JP6754618B2 - Immunochromatography test equipment - Google Patents

Description

The present invention relates to an immunochromatography test apparatus.

A simple measuring device (immunochromatography test device) using an immunochromatography method that can quickly and easily detect a specific substance to be detected in a biological sample such as blood or urine by using a specific reaction such as an antigen-antibody. It is widespread.

For example, in Patent Document 1, the detection of a substance to be detected by the immunochromatography method is performed in a labeled antibody holding member and an antibody immobilizing member for the purpose of increasing the elution rate of the labeled antibody in order to obtain a determination result with high sensitivity and speed. It discloses a form in which a deploying member is arranged between and. In Patent Document 2, for the purpose of suppressing the rise of the background and improving the visibility of the test line, a space is provided between the labeled antibody holding member and the antibody immobilizing member, and a deployable pad is provided as a member for bridging them. The arranged form is disclosed. In Patent Document 3, in order to obtain a highly sensitive and quick determination result, a retention suppressing means for suppressing retention of a labeled antibody in a labeled antibody holding member and a labeling substance adjacent to the region downstream in the development direction. An immunochromatography test apparatus having a backflow suppressing means for suppressing backflow to a region upstream in the developing direction is disclosed.

JP 2006-194678 (Sysmex) JP 2013-205336 (Mitsubishi Chemical Medience) JP-A-2014-178152 (Denka Seiken)

However, all the techniques have a false positive problem, and no means for remedying it is disclosed. Naturally, the problems of high detection signals and false positives have not been solved at the same time.
An object of the present invention is to provide an immunochromatographic test apparatus that obtains a high detection signal while suppressing false positives.

As a result of diligent studies to solve the above problems, the present inventor, in order to improve the problem of false positives while obtaining a high detection signal, between the antigen contained in the test solution and the antibody contained in the apparatus. We focused on the reaction time required for the antigen-antibody reaction and the flowability of the test solution. Then, by arranging a deploying member between the labeled antibody holding member and the antibody-immobilizing member and making the flowability in the detection unit where the antibody is immobilized appropriate, a false detection signal can be obtained. Achieved suppression of positivity and completed the present invention.

That is, the present invention is as follows.
[1] An immunochromatography test device for detecting intracellular antigens of bacteria in a test solution, and the test device is
(1) Test droplet lower member and
(2) A labeled antibody-holding member carrying the labeled first intracellular antigen antibody, and
(3) Deployment members and
(4) At least an antibody-immobilizing member on which a second intracellular antigen antibody against the substance to be detected is immobilized;
Each member is in the form of a substantially sheet long in the flow direction of the test liquid, and the members (1), (2), (1), (2), (1), (2), so that a part of each member directly overlaps a part of the adjacent member. They are arranged in the order of 3) and (4);
The member (2) and the member (4) have a structure that does not overlap directly or indirectly.
The average distance L1 of the portion of the member (3) that does not overlap with the member (2) and the member (4) is 2 mm or more and 12 mm or less;
The ratio (L2 / L1) of the distance L2 and L1 at which the member (2) and the member (3) directly overlap is 0.16 ≦ L2 / L1 ≦ 1.
The ratio (L3 / L1) of the distance L3 and L1 at which the member (3) and the member (4) directly overlap is 0.16 ≦ L3 / L1 ≦ 1.
The ratio (T1 / L1) of the thickness T1 of the member (3) to the L1 of the member (3) is 0.02 ≦ T1 / L1 ≦ 0.2; and the basis weight of the member (3) is 49 g / m. ² or more, 75 g / m ² or less glass fiber material,
Immunochromatography test equipment.
[2] The immunochromatographic test apparatus according to 1, wherein the L1 is 8 mm or more and 12 mm or less.
[3] The immunochromatographic test apparatus according to 1 or 2, wherein the capillary flow time of the member (4) antibody-immobilizing member is 100 seconds / 4 cm or more and 140 seconds / 4 cm or less.
[4] The immunochromatographic test apparatus according to any one of 1 to 3, wherein a gold colloid having an average particle diameter of 75 nm or more and 110 nm or less is used for labeling the first antibody.
[5] In any one of 1 to 4, wherein the bacterium is Streptococcus pneumoniae (Streptococcus pneumoniae), Chlamydia pneumoniae, Legionella pneumophila, Haemophilus influenza, Bordetella pertusis, or Mycoplasma pneumoniae. The immunochromatography test apparatus described.
[6] The immunochromatographic test apparatus according to any one of 1 to 5, wherein the intracellular antigen is a ribosomal protein L7 / L12 antigen.
[7] A kit for detecting bacteria, which comprises the immunochromatography test apparatus according to any one of 1 to 6.
[8] The kit according to 7, further comprising an extract or diluent containing a buffer solution and a surfactant, and a tool for collecting a sample.

According to the present invention, in an immunochromatography test, false positives can be suppressed while obtaining a high detection signal, and results can be obtained with high detection sensitivity.

Sectional drawing of the immunochromatography test apparatus. In the figure, a part of the test droplet lower member, the labeled antibody holding member, and the deploying member is described as if they were floating in the air away from the substrate, but this is a simplification of the illustration. This is because, if the structure has a shape that is flexible to bending, it is adhered to the base material. The same applies to FIG. Sectional drawing of an example of the immunochromatography test apparatus of this invention.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. The drawings are used for convenience of explanation, and the scope of the present invention is not limited to the embodiments shown in the drawings. In the following description, the notation "length" means the distance along the major axis direction of the immunochromatographic test apparatus, and "width" means the distance along the minor axis direction of the immunochromatographic test apparatus. To do.

FIG. 1 is a cross-sectional view of the prior art immunochromatography. FIG. 2 is a cross-sectional view of an example of the immunochromatographic test apparatus of the present invention. This will be described below with reference to FIG.

In this immunochromatography test apparatus, a test droplet lower member 1 and a labeled antibody holding member 2 holding a first antibody against a labeled substance to be detected and a second antibody against the substance to be detected are placed on a substrate 6. The antibody-immobilizing member 4 and the absorbing member 5 are provided. The labeled antibody holding member 2 holds an antibody labeled with colored particles, and this antibody binds to an antigen as a substance to be detected to form a complex. The antibody-immobilizing member 4 includes a line-shaped capture site 7, and an antibody that binds to an antigen is immobilized at this site.

Next, the detection of the substance to be detected by the immunochromatography method by this apparatus will be described. First, when a test solution prepared by diluting a sample such as a body fluid with a developing solvent is dropped onto the test droplet lower member 1, the test solution moves to the labeled antibody holding member 2 due to a capillary phenomenon. In the labeled antibody holding member 2, the antibody labeled with the colored particles elutes into the test solution. When the test solution contains an antigen as a substance to be detected, the antigen-antibody reaction binds the antigen to the antibody to form a complex. Next, the test solution moves to the capture site 7 of the antibody immobilization member 4 due to the capillary phenomenon. At the capture site 7, the antigen is captured by the antibody immobilized at the capture site 7 by the antigen-antibody reaction. Since the antigen forms a complex with the antibody labeled with the colored particles, the color of the colored particles appears in a line at the capture site 7. By visually observing this colored line, the presence or absence of the substance to be detected in the sample is confirmed.

[Bacteria, antigens (substances to be detected), antibodies]
In the present invention, an antibody against the substance to be detected is used.
The substance to be detected in the present invention can be any substance that can be measured by the immunochromatography method, but the effect of the present invention can be exhibited particularly if it is a component of bacteria or a substance secreted by bacteria. It is possible.

The bacteria to be detected are not particularly limited, but the present invention is excellent against Streptococcus pneumoniae (Streptococcus pneumoniae), Chlamydia pneumoniae, Legionella pneumophila, Haemophilus influenza, Bordetella pertusis, and Mycoplasma pneumoniae. Can be effective. In particular, Streptococcus pneumoniae has a remarkable effect.

Further, the component to be detected derived from bacteria to be detected is not particularly limited, but can be, for example, a component of cells, preferably an intracellular antigen. As the intracellular antigen, the ribosomal protein L7 / L12 antigen is preferable. A remarkable effect can be exhibited in the case of L7 / L12 ribosome reprotein of Streptococcus pneumoniae. The L7 / L12 ribosomal protein has high detection sensitivity due to the presence of multiple copies in the cell.

Antibodies to such substances to be detected can distinguish specific bacteria causing bacterial infections from other bacteria by species or genus by the known methods described below, as shown in later examples. The antibody can actually be obtained.
The above antibody can be prepared by the method described in International Publication No. 00/06603. When the bacterial ribosome protein L7 / L12 is used as an antigen, it can be prepared by using the full-length protein of the bacterial ribosome protein L7 / L12 or a partial peptide thereof as an antigen, but the full-length protein is preferable. This partial peptide or full-length protein is contained as it is, or after cross-linking with a carrier protein, it is inoculated into an animal with an adjuvant as needed, and the serum thereof is collected to contain an antibody (polyclonal antibody) that recognizes the Ribosomal Protein L7 / L12 protein. Antiserum can be obtained. It is also possible to purify the antibody from antiserum and use it. The animals to be inoculated include sheep, horses, goats, rabbits, mice, rats and the like, and sheep, rabbits and the like are particularly preferable for producing polyclonal antibodies. Further, as the antibody, it is more preferable to apply a monoclonal antibody obtained by a known method for producing hybridoma cells, but in this case, a mouse is preferable. By screening as the monoclonal antibody, a monoclonal antibody that reacts with the ribosomal protein L7 / L12 of a specific bacterium that causes bacterial infection and does not react with the ribosomal protein L7 / L12 of a bacterium other than the specific bacterium. It can be useful for diagnosing whether or not a person has an infection caused by the bacterium.
In addition, the antibody may be used as F (ab') 2, Fab', or Fab fragment by enzymatic treatment or chemical treatment.

[Immunochromatography test equipment]
The immunochromatographic test apparatus of the present invention is
(1) Test droplet lower member and
(2) A labeled antibody-holding member carrying the labeled first intracellular antigen antibody, and
(3) Deployment members and
(4) At least an antibody-immobilizing member for immobilizing a second intracellular antigen antibody against the substance to be detected is provided.
(5) An absorbing member may be provided.

(1) The member under the test droplet will be described.
The test droplet lower member has a role of absorbing the test solution dropped on the device and ensuring the flowability to the labeled antibody holding member by the capillary phenomenon, and the material used is any material that can perform the immunochromatography method. Although not particularly limited, fiber matrix, filter paper, glass fiber, sponge, cotton and the like using recycled cellulose or cell roll derivative such as rayon or cupra as a raw material are preferable. More preferably, it is cupra.

If the volume of the test droplet lower member is too small, the test solution cannot be received, causing leakage to the outside of the test device or an unintended short circuit of the liquid inside the device, and if it is too large, the test solution is held too much, and the test is performed. Impairs the speed of. From this viewpoint, the length is preferably 8 mm or more, more preferably 10 mm or more, and further preferably 12 mm or more. The length is preferably 28 mm or less, more preferably 24 mm or less, and further preferably 20 mm or less. The width is preferably 2 mm or more, more preferably 3 mm or more, and further preferably 4 mm or more in any case. The width is preferably 10 mm or less, more preferably 8 mm or less, and further preferably 6 mm or less. The thickness is preferably 0.05 mm or more, more preferably 0.10 mm or more, and even more preferably 0.20 mm or more, regardless of the length and width. The thickness is preferably 2.00 mm or less, more preferably 1.00 mm or less, and even more preferably 0.80 mm or less.

(2) A labeled antibody-holding member holding the labeled first antibody will be described.
The labeled antibody holding member holds the antibody labeled inside thereof in a dry state, and in the immunochromatography test, has a role of eluting the labeled antibody into the test solution while moving the test solution by capillarity. ..

Next, labeling and labeling will be described.
(Sign)
In the present invention, the first antibody held on the labeled antibody holding member is labeled in order to obtain a detectable signal. Examples of the label used in the present invention include colored particles, enzymes, radioisotopes and the like, but it is preferable to use colored particles that can be visually detected without the need for special equipment. Examples of the colored particles include metal fine particles such as gold and platinum, non-metal particles, and latex particles.

The colored particles may be of any size as long as they can be transported downstream through the voids of the test piece, but as shown in later examples, the L7 / L12 ribosomal protein of Streptococcus pneumoniae was detected. When used as a substance, a gold colloid having an average particle size of 35 nm or more and 160 nm or less is preferable. The average particle size of the gold colloid is more preferably 55 nm or more and 125 nm or less. More preferably, it is 75 nm or more and 105 nm or less. When the average particle size is 35 nm or less, the particles aggregate with each other, causing clogging when used in a chromatographic medium, or insensitivity when used in a chromatography device. The larger the particle size, the better the visibility, but if it is too large, the color of the gold colloid itself may be faded, and there is a concern that clogging may occur in the chromatographic fee apparatus. In order to improve the sensitivity of the immunochromatography kit, two or more kinds of insoluble carriers having an average particle size may be mixed and used.

The average particle size of the particles referred to in the present invention refers to a dispersion obtained by measuring a dispersion liquid in which particles are dispersed in a liquid with a particle size measuring device. Some particle size measuring devices apply various measuring principles, but in the present invention, unless otherwise specified, a particle size measuring device by a dynamic light scattering method is used. Specifically, "Zeta Sizar Nano ZS" manufactured by Malvern was used. Here, the average particle size is the value of the harmonic mean particle size (diameter) based on the scattered light intensity standard, and the particle size measurement method in a liquid by the dynamic light scattering method is JIS Z8826: 2005 particle size analysis-photon correlation method. It is determined in.

Next, a method of binding colored particles to the first antibody (labeling method) will be described.
Examples of the method for labeling the antibody with colored particles include a method by physical adsorption, a method by covalent bond, and a method by a combination thereof. From the viewpoint of ease of manufacture, a labeling method by physical adsorption is preferable. That is, a dispersion of colored particles prepared to a predetermined concentration is prepared, a buffer solution and an antibody are added, and the antibody is physically adsorbed on the colored particles after a certain period of time. After that, a blocking agent is further added and left for a certain period of time to block the particle surface.
As the blocking agent, various compositions can be used depending on the composition of the substance to be detected, the sample, or the solution for diluting the sample, and the surfactant, sugar, protein, and water-soluble substance can be added to any buffer composition solution. Those in which a synthetic polymer or the like is dissolved are preferable.

Surfactants include Tween20, TritonX100, SDS, Benzalkonium chloride, Zwittergent3-12 and the like, sugars include sucrose, trehalose and dextran, and proteins include BSA, casein, skim milk, fish gelatin and the like. Examples of the molecule include polyvinyl alcohol and polyethylene glycol.

The colored particle dispersion after antibody adsorption and blocking is centrifuged to remove the supernatant, and then suspended in a predetermined dispersion to prepare a labeled antibody solution. The obtained labeled antibody solution can be impregnated into the holding member and dried at room temperature or freeze-dried to obtain a labeled antibody holding member.

The material used for the labeled antibody holding member is a fiber sheet made of glass fiber, polyvinyl alcohol (PVA) fiber, polyester fiber, cell roll fiber or the like, or a mixture of the fibers (for example, a combination of glass fiber and PVA fiber). A fiber sheet or the like made of a combination of a cellulose type and glass) can be used, and a glass fiber sheet is preferable.

The properties of the fiber sheet are represented by the basis weight (unit: g / m ² ) and thickness. When the texture is low and the thickness is large, the internal porosity becomes high and the labeled antibody solution and the test solution are easily retained. However, when the porosity is too high, the flowability and development of the test solution in the holding member are increased. Since the flowability to the working member deteriorates and the test solution stays in the labeled antibody holding member, false positives are likely to occur. On the other hand, when the basis weight is high and the thickness is small, the internal porosity becomes low, the test solution supplied to the apparatus becomes difficult to absorb, the flowability of the test solution is significantly impaired, or the test solution itself becomes It will not flow.

Further, if the volume of the holding member is too small, it becomes difficult to handle the step of impregnating the labeled antibody solution in the manufacturing process. If it is too large, the test solution will be retained too much, which will impair the speed of the test. From this point of view, the length is preferably 5 mm or more, more preferably 6 mm or more, and further preferably 8 mm or more. The length is preferably 20 mm or less, more preferably 17 mm or less, and further preferably 14 mm or less. The width is preferably 2 mm or more, more preferably 3 mm or more, and further preferably 4 mm or more in any case. The width is preferably 10 mm or less, more preferably 8 mm or less, and further preferably 6 mm or less. The thickness is preferably 0.05 mm or more, more preferably 0.10 mm or more, and even more preferably 0.20 mm or more, regardless of the length and width. The thickness is preferably 1.20 mm or less, more preferably 0.80 mm or less, and even more preferably 0.40 mm or less.

From the viewpoint of making the flowability of the labeled antibody holding member appropriate, the basis weight may be specified. The basis weight can be 30 g / m ² or more, preferably 40 g / m ² or more, more preferably 45 g / m ² or more, and 49 g, regardless of the above conditions. It is more preferably / m ² or more. The basis weight is preferably 90 g / m ² or less, more preferably 75 g / m ² or less, and even more preferably 60 g / m ² or less.

(3) The deploying member will be described.
The material used for the developing member is a material that allows the test solution to flow by a capillary phenomenon, and the material is the same as that of the test droplet lower member or the labeled antibody holding member, and among them, a glass fiber material is preferable.
In the present invention, it is presumed that the developing member provides a main field for the antigen-antibody reaction to secure the reaction time and also contributes to ensuring the flowability of the test solution. That is, the reaction in which the labeled antibody eluted in the test solution and the antigen contained in the test solution form a complex by the antigen-antibody reaction proceeds while the test solution moves in the developing member, and thus develops. It is presumed that the long length of the member means that the reaction time is long, which is advantageous for the formation of more complexes.
On the other hand, if the deploying member is too long, the test solution stays in the deploying member, so that false positives are likely to occur.

From the above, the length of the deploying member is preferably 8 mm or more, more preferably 10 mm or more, and further preferably 12 mm or more. The length is preferably 20 mm or less, more preferably 18 mm or less, and even more preferably 16 mm or less. The width is preferably 2 mm or more, more preferably 3 mm or more, and further preferably 4 mm or more in any case. The width is preferably 10 mm or less, more preferably 8 mm or less, and further preferably 6 mm or less. The thickness is preferably 0.05 mm or more, more preferably 0.10 mm or more, and even more preferably 0.20 mm or more, regardless of the length and width. The thickness is preferably 1.20 mm or less, more preferably 0.80 mm or less, and even more preferably 0.40 mm or less. The basis weight can be 30 g / m ² or more, preferably 40 g / m ² or more, more preferably 45 g / m ² or more, and 49 g, regardless of the above conditions. It is more preferably / m ² or more. The basis weight is preferably 90 g / m ² or less, more preferably 75 g / m ² or less, and even more preferably 60 g / m ² or less.

(4) An antibody-immobilizing member on which a second antibody against a substance to be detected is immobilized will be described.
The material used for the antibody-immobilizing member is not particularly limited as long as it can perform an immunochromatography method, but is preferably nitrocellulose, mixed nitrocellulose ester, polyvinylidene fluoride, nylon or the like. Preferably, it is nitrocellulose.

It is preferable to select an antibody-immobilizing member having an appropriate flowability, that is, a capillary flow rate. Capillary flow rate refers to the speed at which the front line of the liquid moves along the material when the liquid is added to one end of the material. The material used for the antibody-immobilizing member has a porous structure in order to allow the test solution to move due to the capillary phenomenon. The capillary flow rate is influenced by the pore size, pore size distribution, and porosity of this porous structure. As the pore size increases or the porosity increases, the capillary flow rate tends to increase. However, it is difficult to measure its value accurately because its speed decreases exponentially as the liquid travels through the material. For this reason, capillary flow time is used as a parameter that is easier to measure. This is a value (seconds / cm) indicating the time during which pure water travels through the target member of a predetermined length and is completely wet. These are described in detail in the "Lateral Flow Test Strip Development Guide (TB500JA00)" published by Merck Millipore. Specifically, the capillary flow time (seconds / 4 cm) is measured as follows: A target member with a length of 4 cm and a width of 1 cm is leaned vertically and leaned against the pure water prepared in advance at the bottom of the leaning ground. Immerse the lower end of the target member. The time from the leaning (start of suction) until the pure water reaches the upper end of the target member is measured and used as the capillary flow time (seconds / 4 cm).

If the flowability of the test solution is too fast, the substance to be detected and the second antibody do not sufficiently react with each other, resulting in a decrease in detection sensitivity. Further, if the flowability of the test solution is too slow, the test solution stays in the antibody-immobilizing member, so that false positives are likely to occur. From this point of view, the capillary flow time is preferably 65 seconds / 4 cm or more, more preferably 80 seconds / 4 cm or more, and further preferably 95 seconds / 4 cm or more. The capillary flow time is preferably 250 seconds / 4 cm or less, more preferably 200 seconds / 4 cm or less, and further preferably 145 seconds / 4 cm or less.

In addition, a second antibody against the substance to be detected is immobilized on the antibody-immobilizing member at a capture site at an arbitrary distance from the end on the side of the start point of chromatographic development. It is known that the capillary flow rate decreases as the distance from the starting end increases. Therefore, if the position of the capture site is too close to the starting end, the flowability is too fast, and the substance to be detected does not sufficiently react with the second antibody, resulting in a decrease in detection sensitivity. Further, in order to make the position of the capture site far from the starting end, it is necessary to increase the length of the antibody-immobilizing member itself, but if it is too long, it takes time to deploy the test solution and the speed of the test is impaired. Is done. From this viewpoint, the length of the antibody-immobilizing member (the length of the entire member) is preferably 13 mm or more, more preferably 15 mm or more, and further preferably 17 mm or more. The length is preferably 50 mm or less, more preferably 40 mm or less, and even more preferably 30 mm or less. The width of the antibody-immobilizing member may be defined, and the width is preferably 2 mm or more, more preferably 3 mm or more, and further preferably 4 mm or more in any case. preferable. The width is preferably 10 mm or less, more preferably 8 mm or less, and further preferably 6 mm or less.

Next, a method for manufacturing the antibody-immobilizing member will be described.
For immobilization of the antibody on the antibody immobilization member, an immobilization method by physical adsorption is preferable from the viewpoint of ease of production. That is, the second antibody can be immobilized on the member by applying the solution containing the antibody to the capture site of the immobilization member in a line shape and then drying the solution.

After that, blocking may be performed by immersing the immobilizing member in a blocking agent. As the blocking agent, various compositions can be used depending on the composition of the substance to be detected, the sample, or the solution for diluting the sample, and the surfactant, sugar, protein, and water-soluble substance can be added to any buffer composition solution. Those in which a synthetic polymer or the like is dissolved are preferable.

Surfactants include Tween20, TritonX100, SDS, Benzalkonium chloride, Zwittergent3-12 and the like, sugars include sucrose, trehalose and dextran, and proteins include BSA, casein, skim milk, fish gelatin and the like. Examples of the molecule include polyvinyl alcohol and polyethylene glycol.

(5) The absorbing member will be described.
The absorbing member has a role of absorbing the test solution after the antibody-immobilizing member is deployed, and the material used is not particularly limited as long as it can perform the immunochromatography method, but cellulose filter paper or the like is preferable.
If the water absorption capacity of the absorbing member is small, the ability to absorb the test solution is weakened, causing the test solution to stagnate in the upstream member, and false positives are likely to occur. From the viewpoint of ensuring the flowability of the entire test apparatus, the length is preferably 16 mm or more, more preferably 18 mm or more, and further preferably 20 mm or more. The length is preferably 28 mm or less, more preferably 26 mm or less, and further preferably 24 mm or less. The width is preferably 2 mm or more, more preferably 3 mm or more, and further preferably 4 mm or more in any case. The width is preferably 10 mm or less, more preferably 8 mm or less, and further preferably 6 mm or less. The thickness is preferably 0.50 mm or more, more preferably 1.00 mm or more, and even more preferably 1.0 mm or more, regardless of the length and width. The thickness is preferably 4.00 mm or less, more preferably 3.00 mm or less, and even more preferably 2.00 mm or less. The water absorption capacity is preferably 90 mg / cm ² or more, more preferably 150 mg / cm ² or more, and further preferably 200 mg / cm ² or more.

The materials, standards, and the like of each member constituting the immunochromatographic test apparatus of the present invention have been described above. In the immunochromatographic test apparatus of the present invention, it is important that the method of arranging each member also satisfies a specific condition. Therefore, the method of arranging each member will be described below.

[(1) Method of arranging test droplet lower member and (2) labeled antibody holding member]
The overlap between the (1) test droplet lower member and the (2) labeled antibody holding member is at least a length that allows the liquid to communicate with each other, and is preferably 1 mm or more from the viewpoint of manufacturing to ensure the overlap. ..

[(2) Labeling antibody holding member, (3) Deployment member, and (4) Antibody immobilization member placement method]
The length (L2) of the direct overlap between the labeled antibody holding member and the deploying member is not particularly limited as long as it is within the range naturally limited from the range of other requirements, but should be 0.5 mm or more. The thickness is preferably 1.0 mm or more, and more preferably 1.8 mm or more. Further, L2 can be 12.0 mm or less, preferably 10.0 mm or less, and more preferably 9.5 mm or less.

The length (L3) of the direct overlap between the deploying member and the antibody-immobilizing member is at least the length at which the liquids communicate with each other, and is 0.5 mm or more from the viewpoint of manufacturing to ensure the overlap. It is preferably 1.0 mm or more, and more preferably 1.8 mm or more. Further, L3 is preferably 4.0 mm or less, more preferably 3.5 mm or less, and further preferably 3.0 mm or less.

Further, the distance (L1) between the downstream end of the labeled antibody holding member and the upstream end of the antibody-immobilizing member, that is, the distance (L1) at which the labeled antibody-holding member and the antibody-immobilizing member do not directly and indirectly overlap is defined. It is preferably 2 mm or more, more preferably 6 mm or more, and further preferably 8 mm or more. Further, L1 is preferably 12 mm or less. If the distance is short, a sufficient reaction time cannot be secured and a sufficient signal improving effect cannot be obtained. If the distance is long, the labeled antibody-holding member becomes long, so that the test solution is held too much, which impairs the speed of the test.

Further, the ratio (L2 / L1) of the distance (L2) and (L1) at which the labeled antibody holding member and the deploying member directly overlap is preferably 0.1 ≦ L2 / L1 ≦ 3.0. It is more preferable that 0.15 ≦ L2 / L1 ≦ 1.0, and further preferably 0.2 ≦ L2 / L1 ≦ 1.5. The ratio (L3 / L1) of the distance (L3) and (L1) at which the deploying member and the antibody-immobilizing member directly overlap is preferably 0.1 ≦ L2 / L1 ≦ 3.0. It is more preferable that 15 ≦ L2 / L1 ≦ 1.0, and further preferably 0.2 ≦ L2 / L1 ≦ 1.5.

The ratio (T1 / L1) of the thickness (T1) of the deploying member to the above (L1) of the deploying member is preferably 0.018 ≦ T1 / L1 ≦ 0.150, preferably 0.020 ≦ T1 /. It is more preferable that L1 ≦ 0.070, and further preferably 0.020 ≦ T1 / L1 ≦ 0.050.

[(4) Method of arranging antibody-immobilizing member and (5) absorbing member]
The overlap between the (4) antibody-immobilizing member and the (5) absorbing member is at least a length that allows the liquid to communicate with each other, and is preferably 1 mm or more and 3 mm or less from the viewpoint of manufacturing to ensure the overlap. ..

The relationship between the arrangement of each member has been described above.
In the present invention, an immunochromatographic test apparatus in which each constituent member is laminated on a substrate may be used as it is as an immunochromatographic test apparatus, or may be stored in a case and used as an immunochromatographic test apparatus.

The base material has a role of fixing the arrangement position of each member and ensuring the connection between the members, and the material may be various materials such as plastic and paper having an adhesive layer on the surface. it can. In addition, the adhesive layer can be selected from those that do not interfere with all the reagents used in the test. A polystyrene sheet having a length of 6.0 or more, 9.0 cm or less, a width of 3 mm or more, 7 mm or less, a thickness of 200 μm or more, and 300 μm or less with an adhesive on the surface is preferable.

The length and width of the immunochromatographic test piece can be appropriately determined according to the sample and purpose, but if the width of the immunochromatographic test piece is too wide, an extra test solution is required, and if it is too narrow, the line The width is preferably 3 mm or more and 8 mm or less because it interferes with the judgment. If the length of the immunochromatographic test piece is too long, it takes time to develop the test solution, and the speed of the test is impaired. On the other hand, if it is too short, the reaction and separation in the immunochromatography method will not be sufficiently performed, and the detection sensitivity will be lowered. Therefore, it is preferably 4 cm or more and 15 cm or less.

[kit]
The present invention provides a kit including an immunochromatography test apparatus. In addition to the immunochromatography device, the kit may include a test solution (diluted solution or extract), a tool for collecting a sample, such as a cotton swab, and / or a suction catheter.

(Test solution)
The test solution will be described.
In the present invention, the substance to be detected (intracellular antigen) is detected using the above-mentioned test apparatus, and the test solution containing the substance to be detected is a test solution obtained by mixing a sample and a diluted solution at an arbitrary ratio. Can be. As the sample, urine, blood, serum, plasma, and upper respiratory tract sample (for example, pharyngeal swab, nasal swab, nasal suction, saliva, sputum, etc.) can be used. It is also possible to extract the substance to be detected with an extract from a collection tool such as a cotton swab that has scraped the affected area, and use it as a test solution to be provided to the test apparatus. In addition, a diluent may be added after the extraction operation.

The composition of the test solution affects the speed at which it moves through each member in the immunochromatography test apparatus by capillarity. In general, the faster the moving speed, the shorter the time to complete the test, but the lower the detection signal tends to be. On the other hand, if the moving speed is slow, the detection signal tends to be high, but if it is too slow, false positives may occur due to the retention of the test solution.

According to the study by the present inventors based on this point, an extract or a diluent obtained by dissolving a surfactant, a sugar, a protein, a water-soluble synthetic polymer, or the like in an arbitrary buffer composition solution is used. Is preferable. With any buffer composition, the pH of the sample can be a factor that affects the reactivity of the antigen-antibody reaction, so the solution for diluting the sample has a pH of 5.5 or more and 8.5 or less. It is preferable that the buffer is buffered so as to be. In order to have a buffering action, the diluted solution of the sample preferably contains a buffer solution such as various Good's buffers. Further, from the viewpoint of the influence on the antigen-antibody reaction, a salt may be added for the purpose of adjusting the ionic strength. Those skilled in the art can appropriately design the selection of the buffer solution and the adjustment of the ionic strength.

Examples of the surfactant include Tween20, TritonX100, SDS, Benzalkonium chloride, Zwittergent3-12 and the like. The lower limit of the concentration of the surfactant can be such that the final concentration when mixed with the sample is 0.03% or more, preferably 0.05% or more, in order to ensure the flowability of the test solution. It is more preferably 0.075% or more, and particularly preferably 0.1% or more. The upper limit of the concentration of the surfactant may be such that it does not significantly inhibit the antigen-antibody reaction, and in any case, it can be 10% or less, preferably 7.5% or less. , More preferably 5% or less, and particularly preferably 3% or less. Examples of sugars include sucrose, trehalose, dextran and the like, examples of proteins include BSA, casein, skim milk and fish gelatin, and examples of water-soluble synthetic polymers include polyvinyl alcohol and polyethylene glycol. The concentration of the lysate selected from sugars, proteins, water-soluble polymers and the like can be such that the total of them has a final concentration of 0.03% or more when mixed with the sample, and is preferably 0. It is 0.05% or more, more preferably 0.075% or more, and particularly preferably 0.1% or more. Further, the upper limit value may be such that it does not significantly inhibit the flowability of the liquid and the antigen-antibody reaction, and in any case, it can be 15% or less, preferably 10% or less, more preferably. Is 8% or less, and particularly preferably 5% or less.

The present invention will be further described with reference to the following examples, but the present invention is not limited to the examples.
[1: Examination of dimensions and overlap of each member in immunochromatography test equipment]
An immunochromatography test device was prepared as follows, and the dimensions and overlap of each member were examined.
(1) Preparation of immunochromatography test apparatus (a) Preparation of a member holding a labeled antibody A monoclonal antibody derived from the REAMSP-8 strain was used as the gold colloid-labeled Streptococcus pneumoniae ribosome protein L7 / L12 antibody.
Colloidal gold solution manufactured by Tanaka Kikinzoku Co., Ltd. (particle size 80 nm. The average value of three values measured using Zeta Nanosizer Nano ZS manufactured by Malvern is the standard value of 80.0 ± 4.0) 0 in 0.9 mL .1M CHES buffer pH 9.0 was mixed, 80 μg / mL of anti-REAMSP-8 ribosome protein L7 / L12 antibody labeled with gold colloid was added, and the mixture was allowed to stand at room temperature for 10 minutes to bind this antibody to the surface of gold colloid particles. , A 1% aqueous solution of caseine was added so that the final concentration in the gold colloid solution was 0.1%, and the remaining surface of the gold colloid particles was blocked with casein to obtain a gold colloid-labeled antiribosome protein L7 / L12 antibody (hereinafter , “Gold colloid labeled antibody”) solution was prepared. This solution was centrifuged (15000 × rpm, 5 minutes) to precipitate a gold colloid-labeled antibody, and the supernatant was removed to obtain a gold colloid-labeled antibody. This gold colloid-labeled antibody was suspended in a 20 mm Tris-hydrogen buffer (pH 8.2) containing 0.25% casein and 2.5% sculose to obtain a gold colloid-labeled antibody solution.
A 10 mm × 150 mm strip-shaped glass fiber sheet # 8975 (manufactured by Paul, 49 g / m ² ) is impregnated with 0.63 mL of a gold colloid-labeled antibody solution, dried under reduced pressure at room temperature, and a gold colloid-labeled antibody holding member. And said.

(B) Preparation of antibody-immobilizing member A nitrocellulose membrane UniSart CN95 (manufactured by Sartorius, capillary flow time 100 seconds / 4 cm) having a width of 25 mm and a length of 300 mm was prepared as a raw material for the antibody-immobilizing member.
A solution containing 2.0 mg / mL of Streptococcus pneumoniae ribosomal protein L7 / L12 monoclonal antibody derived from the REAMPP-5 strain was placed at 1 μL / cm at a position 10 mm from the end of the antibody-immobilized member on the side of the starting point of chromatographic development. It was applied in a line and dried at 50 ° C. for 60 minutes, then dipped in a 0.1% casein solution and dried overnight at room temperature. The capture site 7 of the complex of Streptococcus pneumoniae ribosomal protein L7 / L12 antigen and colloidal gold-labeled antibody was used.

(C) Assembly of Immunochromatography Test Equipment In addition to the antibody-immobilizing member and the labeled antibody-holding member, regenerated cellulose non-woven fabric UR601 (manufactured by Asahi Kasei Fibers) as a test droplet lower member and glass fiber sheet # 8975 (manufactured by Asahi Kasei Fibers) as a developing member. (Manufactured by Paul) and cellulose filter paper CF7 (manufactured by GE Healthcare) were prepared as an absorbent member. Then, these members were attached to the base material ARcare9020 (manufactured by Adhesives Research, Inc.) and then cut into a width of 5 mm to prepare an immunochromatographic test apparatus similar to that in FIG.
For comparison, an immunochromatography test device similar to that shown in FIG. 1 was manufactured as a general immunochromatographic test device that does not use a developing member.

(2) Test method immunochromatography test as an antigen, a pneumococcal (about 1 × 10 ⁵ CFU / ml) prepared mock samples diluted 10-fold with PBS, specimen diluent (TritonX-100 0.5 %, 0.25% casein, 0.5% dextran, NaCl 0.2M) and 1: 1 mixture was used as a test solution. Here, CFU indicates a Colony Forming Unit: a colony forming unit, and indicates the number of living cells in a target sample.

Then, 120 μl of the test solution was dropped onto the test droplet lower member 5 of the immunochromatographic test apparatus obtained in (1) above with a micropipette to start the immunochromatographic test, and after 30 minutes at room temperature, the capture site 7 was described. The presence or absence of capture of the complex of the ribosome protein L7 / L12 antigen captured in 1 and the gold colloid-labeled antibody was determined by the density of the magenta line that increased or decreased in proportion to the capture amount.
The test line intensity was evaluated semi-quantitatively from "+" to "7+" from light to dark red-purple lines.
In addition, if the test line color was observed in the test on the sample containing no antigen, it was considered as a false positive.

(Test 1)
Table 1 shows the results of the immunochromatography test when the length of the member under the test droplet, the length of the developing member, and the combination of L2 and L1 were changed.

The distance (L1) at which the labeled antibody-holding member and the antibody-immobilizing member do not directly or indirectly overlap is determined.
The effect was obtained at 2 mm or more and 12 mm or less, and a higher effect was obtained at 8 mm or more and 12 mm or less.

This is because when the labeled first labeled antibody is eluted in the test solution and then reacts with the antigen contained in the test solution, the test solution moves in the developing member and undergoes an antigen-antibody reaction. As the reaction progresses, the reaction time is lengthened in proportion to the length of the developing member, and as a result, a sufficient amount of the complex can be formed between the antigen and the first antibody, and these are sequentially fixed to the antibody. When it moved to the capture site of the chemical member, it was captured by the immobilized second antibody, and a high detection signal could be obtained at the time of determination. This is consistent with the increase in test line strength as L1 increases.
On the other hand, when L1 was 14 mm or more, false positives due to retention of the test solution were observed in the test using the sample containing no antigen.
Further, the length (L2) of the portion where the deploying member and the labeled antibody holding member directly overlap each other showed the same effect in the range of 2 mm or more and 9 mm or less.

[2: Examination of materials of each member in immunochromatography test equipment]
An immunochromatography test device was prepared as follows, and the materials of each member were examined.
(1) Preparation of Immunochromatography Test Device An immunochromatography test device was prepared in the same manner as in Example 1 except that the material of each member was changed. In addition, the length and overlapping arrangement of each member are unified as follows.
Length of test droplet lower member 14 mm
Length of labeled antibody holding member 10 mm
Deployment member length 14 mm
L2 = 2mm, L3 = 2mm, L1 = 10mm

(2) Test method The immunochromatographic test was carried out in the same manner as in [1].

(Test 2)
Table 2 shows the results of the immunochromatography test when the developing member was changed.

The effect was shown in the range where the thickness of the deploying member was 0.28 mm or more and 0.44 mm or less, and the basis weight was 49 g / m ² or more and 75 g / m ² or less.

(Test 3)
Table 3 shows the results of the immunochromatography test when the antibody-immobilizing member was changed.

The effect was shown in the range of capillary flow time of 100 seconds / 4 cm or more and 140 seconds / 4 cm or less.

[3: Examination of colloidal gold particle size in immunochromatography test equipment]
An immunochromatography test device was prepared as follows, and the particle size of gold colloid was examined.
(1) Preparation of Immunochromatography Test Device An immunochromatography test device was prepared in the same manner as in Example 1 except that the particle size of the gold colloid used for labeling was changed. In addition, the length and overlapping arrangement of each member are unified as follows.
Length of test droplet lower member 14 mm
Length of labeled antibody holding member 10 mm
Deployment member length 14 mm
L2 = 2mm, L3 = 2mm, L1 = 10mm

(2) Test method immunochromatography test as antigens, pneumococcus (approximately 1 × 10 ⁵ CFU / ml) prepared mock samples serially diluted in PBS, sample dilution buffer (TritonX-100 0.5 %, 0.25% casein, 0.5% dextran, NaCl 0.2M) and 1: 1 mixture was used as a test solution. Antigen concentrations were tested at 8 × 10 ³ CFU / ml, 4 × 10 ³ CFU / ml, 2 × 10 ³ CFU / ml, and 1 × 10 ³ CFU / ml, respectively.

Then, 120 μl of the test solution was dropped onto the test droplet lower member 5 of the immunochromatographic test apparatus obtained in (1) above with a micropipette to start the immunochromatographic test, and after 15 minutes at room temperature, the capture site 7 was described. The presence or absence of capture of the complex of the ribosome protein L7 / L12 antigen captured in 1 and the gold colloid-labeled antibody was determined by whether or not the determination line was observed.

(Test 4)
Table 4 shows the results of the immunochromatography test when the particle size of the gold colloid used for labeling was changed.

Since the color tone changes between gold colloids of different particle sizes, the evaluation was performed based on the detection limit when the amount of antigen was gradually reduced, not on the comparison of the strength of the test line. As a result, 80 nm and 100 nm (80 nm) Similarly, using the Zeta Nanosizer Nano ZS manufactured by Malvern, the average value of the three measured values was the standard value of 100.0 ± 5.0), and the same minimum detection sensitivity was shown.

By using the immunochromatographic test apparatus of the present invention, results can be obtained with high detection sensitivity.

1. 1. Test droplet lower member 2. Labeled antibody holding member 3. Deployment member 4. Antibody immobilization member 5. Absorbent member 6. Base material 7. Capture site

Claims (8)

An immunochromatography test device for detecting intracellular antigens of bacteria in a test solution, the test device is
(1) Test droplet lower member and
(2) A labeled antibody-holding member carrying the labeled first intracellular antigen antibody, and
(3) Deployment members and
(4) At least an antibody-immobilizing member on which a second intracellular antigen antibody against the substance to be detected is immobilized;
Each member is in the form of a substantially sheet long in the flow direction of the test liquid, and the members (1), (2), (1), (2), (1), (2), so that a part of each member directly overlaps a part of the adjacent member. They are arranged in the order of 3) and (4);
The member (2) and the member (4) have a structure that does not overlap directly or indirectly.
The average distance L1 of the portion of the member (3) that does not overlap with the member (2) and the member (4) is 2 mm or more and 12 mm or less;
The ratio (L2 / L1) of the distance L2 and L1 at which the member (2) and the member (3) directly overlap is 0.16 ≦ L2 / L1 ≦ 1.
The ratio (L3 / L1) of the distance L3 and L1 at which the member (3) and the member (4) directly overlap is 0.16 ≦ L3 / L1 ≦ 1.
The ratio (T1 / L1) of the thickness T1 of the member (3) to the L1 of the member (3) is 0.02 ≦ T1 / L1 ≦ 0.150 ; and the basis weight of the member (3) is 49 g / m. ² or more, 75 g / m ² or less glass fiber material,
Immunochromatography test equipment. The immunochromatographic test apparatus according to claim 1, wherein the L1 is 8 mm or more and 12 mm or less. The immunochromatographic test apparatus according to claim 1 or 2, wherein the capillary flow time of the member (4) antibody-immobilizing member is 100 seconds / 4 cm or more and 140 seconds / 4 cm or less. The immunochromatographic test apparatus according to any one of claims 1 to 3, wherein a gold colloid having an average particle diameter of 75 nm or more and 110 nm or less is used for labeling the first antibody. 6. The bacterium according to any one of claims 1 to 4, wherein the bacterium is Streptococcus pneumoniae (Streptococcus pneumoniae), Chlamydia pneumoniae, Legionella pneumophila, Haemophilus influenza, Bordetella pertusis, or Mycoplasma pneumoniae. Immunochromatography test equipment. The immunochromatographic test apparatus according to any one of claims 1 to 5, wherein the intracellular antigen is a ribosomal protein L7 / L12 antigen. A kit for detecting bacteria, which comprises the immunochromatography test apparatus according to any one of claims 1 to 6. The kit according to claim 7, further comprising a diluent or extract containing a buffer solution and a surfactant, and a tool for collecting a sample.

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