / cm ² , preferably 0.3 to 0.65 μg / cm ² per unit area of the labeling substance holding part, Preferably, it is 0.4 to 0.6 μg / cm ² . This is because, by setting the range, the labeled particles are developed in a dispersed state, and the recognition site of the antibody is not hindered and the sensitivity can be increased.

The chromatographic medium part (3) is a developed part of the chromatograph. The chromatographic medium part (3) is an inert film made of a microporous material that exhibits capillary action. From the viewpoint of not having reactivity with a detection reagent, an immobilization reagent or a substance to be detected used in a chromatograph, and from the viewpoint of improving the effect of the present invention, for example, a membrane made of nitrocellulose (hereinafter, Nitrocellulose membrane) and cellulose acetate membrane (hereinafter also referred to as cellulose acetate membrane) are preferred, and nitrocellulose membrane is more preferred. Cellulose membranes, nylon membranes and porous plastic cloth (polyethylene, polypropylene) can also be used.

As the nitrocellulose membrane, it is only necessary to mainly contain nitrocellulose, and a membrane mainly composed of nitrocellulose such as a pure product or a nitrocellulose mixed product can be used.

The nitrocellulose membrane may further contain a substance that promotes capillary action. As the substance, a substance that lowers the surface tension of the film surface and brings about hydrophilicity is preferable. For example, a substance having an amphipathic action such as saccharides, amino acid derivatives, fatty acid esters, various synthetic surfactants or alcohols, which does not affect the movement of the detected substance and does not affect the coloring of the labeling substance. No material is preferred.

Nitrocellulose membrane is porous and exhibits capillary action. The index of the capillary phenomenon can be confirmed by measuring the water absorption rate (water absorption time: capillary flow time). The water absorption rate affects detection sensitivity and inspection time.

The form and size of the chromatographic medium part (3) represented by the above nitrocellulose membrane and cellulose acetate membrane are not particularly limited, and are appropriate in terms of actual operation and reaction results. If it is.

In order to further simplify the operation, it is preferable to provide a support made of plastic or the like on the back surface of the chromatographic medium part (3). The properties of the support are not particularly limited, but when the measurement result is observed by visual judgment, the support preferably has a color that is not similar to the color caused by the labeling substance. Usually, it is preferably colorless or white.

The detection part (4) is formed on the chromatographic medium part (3). That is, the antibody P30 (A) that binds to the substance to be detected Mycoplasma pneumoniae is immobilized at an arbitrary position on the chromatographic medium part (3). Immobilization of the antibody P30 (A) can be performed according to a conventional method.

The content of antibody P30 (A) in the detection section (4) is usually 0.1 to 2.5 μg / device, preferably 0.3 to 2.0 μg / device, more preferably 0.3 to 1.0 μg / device. Further, the content of the antibody P30 (A) per unit area of the detection unit (4) is usually 0.04 to 1.0 g / cm ² , preferably 0.125 to 0.8 μg / cm ² . More preferably, it is 0.125 to 0.42 μg / cm ² .

Moreover, on the chromatographic medium part (3), in order to prevent the accuracy of analysis from being reduced due to non-specific adsorption, the chromatographic medium part (3) is blocked by a known method as necessary. Processing can be performed. Generally, proteins such as bovine serum albumin, skim milk, casein or gelatin are preferably used for the blocking treatment. After such blocking treatment, if necessary, for example, one or a combination of two or more surfactants such as Tween 20, Triton X-100, or SDS may be washed.

The absorption part (5) is installed at the end of the chromatographic medium part (3) in order to absorb a liquid such as a specimen or a developing solution that has passed through the detection part (4). In the immunochromatography analyzer of the present invention, the absorption part (5) contains, for example, glass fiber, pulp, cellulose fiber, or the like, or a non-woven fabric containing a polymer such as an acrylic acid polymer, a hydrophilic drug having an ethylene oxide group or the like. What was made to use is used, Especially preferably, it is a glass fiber. When the absorption part (5) is made of glass fiber, the return of the sample liquid can be greatly reduced.

The backing sheet (6) is a base material. By applying an adhesive on one side or sticking an adhesive tape, one side has adhesiveness, and the sample addition part (1), labeling substance holding part (2), chromatographic medium part ( 3) A part or all of the detection unit (4) and the absorption unit (5) are provided in close contact with each other. The backing sheet (6) is not particularly limited as long as the backing sheet (6) becomes impermeable and impermeable to the sample solution by the adhesive.

The immunochromatographic analyzer produced as described above is usually subjected to a drying process before commercialization. The drying temperature is, for example, 20 to 50 ° C., and the drying time is 0.5 to 1 hour.

The immunochromatography analysis kit of the present invention includes the above immunochromatography analyzer and a sample diluent for diluting and developing the sample.

In the immunochromatography analysis kit of the present invention, the sample diluent can also be used as a developing solution, but usually water is used as a solvent, to which a buffer solution, salt, and nonionic surfactant are further added. , For example, one or more of proteins, polymer compounds (PVP, etc.), ionic surfactants or polyanions, or antibacterial agents, chelating agents, etc. for promoting antigen-antibody reactions or suppressing nonspecific reactions May be added.

In particular, P30 was isolated from the protein complex (P1, P90, P40, P30) present in the cell adhesion organ of Mycoplasma pneumonia, and a nonionic surfactant was diluted with the sample to expose the antigen recognition site of the anti-P30 antibody. It is preferable to make it contain in a liquid. Nonionic surfactants include, for example, Triton X-100 (trade name, polyethylene glycol mono-p-isooctylphenyl ether), Tween 20 (trade name, polyoxyethylene sorbitan monolaurate), NP-40 (product) Name Nonit 40) and Brij 35 may be mentioned, and one or more may be added.

Preferably, 50% or more of the nonionic surfactant contained in the sample diluent is a nonionic surfactant having an HLB value of 13 to 18. More preferably, 60% or more of the nonionic surfactant contained in the sample diluent is a nonionic surfactant having an HLB value of 13 to 17, and particularly preferably the nonionic surfactant contained in the sample diluent. 100% of the ionic surfactant is a nonionic surfactant having an HLB value of 13-17. Specifically, it is preferable to contain two types of Triton X-100 (HLB value: 13.7) and Tween 20 (HLB value: 16.7) at a mass ratio of 1: 1.

When using the sample diluent as the developing solution, the sample and developing solution mixed in advance can be supplied and dropped onto the sample addition unit, or the sample can be supplied to the sample addition unit first. -After dripping, you may make it expand | deploy by supplying and dripping a developing solution on a sample addition part.

The immunochromatographic analysis method of the present invention includes the following steps (1) to (4), and detects Mycoplasma pneumoniae to be detected contained in a specimen using the above immunochromatographic analyzer.
(1) A step of adding a sample-containing solution obtained by diluting a sample with a sample dilution solution to a sample addition portion (2) P30 protein of Mycoplasma pneumoniae having the amino acid sequence of SEQ ID NO: 2 held in the labeling substance holding portion A step of recognizing Mycoplasma pneumoniae by an antibody that strongly recognizes domain III (hereinafter referred to as antibody P30 (A)) (3) a step of developing the specimen and antibody P30 (A) as a mobile phase in a chromatographic medium part (4) Process of detecting Mycoplasma pneumoniae in the developed mobile phase with antibody P30 (A) contained in the detection unit Each process will be described below.

(1) Step of adding a specimen-containing solution obtained by diluting a specimen with a specimen diluent to the sample addition section In step (1), first, the specimen is smoothly passed through the immunochromatographic medium without degrading measurement accuracy. It is preferable to adjust or dilute with a sample diluent to a concentration that allows the sample to be transferred to a concentration of 1). The specimen dilution liquid described above can be used. Second, a predetermined amount (usually 0.1 to 2 mL) of the specimen-containing liquid is dropped onto the sample addition part (1). When the specimen-containing liquid is dropped, the specimen-containing liquid starts to move in the sample addition part (1).

The specimen used in the present invention is a specimen that may contain Mycoplasma pneumoniae to be detected. For example, among biological samples, pharyngeal wiping liquid, nasal wiping liquid, nasal aspirate, nasal washing liquid, sputum, Although alveolar lavage fluid etc. are mentioned, it is not limited to these.

(2) An antibody that strongly recognizes domain III of the P30 protein of Mycoplasma pneumoniae consisting of the amino acid sequence of SEQ ID NO: 2 held in the labeling substance holding part (hereinafter referred to as antibody P30 (A)). Step of recognizing pneumoniae In step (2), the sample-containing liquid added to the sample addition part in step (1) is moved to the labeling substance holding part (2), and the label held in the labeling substance holding part This is a step of recognizing the domain III of the P30 protein in Mycoplasma pneumoniae, which is the substance to be detected in the specimen, by the antibody P30 (A) to which the substance is bound.

The above-mentioned labeling substances can be used. The antibody P30 (A) to which the labeling substance is bound recognizes and binds to a part of the specific repeating sequence in the amino acid sequence of domain III of the P30 protein.

(3) Step of developing the sample and antibody P30 (A) as a mobile phase in a chromatographic medium part In step (3), Mycoplasma pneumoniae, which is a substance to be detected in step (2), is labeled in the labeling substance holding part. Is recognized by the antibody P30 (A) bound to the sample, and then the specimen and the antibody P30 (A) are passed over the chromatographic medium as a mobile phase.

(4) A step of detecting Mycoplasma pneumoniae in the developed mobile phase with the antibody P30 (A) contained in the detection unit Step (4) is a step of detecting the mycoplasma in the sample that has passed over the chromatographic medium as the mobile phase. Pneumonier is sandwiched between antibody P30 (A) immobilized on the detection part and antibody P30 (A) to which the labeling substance is bound in the above step (2) by a specific antigen-antibody binding reaction. This is a step of specifically reacting and binding to color the detection part.

In this step (4), the antibody P30 (A) immobilized on the detection part is sandwiched between the same repetitive sequences present at a different place from the place where the antibody P30 (A) held in the labeling substance holding part is bound. Combined so as to be sandwiched in a shape.

When the detection target Mycoplasma pneumoniae does not exist, the labeling reagent dissolved in the sample water does not cause a specific binding reaction even if it passes through the detection part on the chromatographic medium part. Do not color.

Finally, the moisture in the specimen-containing liquid moves to the absorption part (5).

Hereinafter, the present invention will be further described with reference to examples, but the present invention is not limited to the following examples.

[Test Example 1]
In this test, two different types of antibodies recognizing the domain III of the P30 protein of Mycoplasma pneumoniae were prepared, and it was examined which part of the domain III of the P30 protein each antibody recognizes. The antibodies used were Fucal Research, product name: Mp-P30-3-AB (hereinafter referred to as antibody P30 (a)), Fucal Research, product name: Mp-P30-9-AB (hereinafter, referred to as antibody P30 (a)). Antibody P30 (b)). It was verified by the following immunochromatographic analysis to which part of domain III of P30 protein of Mycoplasma pneumoniae this antibody P30 (a) and antibody P30 (b) bind.
First, the following two types of peptides containing any of the three types of amino acid repetitive sequences (SEQ ID NOs: 3 to 5) in the above domain III are prepared by peptide solid-phase synthesis, which is a conventional method for peptide chemical synthesis. Produced.
Peptide 1: PGMAPRPGMPPHPPGAPR (SEQ ID NO: 6)
Peptide 2: PGMAPRPGFPPQPGMAPR (SEQ ID NO: 7)

The above peptide 1 is a peptide having the amino acid sequence shown in SEQ ID NO: 4 (PGMPPH) and the amino acid sequence shown in SEQ ID NO: 3 (PGMAPR). Peptide 2 is a peptide having the amino acid sequence shown in SEQ ID NO: 5 (PGGFPPQ) and the amino acid sequence shown in SEQ ID NO: 3 (PGMAPR).

It can be determined as follows to which part of domain III the antibody P30 (a) or the antibody P30 (b) binds.
(1) Preparation of sample addition part The nonwoven fabric (Millipore company_made: 300 mm x 30 mm) which consists of glass fiber was used as a sample addition part.
(2) Preparation of labeling substance holding part 0.5 ml of colloidal gold suspension (manufactured by Tanaka Kikinzoku Kogyo Co., Ltd .: LC 40 nm) diluted with phosphate buffer (pH 7.4) to a concentration of 0.05 mg / mL 0.1 mL of the prepared antibody P30 (a) (Fucal Research, trade name: Mp-P30-3-AB) was added and allowed to stand at room temperature for 10 minutes.
Next, 0.1 mL of a phosphate buffer solution (pH 7.4) containing 1% by mass of bovine serum albumin (BSA) was added, and the mixture was allowed to stand at room temperature for 10 minutes. Then, after sufficiently stirring, centrifugation was performed at 8000 × g for 15 minutes, and the supernatant was removed. Then, 0.1 mL of a phosphate buffer (pH 7.4) containing 1% by mass of BSA was added. The labeling substance solution was prepared by the above procedure.
After adding 300 μL of a 10% by mass trehalose aqueous solution and 1.8 mL of distilled water to 300 μL of the prepared labeling substance solution, uniformly added to a 12 mm × 300 mm glass fiber pad (Millipore), vacuum is added. The labeling substance holding part was produced by drying with a dryer.

(3) Preparation of chromatographic medium part and detection part As a membrane, a sheet made of nitrocellulose (manufactured by Millipore, trade name: HF120, 300 mm × 25 mm) was used.
Next, 150 μL of a solution obtained by diluting the antibody P30 (a) with a phosphate buffer solution (pH 7.4) containing 5% by mass of isopropyl alcohol so as to have a concentration of 1.0 mg / mL was dried with a membrane. An upper chromatographic dispenser “XYZ3050” (manufactured by BIODOT) was applied to the upper detection site (detection line) at a width of 1 mm in an amount of 1 μL / mm (25 μL per sheet).
Further, in order to confirm the presence / absence and development speed of the gold particle labeling reagent, dilute a goat-derived antiserum having a wide affinity for the gold particle labeling substance with a phosphate buffer (pH 7.4) downstream of the detection site. The solution was applied to the control site (control line). Then, it was dried at 50 ° C. for 30 minutes and dried overnight at room temperature to prepare a chromatographic medium part and a detection part.
(4) Production of immunochromatography analyzer Next, a substrate consisting of a backing sheet, a sample addition part, a labeling substance holding part, a chromatographic medium part having a detection part, absorption for absorbing the developed sample and labeling substance Glass fiber non-woven fabrics were bonded together as a part. And it cut | judged so that a width | variety might be set to 5 mm with the cutter, and it was set as the immunochromatography analyzer. The length of the labeling substance holding part in the sample developing direction was 12 mm.

(5) Specimen diluent 1% by mass of nonionic surfactant NP-40 (trade name: Nonidet P-40, HLB value 17.7) manufactured by Nacalai Tesque Co., Ltd., trade name Nonidet MN-811 (manufactured by NOF Corporation) A 50 mM HEPES buffer solution (pH 7.5) containing a 1: 1 mixture of HLB values 9.3) was prepared, and used as a sample diluent for subjecting the sample to dilution treatment.
(6) Measurement Using a sample sample containing Mycoplasma pneumoniae and Peptide 1 or Peptide 2, the color intensity at the detection part was measured when the prepared immunochromatography analyzer was used. As a specimen sample containing Mycoplasma pneumoniae, Mycoplasma pneumonia P30 ((amino acid 96-274) was expressed and purified by a conventional method using Escherichia coli P30Ag) was used. This P30Ag was adjusted to the target concentration of 10 pg / mL with the above extract, and peptide 1 or peptide 2 was prepared so that 400 ng of peptide 1 or peptide 2 was developed, and used as a sample sample.
150 μL of the sample sample was placed on the sample addition part of the immunochromatography analyzer and developed, and the degree of coloring (coloring intensity) of the detection part was measured with a densitometer (the unit in the table is mAbs).

<Judgment method>
When inhibited with peptide 1 but not with peptide 2: The antibody binds to the amino acid sequence shown in SEQ ID NO: 4 (PGMPPH).
When not inhibited by peptide 1 but inhibited by peptide 2: The antibody binds to the amino acid sequence shown in SEQ ID NO: 5 (PGGFPPQ).
When inhibited with peptide 1 and also with peptide 2: The antibody binds to the amino acid sequence shown in SEQ ID NO: 3 (PGMAPR).

In addition, the mechanism considered that inhibition has arisen by developing peptide 1 or peptide 2 with P30Ag of a specimen is demonstrated below. Since Peptide 1 and Peptide 2 have a much smaller molecular weight than P30Ag of the specimen and a much larger amount to be developed, they reach the labeling substance holding part earlier than P30Ag and bind to the antibody held at the same site. Then, it is expanded toward the detection unit. However, since the antibody binding sites of Peptide 1 and Peptide 2 are much smaller than P30Ag (1 to 2 sites), many of the above peptides already bound by the antibody have antibodies immobilized on the detection part. The number of binding sites is small or nonexistent, and a large amount of peptide flows to the absorption unit without being captured by the detection unit. As a result, the amount of labeling substance that is captured and deposited by the detection unit decreases, thereby reducing the color intensity. . On the other hand, the antibody held in the labeling substance holding part binds to Peptide 1 and Peptide 2 prior to P30Ag, so that the amount of the antibody that binds to P30Ag developed later is present in Peptide 1 or Peptide 2. Therefore, the amount of P30Ag captured by the detection unit is relatively higher when the antibody is not bound, and as a result, the color intensity caused by P30Ag detected by the detection unit is low. It will be a hindrance.

In this test, when antibody P30 (a) is used and neither of the

above peptides

1 and 2 is used, antibody P30 (a) is used and when peptide 1 is used, antibody P30 (a) is used. Table 3 shows the results of immunochromatographic analysis and measurement of color intensity for the three patterns when peptide 2 was used.
Further, Table 2 shows the results of a similar test performed by changing the antibody P30 (a) to the antibody P30 (b).

As can be seen from the results in Table 1, with respect to antibody P30 (a), compared to the case without peptide, the chromogenic intensity was decreased in both cases where peptide 1 was used and peptide 2 was used. It can be seen that antibody P30 (a) is inhibited by both peptide 1 and peptide 2. Therefore, it was found that antibody P30 (a) recognizes the amino acid sequence shown in SEQ ID NO: 3 (PGMAPR).

In addition, as can be seen from the results in Table 2, when the peptide 1 was used for the antibody P30 (b) compared to the case without the peptide, the color intensity was not changed, but the peptide 2 was used. In this case, the color intensity was decreased, and it was found that antibody P30 (b) was not inhibited by peptide 1 but was inhibited by peptide 2. Therefore, it was found that antibody P30 (b) recognizes the amino acid sequence shown in SEQ ID NO: 5 (PGGFPPQ).

[Test Example 2]
In this test, an antibody (antibody P30 (a)) that recognizes the domain III of the P30 protein of Mycoplasma pneumoniae used in the immunochromatography apparatus of the present invention prepared in Test Example 3 described below is “strong”. In order to confirm whether the antibody was a recognized antibody, the ELISA test described in paragraphs [0025] to [0030] and the competitive inhibition ELISA test described in paragraphs [0032] to [0039] were performed.

First, in order to confirm whether or not the antibody P30 (a) used in Test Example 1 is an antibody that strongly recognizes domain III of the P30 protein of Mycoplasma pneumoniae, according to the protocol described in paragraphs [0025] to [0030] The test by the ELISA method was performed. Specifically, whether or not the antibody P30 (a) was an antibody having an absorbance at 450 nm of 0.2 Abs or more with a blank drawn was examined as follows.

First, Nunc Immunomodules (Thermo Fisher Scientific, code 469949) Mycoplasma pneumonia P30 ((amino acids 96-274) was expressed and purified in an ordinary manner using E. coli 4 g / n (4 Pg / mL). 100 μL of in 50 mM carbonate buffer pH 9.5 was added and incubated at 4 ° C. for 16 hours. After 16 hours, the P30 solution was removed, the wells were washed 3 times with 300 μL PBST (0.05% Tween 20 in PBS), and the liquid remaining in the wells was removed by tapping on a paper towel. As a blocking solution, 300 μL of 5% BSA in PBST (BSA: manufactured by Oriental Yeast) was added and incubated at 37 ° C. for 1 hour. Thereafter, the BSA solution was removed, and the wells were washed three times with 300 μL PBST (0.05% Tween 20 in PBS), and the liquid remaining in the wells was removed by tapping on a paper towel.

As a primary antibody, 100 μL of the antibody P30 (a) 5 μg / mL in 50% blocking solution (primary antibody solution) was added to the well, incubated at 37 ° C. for 1 hour, the primary antibody solution was removed, and the well was removed with 300 μL PBST (0.05 % Tween20 in PBS).
As a secondary antibody, 1 mg / mL of Anti Mouse IgG (H + L), Rabbit, IgG Whole, Peroxidase Cojugated (manufactured by Wako Pure Chemical Industries, Ltd., code 04-117611) was added to 100 μL of the well and incubated at 37 ° C. for 1.5 hours. Thereafter, the BSA solution was removed, and the wells were washed three times with 300 μL PBST (0.05% Tween 20 in PBS), and the liquid remaining in the wells was removed by tapping on a paper towel.
100 μL of Sure Blue Reserve TMB Microwell Peroxidase Substrate (1-Component) (KPL, code 53-00-01) was added to the well as a chromogenic substrate, reacted for 15 minutes, and then the reaction was stopped by adding 100 μL of 2N sulfuric acid. Then, absorbance at 450 nm was measured with a microplate reader (manufactured by BIORAD).

When the ELISA test was performed by the above method, the absorbance at 450 nm obtained by subtracting the absorbance of the blank (secondary antibody without adding the primary antibody, well in which the color reaction was performed) was 0.403 Abs, which was 0.2 Abs or more. I was able to confirm. That is, it was confirmed that the antibody P30 (a) “recognizes strongly the domain III of the P30 protein of Mycoplasma pneumoniae”.

Subsequently, in order to further confirm whether or not this antibody P30 (a) is an antibody that "recognizes strongly domain III of the P30 protein of Mycoplasma pneumoniae", the protocol described in paragraphs [0032] to [0039] is followed. Competition inhibition ELISA test (indirect competition method) was performed. Specifically, as a result of competitive inhibition ELISA test using antibody P30 (a), whether or not the absorbance decreased by 30% or more was tested as follows.

Nunc Immuno modules (manufactured by Thermo Fisher Scientific, code 469949) ELISA 96-well plate, full length protein Mycoplasma pneumonia P30 (amino acid 1-274: SEQ ID NO: 1) 4 ng / mL in 50 mM carbonate buffer L9. Incubated for 16 hours at 4 ° C. After 16 hours, the P30 solution was removed, and the wells were washed 3 times with 300 μL PBST (0.05% Tween20 in PBS). The liquid remaining in the well was removed by tapping on a paper towel.

As a blocking solution, add 300 μL of 5% BSA in PBST (BSA: manufactured by Oriental Yeast), and incubate at 37 ° C. for 1 hour. The BSA solution was then removed, and the wells were washed 3 times with 300 μL PBST (0.05% Tween 20 in PBS). The liquid remaining in the well was removed by tapping on a paper towel to create a full-length protein-fixed well.

Next, 100 μL of an anti-Mycoplasma pneumoniae P30 antibody (antibody P30 (a)) 5 μg / mL in 50% blocking solution was added to the well as a primary antibody in the full-length protein-fixed well and incubated at 37 ° C. for 1 hour. Thereafter, the primary antibody solution was removed, and the wells were washed 3 times with 300 μL PBST (0.05% Tween 20 in PBS).

In addition to the addition of the primary antibody alone, the primary antibody anti-Mycoplasma pneumoniae P30 antibody (antibody P30 (a)) 5 μg / mL and 40 times the amount of the primary antibody P30 (a) 100 μL of a 50% blocking solution containing the peptide 1 (SEQ ID NO: 6: PGMAPRPGMPPHPGMAPR) or peptide 2 (SEQ ID NO: 7: PGMAPRPGFPPQPGMAPR) used in 1 was added to the well and incubated at 37 ° C. for 1 hour. Thereafter, the primary antibody solution was removed, and the wells were washed 3 times with 300 μL PBST (0.05% Tween 20 in PBS).

As a secondary antibody, Anti Mouse IgG (H + L), Rabbit, IgG Whole, Peroxidase Cojugated (manufactured by Wako Pure Chemicals Co., Ltd., code 04-117611) 1 mg / mL was added to 100 μL of the two wells prepared above, 37 Incubated for 1.5 hours at ° C. The BSA solution was then removed and the wells were washed 3 times with 300 μL PBST (0.05% Tween 20 in PBS). The liquid remaining in the well was removed by tapping on a paper towel.
100 μL of Sure Blue Reserve TMB Microwell Peroxidase Substrate (1-Component) (KPL, code 53-00-01) was added to the well as a chromogenic substrate, reacted for 15 minutes, and 100 μL of 2N sulfuric acid was added to stop the reaction. .
Absorbance at 450 nm was measured with a microplate reader (BIORAD).

As a result, it was confirmed that the absorbance of the well added with the primary antibody and the above peptide 1 or peptide 2 decreased by 30% or more compared to the absorbance of the well added with only the primary antibody. That is, it was confirmed that the antibody P30 (a) “recognizes strongly the domain III of the P30 protein of Mycoplasma pneumoniae”.

[Test Example 3]
<Preparation of immunochromatography analyzer of the present invention>
[Example 1]
(1) Preparation of sample addition part The nonwoven fabric (Millipore company_made: 300 mm x 30 mm) which consists of glass fiber was used as a sample addition part.

(2) Preparation of labeling substance holding part 0.5 ml of colloidal gold suspension (manufactured by Tanaka Kikinzoku Kogyo Co., Ltd .: LC 40 nm) diluted with phosphate buffer (pH 7.4) to a concentration of 0.05 mg / mL 0.1 mL of the prepared antibody P30 (a) (Fucal Research, trade name: Mp-P30-3-AB) was added and allowed to stand at room temperature for 10 minutes.

Next, 0.1 mL of a phosphate buffer solution (pH 7.4) containing 1% by mass of bovine serum albumin (BSA) was added, and the mixture was allowed to stand at room temperature for 10 minutes. Then, after sufficiently stirring, centrifugation was performed at 8000 × g for 15 minutes, and the supernatant was removed. Then, 0.1 mL of a phosphate buffer (pH 7.4) containing 1% by mass of BSA was added. The labeling substance solution was prepared by the above procedure.
After adding 300 μL of a 10% by mass trehalose aqueous solution and 1.8 mL of distilled water to 300 μL of the prepared labeling substance solution, uniformly added to a 12 mm × 300 mm glass fiber pad (Millipore), vacuum is added. The labeling substance holding part was produced by drying with a dryer.

(3) Preparation of chromatographic medium part and detection part As a membrane, a sheet made of nitrocellulose (manufactured by Millipore, trade name: HF120, 300 mm × 25 mm) was used.
Next, 150 μL of a solution obtained by diluting the antibody P30 (a) with a phosphate buffer solution (pH 7.4) containing 5% by mass of isopropyl alcohol so as to have a concentration of 1.0 mg / mL was dried with a membrane. An upper chromatographic dispenser “XYZ3050” (manufactured by BIODOT) was applied to the upper detection site (detection line) at a width of 1 mm in an amount of 1 μL / mm (25 μL per sheet).
Further, in order to confirm the presence / absence and development speed of the gold particle labeling reagent, dilute a goat-derived antiserum having a wide affinity for the gold particle labeling substance with a phosphate buffer (pH 7.4) downstream of the detection site. The solution was applied to the control site (control line). Then, it was dried at 50 ° C. for 30 minutes and dried overnight at room temperature to prepare a chromatographic medium part and a detection part.

(4) Production of immunochromatography analyzer Next, a substrate consisting of a backing sheet, a sample addition part, a labeling substance holding part, a chromatographic medium part having a detection part, absorption for absorbing the developed sample and labeling substance Glass fiber non-woven fabrics were bonded together as a part. And it cut | judged so that a width | variety might be set to 5 mm with the cutter, and it was set as the immunochromatography analyzer. The length of the labeling substance holding part in the sample developing direction was 12 mm.

(5) Specimen diluent 1% by mass of nonionic surfactant NP-40 (trade name: Nonidet P-40, HLB value 17.7) manufactured by Nacalai Tesque Co., Ltd., trade name Nonidet MN-811 (manufactured by NOF Corporation) A 50 mM HEPES buffer solution (pH 7.5) containing a 1: 1 mixture of HLB values 9.3) was prepared, and used as a sample diluent for subjecting the sample to dilution treatment.

(6) Measurement Using a specimen sample containing Mycoplasma pneumoniae, the color intensity at the detection part was measured when the prepared immunochromatography analyzer was used. As a specimen sample containing Mycoplasma pneumoniae, a commercially available inactivated Mycoplasma pneumoniae (manufactured by Meridian, trade name Mycoplasma pneumoniae Antigen (FH)), or a pharyngeal wipe from a pneumonia mycoplasma infected person was used. The pharyngeal swab was collected from two subjects with pneumonia mycoplasma infection by wiping the pharynx with a commercially available cotton swab.
A commercially available inactivated mycoplasma pneumoniae was adjusted to the target concentration with the above extract and used as a specimen sample (described as a commercially available specimen in Table 3).
Further, the collected pharyngeal wiping solution was diluted 20-fold with the above-described sample diluent, and used as sample samples (described as subject 1 and subject 2 in Table 3).

150 μL of each specimen sample was placed on the sample addition part of the immunochromatography analyzer and developed, and the degree of coloring (color intensity) of the detection part was measured with a densitometer (the unit in the table is mAbs). The results are shown in Table 3 and FIG.

[Comparative Example 1]
Except that the antibody P30 (a) in the antibody dilution solution applied to the detection part was changed to the antibody P30 (b) (Future Research, trade name: Mp-P30-9-AB) used in Test Example 1. Example 1 was repeated. The results are shown in Table 3 and FIG.
In order to confirm whether or not this antibody P30 (b) “recognizes strongly the domain III of the P30 protein of Mycoplasma pneumoniae”, the test by the ELISA method described in paragraphs [0025] to [0030] and the paragraph [ The competitive inhibition ELISA test described in [0032] to [0039] was performed.
First, in order to confirm whether or not the antibody P30 (b) is an antibody that strongly recognizes domain III of the P30 protein of Mycoplasma pneumoniae, an ELISA test was performed according to the protocol described in paragraphs [0025] to [0030]. It was. Specifically, whether or not the antibody P30 (b) was an antibody having an absorbance at 450 nm with a blank drawn of 0.2 Abs or more was examined as follows.

Nunc Immuno modules (manufactured by Thermo Fisher Scientific, code 469949) In a 96-well plate for ELISA, Mycoplasma pneumonia P30 ((amino acids 96-274) was expressed and purified in a conventional manner using E. coli / P30Ag) 100 μL of 50 mM carbonate buffer pH 9.5 was added and incubated at 4 ° C. for 16 hours. After 16 hours, the P30 solution was removed, the wells were washed 3 times with 300 μL PBST (0.05% Tween 20 in PBS), and the liquid remaining in the wells was removed by tapping on a paper towel. As a blocking solution, 300 μL of 5% BSA in PBST (BSA: manufactured by Oriental Yeast) was added and incubated at 37 ° C. for 1 hour. Thereafter, the BSA solution was removed, and the wells were washed three times with 300 μL PBST (0.05% Tween 20 in PBS), and the liquid remaining in the wells was removed by tapping on a paper towel.

The antibody P30 (b) 5 μg / mL in 50% blocking solution 100 μL was added to the well as a primary antibody and incubated at 37 ° C. for 1 hour. Then, the primary antibody solution antibody P30 (b) was removed, and the well was removed with 300 μL PBST (0.05 % Tween20 in PBS).
As a secondary antibody, 1 mg / mL of Anti Mouse IgG (H + L), Rabbit, IgG Whole, Peroxidase Cojugated (manufactured by Wako Pure Chemical Industries, Ltd., code 04-117611) was added to 100 μL of the well and incubated at 37 ° C. for 1.5 hours. Thereafter, the BSA solution was removed, and the wells were washed three times with 300 μL PBST (0.05% Tween 20 in PBS), and the liquid remaining in the wells was removed by tapping on a paper towel.
100 μL of Sure Blue Reserve TMB Microwell Peroxidase Substrate (1-Component) (KPL, code 53-00-01) was added to the well as a chromogenic substrate, reacted for 15 minutes, and then the reaction was stopped by adding 100 μL of 2N sulfuric acid. Then, absorbance at 450 nm was measured with a microplate reader (manufactured by BIORAD).

When the ELISA test was performed by the above method, the absorbance at 450 nm obtained by subtracting the absorbance of the blank (secondary antibody without adding the primary antibody, well in which the color reaction was performed) was 0.061 Abs and less than 0.2 Abs. I was able to confirm. That is, it was confirmed that the antibody P30 (b) was not an antibody that “recognizes strongly the domain III of the P30 protein of Mycoplasma pneumoniae”.

Subsequently, in order to further confirm whether or not this antibody P30 (b) is an antibody “recognizes domain III of P30 protein of Mycoplasma pneumoniae”, the protocol described in paragraphs [0032] to [0039] is followed. A competitive inhibition ELISA test (indirect competitive method) was performed, and whether or not the absorbance decreased to 30% or less was examined as follows.

Nunc Immuno modules (manufactured by Thermo Fisher Scientific, code 469949) ELISA 96-well plate, full length protein Mycoplasma pneumonia P30 (amino acid 1-274: SEQ ID NO: 1) 10 ng / mL in 50 mM carbonate buffer L9. Incubated for 16 hours at 4 ° C. After 16 hours, the P30 solution was removed, and the wells were washed 3 times with 300 μL PBST (0.05% Tween20 in PBS). The liquid remaining in the well was removed by tapping on a paper towel.

In addition to the addition of the primary antibody alone, the primary antibody anti-Mycoplasma pneumoniae P30 antibody (antibody P30 (b)) 5 μg / mL and the amount that is 40 times the amount of the primary antibody P30 (b), test 100 μL of a 50% blocking solution containing Peptide 1 (SEQ ID NO: 6: PGMAPRPGMPPHPGMAPR) or Peptide 2 (SEQ ID NO: 7: PGMAPRPGFPPQPGMAPR) used in Example 1 was added to the well and incubated at 37 ° C. for 1 hour. Thereafter, the primary antibody solution was removed, and the wells were washed 3 times with 300 μL PBST (0.05% Tween 20 in PBS).

As a result, the absorbance of the well added with the primary antibody and the peptide 1 or peptide 2 was not decreased in the peptide 1 compared with the absorbance of the well added only with the primary antibody, and only about 22% in the peptide 2 It was confirmed that the decrease rate was less than 30%. That is, it was confirmed that the antibody P30 (b) was not an antibody that “recognizes strongly the domain III of the P30 protein of Mycoplasma pneumoniae”.

[Comparative Example 2]
Except that both the antibody P30 (a) in the labeling substance solution added to the labeling substance holding part and the antibody P30 (a) in the antibody diluent applied to the detection part were changed to the antibody P30 (b), Example 1 was repeated. The results are shown in Table 3 and FIG.

[Comparative Example 3]
The antibody P30 (a) in the antibody dilution solution applied to the detection part was expressed as an antibody against Mycoplasma pneumoniae P1 protein (Meridian, trade name MAb to Mycoplasma pneumonia P1 (clone B1947M): hereinafter, antibody P1 (b) and Example 1 was repeated except that it was changed to (indicated). The results are shown in Table 3 and FIG.

[Comparative Example 4]
The antibody P30 (a) in the labeling substance solution added to the labeling substance holding part was changed to an antibody against P1 protein of Mycoplasma pneumoniae (Merdian, trade name MAb to Mycoplasma pneumonia P1 (clone B1948M): hereinafter, antibody P1 (a Example 1 was repeated except that it was changed to)). The results are shown in Table 3 and FIG.

[Comparative Example 5]
The antibody P30 (a) in the labeling substance solution added to the labeling substance holding part is changed to the antibody P1 (a), and the antibody P30 (a) in the antibody diluent applied to the detection part is changed to the antibody P1 (b). Example 1 was repeated except that. The results are shown in Table 3 and FIG.

[Example 2]
The composition of the sample diluent was Triton X-100 (trade name, polyethylene glycol mono-p-isooctylphenyl ether, HLB value: 13.7, manufactured by Wako Pure Chemical Industries, Ltd.), Tween 20 (trade name, manufactured by Wako Pure Chemical Industries, Ltd.). Example 1 was repeated except that polyoxyethylene sorbitan monolaurate, HLB value: 16.7) was changed. The results are shown in Table 4 and FIG.
[Comparative Example 6]
Example 2 was repeated except that the antibody P30 (a) in the antibody diluent applied to the detection unit was changed to the antibody P30 (b). The results are shown in Table 4 and FIG.

[Comparative Example 7]
Implemented except that both antibody P30 (a) in the labeling substance solution added to the labeling substance holding part and antibody P30 (a) in the antibody diluent applied to the detection part were changed to antibody P30 (b) Example 1 was repeated. The results are shown in Table 4 and FIG.

[Example 3]
The detection sample was changed to P30 protein ((amino acid 96-274) was expressed and purified by a conventional method using E. coli P30Ag), and the composition of the sample dilution was changed to Triton.
X-100 (trade name, polyethylene glycol mono-p-isooctylphenyl ether, HLB value: 13.7), manufactured by Wako Pure Chemical Industries, Ltd., Tween 20 (trade name, polyoxyethylene sorbitan monolaurate, manufactured by Wako Pure Chemical Industries, Ltd.) , HLB value: 16.7) Example 1 was repeated except that it was changed to a 1: 1 mixture. The results are shown in Table 5 and FIG.

[Comparative Example 8]
Example 3 was repeated except that the antibody P30 (a) in the antibody diluent applied to the detection unit was changed to the antibody P30 (b). The results are shown in Table 5 and FIG.

[Comparative Example 9]
Implemented except that both antibody P30 (a) in the labeling substance solution added to the labeling substance holding part and antibody P30 (a) in the antibody diluent applied to the detection part were changed to antibody P30 (b) Example 3 was repeated. The results are shown in Table 5 and FIG.

[Comparative Example 10]
Example 3 was repeated except that the antibody P30 (a) in the antibody dilution solution applied to the detection unit was changed to the antibody P1 (b). The results are shown in Table 5 and FIG.

[Comparative Example 11]
Example 3 was repeated except that the antibody P30 (a) in the labeling substance solution added to the labeling substance holding part was changed to the antibody P1 (a). The results are shown in Table 5 and FIG.

[Comparative Example 12]
The antibody P30 (a) in the labeling substance solution added to the labeling substance holding part is changed to the antibody P1 (a), and the antibody P30 (a) in the antibody diluent applied to the detection part is changed to the antibody P1 (b). Example 3 was repeated except that. The results are shown in Table 5 and FIG.

From the above results, when both the labeling substance holding part and the detection part contain the antibody P30 (a) that strongly recognizes the domain III of the P30 protein of Mycoplasma pneumoniae (Examples 1 to 3), otherwise It was found that the detection sensitivity of Mycoplasma pneumoniae or P30 protein was remarkably higher than in the case of (Comparative Examples 1 to 12).

[Test Example 4]
In order to confirm whether the antibody P30 (a) in Test Example 2 and the antibody P30 (b) in Comparative Example 1 are antibodies that “recognize domain III of the P30 protein of Mycoplasma pneumoniae”, a competitive inhibition ELISA test (indirect Competition method). In this test, either the peptide 1 or the peptide 2 used in Test Example 1 was used as a domain III fragment to be competed, and the concentration of the peptide was changed as shown in Tables 6 and 7 below (× 1 to x640), a competitive inhibition ELISA test (indirect competitive method) was performed in the same manner as in Test Example 2 or Comparative Example 1. The results are shown in Tables 6 and 7. Table 6 shows the results of using antibody P30 (a) as an antibody, and Table 7 shows the results of using antibody P30 (b) as an antibody. Moreover, the result of Table 6 is shown in FIG. 5, and the result of Table 7 is shown in FIG.

From the results of Table 6 and FIG. 5, when the peptide concentration is 40 times the primary antibody anti-P30 (a) antibody concentration, the well absorbance is reduced by 30% or more compared to the control, and the amount is 80 times the amount. However, it was confirmed that it decreased by 50% or more. Since antibody P30 (a) is inhibited by both peptide 1 and peptide 2, it was confirmed in this test that antibody P30 (a) recognizes the amino acid sequence (PGMAPR) shown in SEQ ID NO: 3. .

Further, from the results shown in Table 7 and FIG. 6, even when the peptide concentration was 40 times the amount of the primary antibody P30 (b), the absorbance of the well decreased only about 22% compared to the control, and was less than 30%. there were. That is, it was confirmed that the antibody P30 (b) was not an antibody that “recognizes strongly the domain III of the P30 protein of Mycoplasma pneumoniae”. Since antibody P30 (b) was not inhibited by peptide 1 but was inhibited by peptide 2, it was confirmed in this test that the amino acid sequence (PGGFPPQ) shown in SEQ ID NO: 5 was recognized.

Although the present invention has been described in detail using specific embodiments, it will be apparent to those skilled in the art that various modifications and variations can be made without departing from the spirit and scope of the invention. This application is based on a Japanese patent application filed on June 1, 2015 (Japanese Patent Application No. 2015-111732) and a Japanese patent application filed on August 31, 2015 (Japanese Patent Application No. 2015-171163). Which is incorporated by reference in its entirety.

According to the present invention, Mycoplasma pneumoniae can be detected quickly and easily with high sensitivity by using an immunochromatography analyzer using an antibody that binds to a specific site of the P30 protein of Mycoplasma pneumoniae. It can be used for early diagnosis.

1 Sample addition part (sample pad)
2 Labeling substance holding part 3 Chromatographic medium part 4 Detection part 5 Absorption part 6 Backing sheet

Claims

An immunochromatography analyzer for detecting Mycoplasma pneumoniae, comprising a sample addition part, a labeling substance holding part, a chromatographic medium part having a detection part and an absorption part,
The immunochromatographic analyzer, wherein the labeling substance holding part and the detection part contain an antibody that strongly recognizes the domain III of the P30 protein of Mycoplasma pneumoniae consisting of the amino acid sequence of SEQ ID NO: 2.
The immunochromatographic analyzer according to claim 1, wherein the antibody recognizes the amino acid sequence of SEQ ID NO: 3 in the domain III.
The immunochromatography according to claim 1 or 2, wherein the labeling substance contained in the labeling substance holding part is gold particles, and the labeling substance holding part contains 0.25 to 0.7 µg / cm 2 of the gold particles. Analysis equipment.
An immunochromatography analysis kit comprising the immunochromatography analyzer according to any one of claims 1 to 3 and a sample diluent for diluting and developing the sample.
The immunochromatographic analysis kit according to claim 4, wherein the specimen diluent contains at least one nonionic surfactant.
6. The immunochromatographic analysis kit according to claim 5, wherein 50% or more of the nonionic surfactant contained in the specimen diluent is a nonionic surfactant having an HLB value of 13 to 18.
A method for detecting Mycoplasma pneumoniae in a specimen using an immunochromatographic analyzer including a sample addition unit, a labeled substance holding unit, a chromatographic medium unit having a detection unit, and an absorption unit, the following step (1) An immunochromatographic analysis method comprising (4).
(1) A step of adding a sample-containing solution obtained by diluting a sample with a sample dilution solution to a sample addition portion (2) P30 protein of Mycoplasma pneumoniae having the amino acid sequence of SEQ ID NO: 2 held in the labeling substance holding portion A step of recognizing Mycoplasma pneumoniae by an antibody that strongly recognizes domain III (hereinafter referred to as antibody P30 (A)) (3) a step of developing the specimen and antibody P30 (A) as a mobile phase in a chromatographic medium part (4) A step of detecting Mycoplasma pneumoniae in the developed mobile phase with the antibody P30 (A) contained in the detection unit.