JP2021004810A - Immuno-chromatography measuring device - Google Patents

Abstract

To provide an immuno-chromatography measuring device which is capable of operation to add liquid sample to a sample addition part one-step and of detection with high sensitivity.SOLUTION: An immuno-chromatography measuring device 10 includes at least a chromatographic developing part A which is provided with a membrane carrier 2 having a capture region 21 formed by solidifying a material which can be combined with a subject immunologically and a sample addition part B which communicates with the membrane carrier so as to perform chromatographic development in the membrane carrier to the capture region by supplying liquid sample for the membrane carrier. The sample addition part includes: a receiving part to which the liquid sample is added; a plurality of flow passages communicating with the receiving part; and a discharge part communicating with the plurality of flow passages so as to supply the liquid sample for the membrane carrier. The plurality of flow passages are configured so that the liquid sample passing different flow passages reach the capture region at mutually different times.SELECTED DRAWING: Figure 1

Description

The present invention relates to an immunochromatography measuring device capable of high-sensitivity detection in one step.

In a conventional immunochromatography measuring device, a chromatographic development unit having a membrane carrier having a capture site formed by immobilizing a substance that can be immunologically bound to a subject and a liquid sample on the membrane carrier are used. It is provided with a sample addition section that communicates with the membrane carrier so as to supply and chromatographically develop the inside of the membrane carrier toward the capture site, and specifically binds to the subject between the sample addition section and the chromatographic development section. It is configured by arranging an impregnated member impregnated with a possible labeled antibody. Then, a liquid sample is added to the sample addition portion to form a complex of the subject and the antibody when the liquid sample passes through the impregnated member, and then this complex is captured by the capture site. The subject was detected by developing a color at the capture site (see Patent Document 1).

International Publication No. 2007/074812

However, the conventional immunochromatography measuring device has a problem that the detection sensitivity cannot be sufficiently obtained when a large amount of a subject reacting with the labeled antibody is present in the liquid sample. Specifically, when the subject is a specific IgG antibody in serum, the labeled antibody is consumed in the reaction with the IgG antibody other than the subject contained in the serum, and the specific IgG antibody that is the subject is used. The amount of capture of the complex of the antibody and the labeled antibody at the capture site was reduced, and sufficient sensitivity could not be obtained, and in some cases, even detection was not possible. Further, even if the subject is an antigen, when it is present in a large amount in the liquid sample, the labeling rate of the antigen by the labeled antibody decreases, so that sufficient sensitivity cannot be obtained.

In order to solve the above problem, it is conceivable to first develop only the liquid sample on the membrane carrier to capture the subject on the capture site, and then deploy the labeled antibody on the membrane carrier. Since a two-step process of addition and addition of the labeled antibody is required, there arises a problem that the operation becomes complicated and the convenience of the operation is impaired.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide an immunochromatography measuring apparatus capable of performing an operation in one step of adding a liquid sample to a sample addition portion and performing detection with high sensitivity. To do.

As a result of diligent research, the present inventors provided a plurality of channels leading to the membrane carrier in the sample addition section of the immunochromatography measuring device, and captured the liquid sample added to the sample addition section from the plurality of channels with a time lag. It was found that the above object was achieved by reaching the above, and the present invention was completed.

That is, one aspect of the present invention is a chromatographic development unit having a membrane carrier having a capture site formed by immobilizing a substance that can be immunologically bound to a subject, and a liquid sample as the membrane. An immunochromatography measuring apparatus including at least a sample addition portion that is supplied to a carrier and communicated with the membrane carrier so as to chromatographically develop the inside of the membrane carrier toward the capture site, wherein the sample addition portion is the liquid sample. The plurality of flow paths include a receiving site to which the sample is added, a plurality of flow paths communicating with the receiving site, and a discharge site communicating with the plurality of flow paths so as to supply the liquid sample to the membrane carrier. Provides an immunochromatography measuring device configured such that the liquid samples that have passed through different channels reach the capture site at different times.

According to a preferred embodiment, the plurality of channels have different distances from the receiving site to the trapping site.

According to another preferred embodiment, the sample addition section comprises a liquid impermeable sheet disposed at the receiving site, and the added liquid sample is dispensed from one end of the liquid impermeable sheet. A first flow path leading to the membrane carrier and a second flow path leading to the membrane carrier from the other end of the liquid impermeable sheet are formed.

According to another preferred embodiment, the liquid permeable sheet is disposed on the upper surface of the first liquid permeable sheet articulated to one end of the membrane carrier, and the added liquid sample is subjected to the above. The first flow path leading from the end portion of the liquid impermeable sheet on the membrane carrier side to the membrane carrier, and the added liquid sample from the end portion on the side opposite to the end portion of the liquid impermeable sheet. It forms a second flow path leading to the liquid permeable sheet.

According to another preferred embodiment, the sample addition section further comprises a second liquid permeable sheet that covers the liquid permeable sheet.

According to another preferred embodiment, the subject is an antibody contained in the liquid sample, and the immobilized substance is an antigen to which the antibody specifically binds, preferably the capture site. The antibody that specifically binds to the antibody accumulated in is arranged in the second flow path so as to be chromatographically developed.

According to another preferred embodiment, the subject is an antigen contained in the liquid sample, and the immobilized substance is a first antibody that specifically binds to the antigen, preferably. A second antibody that specifically binds to the antigen accumulated at the capture site is arranged in the second flow path so that it can be chromatographically developed. The second antibody is preferably pre-labeled with a color labeling substance. According to another preferred embodiment, the enzyme-labeled antibody that specifically binds to the antigen accumulated at the capture site is arranged in the first flow path so as to be chromatographically developed, and the enzyme-labeled antibody is arranged. The color-developing substrate to be colored is arranged in the second flow path so as to be chromatographically developed.

According to the present invention, a plurality of flow paths from a receiving site to which a liquid sample is added to a membrane carrier are provided in a sample addition portion of an immunochromatography measuring device, and the liquid passing through the plurality of flow paths is different. Since the samples are configured to reach the discharge site at different times and similarly reach the capture site at different times, the reaction that had to be performed in two steps in the past can be performed in one step.

FIG. 1 is a sectional view in the length direction showing an example of the immunochromatography measuring apparatus of the present invention. FIG. 2A is a sectional view in the length direction showing a part of the sample addition portion and the chromatographic development portion connected to the sample addition portion of the immunochromatography measuring apparatus of FIG. 1, and FIG. 2B is FIG. 2A. 2 (C) is a similar diagram showing a modified example of the sample addition portion of FIG. 2 (B), and FIG. 2 (D) is a similar diagram showing a modified example of the sample addition portion of FIG. 2 (B). It is a similar figure which shows the modification of the sample addition part of 2 (C). FIG. 3 is a diagram showing the correlation of sensitivity between the immunochromatography measuring device obtained in Example 1 and the conventional product. FIG. 4 is a graph showing the results of Example 2, the solid line shows the results of the immunochromatography measuring apparatus of the present invention, and the broken lines show the results of the control product. FIG. 5 is a graph showing the results of Example 3, in which the solid line, the dotted line, and the broken line indicate that the impregnated member 31 has an upstream end of 10 mm, 13 mm, and 20 mm from the upstream end of the adhesive sheet 1 to the downstream side, respectively. The result when it is arranged so as to be located at the location is shown, and the alternate long and short dash line shows the result of the control product.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In the present specification, it is assumed that chromatographic development is performed from the right side to the left side of the drawing, and the chromatographic start point side, that is, the right side of FIG. 1 is described as "upstream side", and the opposite side, that is, the left side of FIG. Is described as "downstream side".

FIG. 1 shows an embodiment of the immunochromatography measuring device of the present invention. In the immunochromatography measuring device 10 of FIG. 1, an elongated strip-shaped membrane carrier 2 is attached to the middle of an elongated strip-shaped adhesive sheet 1, and a liquid permeable sheet 3 is attached to a margin on the upstream side of the adhesive sheet 1. Then, the liquid absorbing member 4 is attached to the downstream side of the adhesive sheet 1. As shown in FIG. 1, the downstream end of the liquid permeable sheet 3 is arranged so as to abut against the upstream end of the membrane carrier 2, whereby the liquid permeable sheet 3 can be formed from the membrane. The carrier 2 is connected so that a liquid can be supplied. As shown in FIG. 1, the upstream portion of the liquid absorbing member 4 is laminated on the downstream end portion of the membrane carrier 2, and the downstream portion of the liquid absorbing member 4 is the adhesive sheet 1. It is attached on the margin on the downstream side, whereby the liquid absorbing member 4 is connected so that the liquid contained on the downstream side of the membrane carrier 2 can be absorbed.

As is known, the membrane carrier 2 has a capture site 21 formed by immobilizing a substance immunologically bindable to the subject on the membrane carrier 2. Further, between the liquid permeable sheet 3 and the adhesive sheet 1, an impregnating member 31 is provided so as to prepare a reagent such as a labeled antibody that reacts with the subject accumulated in the capture site 21 so that it can be chromatographically developed. Have been placed. Further, as is known, a control line 22 for confirming that the reaction is completed regardless of the presence or absence of the subject is provided at a position downstream of the capture site 21 of the membrane carrier 2. The control line 22 is provided as needed, but is usually formed by a substance that reacts with the reagent contained in the impregnating member 31, and the labeled antibody that specifically reacts with the subject as the reagent is used. When used, it can be formed by immobilizing an antibody that specifically binds to the labeled antibody on the membrane carrier 2.

The immunochromatography measuring apparatus of FIG. 1 is characterized in that a liquid permeable sheet 5 is placed on the upper surface of the liquid permeable sheet 3 and a liquid sample is added onto the liquid permeable sheet 5. It is said. The length of the liquid permeable sheet 5 is slightly shorter than the length of the liquid permeable sheet 3, and the downstream end of the liquid permeable sheet 5 is the liquid permeable sheet 3 as shown in FIG. It is arranged so as to coincide with the downstream end of the. Therefore, the liquid permeable sheet 3 has its upstream end protruding from the upstream end of the liquid permeable sheet 5 to the upstream side, and the upper surface thereof is exposed. In FIG. 1, the membrane carrier 2 and the liquid absorbing member 4 constitute the chromatographic developing portion A of the present invention, and the liquid permeable sheet 3 and the liquid impermeable sheet 5 located upstream of the chromatographic developing portion A of the present invention are the present invention. It constitutes the sample addition part B. The impregnating member 31 can be provided at an arbitrary position in the flow path through which the liquid sample passes, depending on the purpose, and the position where the impregnating member 31 is arranged is not limited to the position shown in FIG.

In the case of the immunochromatography measuring device of FIG. 1, since the liquid sample is added on the liquid impermeable sheet 5, the liquid sample is moved in both the upstream side and the downstream side as shown in FIG. 2 (A). It branches and flows. As shown by the triangular arrow in FIG. 2A, the fraction of the liquid sample flowing to the downstream side immediately permeates into the membrane carrier 2 from the downstream end of the liquid impermeable sheet 5 and is contained in the membrane carrier 2. Is chromatographically developed, and the subject contained therein is accumulated at the capture site 21. On the other hand, as shown by the arrowhead arrow in FIG. 2A, the fraction of the liquid sample flowing to the upstream side passes through the liquid permeable sheet 3 from the upstream end of the liquid permeable sheet 5 and is described above. Similarly, it penetrates into the membrane carrier 2. That is, the sample addition portion B of the apparatus of FIG. 1 has a first flow path from the downstream side of the liquid impermeable sheet 5 to the membrane carrier 2 and a liquid permeable sheet 3 from the upstream side of the liquid impermeable sheet 5. It is provided with a second flow path leading to the membrane carrier 2 via the above. Since the distance of the first flow path to the membrane carrier 2 is shorter than that of the second flow path, the liquid sample that has passed through the first flow path first permeates the membrane carrier 2 and reaches the capture site 21. After that, the liquid that has passed through the second flow path permeates the membrane carrier 2 with a delay and reaches the capture site 21. As described above, the liquid sample added to the sample addition section B of the apparatus of FIG. 1 is automatically divided into two fractions and is configured to reach the capture site 21 with a time lag.

In the apparatus of FIG. 1, the position indicated by the downward white arrow above the liquid opaque sheet 5 corresponds to the “reception site to which the liquid sample is added” in the present invention, and the first above. And the second path correspond to the "plurality of flow paths communicating with the receiving site" in the present invention, and the downstream end portions of the liquid impermeable sheet 5 and the liquid permeable sheet 3 are the present invention. Corresponds to the "discharge site" in. In the case of the apparatus of FIG. 1, since the distances from the receiving site to the capturing site 21 or the discharging site are different between the first flow path and the second path, the fractions of the liquid sample passing through each path are captured with a time lag. Reach site 21 or discharge site.

In the apparatus of FIG. 1, in order to facilitate the acceptance of the liquid sample, grooves are provided on the upper surface of the liquid impermeable sheet 5 toward both ends, or the liquid impermeable sheet 5 is added to the surface. It is preferable to perform a treatment for preventing the liquid sample from scattering, and as a preferable embodiment, for example, as in the configuration of FIG. 2B described later, the upper surface of the liquid impermeable sheet 5 is used. Covering with a liquid permeable sheet 6 can be mentioned.

In the immunochromatography measuring apparatus of FIG. 1, when a specific IgG antibody in serum is used as a subject, the capture site 21 is immobilized by immobilizing the antigen recognized by the specific IgG antibody as the subject on the membrane carrier 2. The impregnated member 31 may be impregnated with a labeled antibody that is formed and labeled with an appropriate labeling substance with an antibody that binds to the constant region of the specific IgG antibody that is the subject. In this case, the fraction of the liquid sample that has passed through the first flow path first reaches the capture site 21, the unlabeled subject accumulates at the capture site 21, and then the liquid that has passed through the second flow path. Since the fraction of the sample reaches the capture site 21 together with the labeled antibody and binds to the subject accumulated therein, the labeling rate of the sample at the capture site 21 is increased, and highly sensitive detection can be performed.

Further, in the immunochromatography measuring apparatus of FIG. 1, when an antigen such as a microorganism such as a virus or a bacterium or a protein is used as a subject, an antibody that recognizes the first epitope of the antigen as the subject is solid-phased on the membrane carrier 2. The capture site 21 may be formed by the formation of the antigen, and the impregnated member 31 may be impregnated with a labeled antibody labeled with an appropriate labeling substance with an antibody that recognizes the second epitope of the antigen so that it can be chromatographed. In this case, the fraction of the liquid sample that has passed through the first flow path first reaches the capture site 21, where the unlabeled antigen accumulates, and then the liquid sample that has passed through the second flow path. Since the fraction reaches the capture site 21 together with the labeled antibody and binds to the sample accumulated therein, the labeling rate of the sample at the capture site 21 is increased, and highly sensitive detection can be performed.

The labeling substance may be any substance as long as it can be used, but it is preferable to use a color labeling substance from the viewpoint that it can be measured in one step with the apparatus shown in FIG. Examples of the color-developing labeling substance include metal colloids such as gold colloid and platinum colloid, synthetic latex such as polystyrene latex colored with each pigment such as red and blue, and latex such as natural rubber latex. Of these, metal colloids such as gold colloids are particularly preferable.

In the present invention, the time difference between the fraction of the liquid sample passing through the first flow path of the sample addition section B and the fraction of the liquid sample passing through the second flow path reaching the capture site 21 is 5 for the detection time. In the case of an immunochromatography measuring device set to about 30 minutes, it is preferably about 3 to 15 minutes. This time difference can be adjusted by appropriately changing the length of the liquid permeable sheet 3, the length and arrangement of the liquid permeable sheet 5, and the physical property values such as the average pore diameter, density, and basis weight of the liquid permeable sheet 3. can do. In FIG. 2A, the downstream end of the liquid permeable sheet 5 coincides with the downstream end of the liquid permeable sheet 3, that is, the upstream end of the membrane carrier 2, but is limited to this. Other configurations may be adopted as long as the above time difference is achieved. Further, in FIG. 2A, the upstream end portion of the liquid permeable sheet 3 is in a state of projecting from the upstream end of the liquid permeable sheet 5 to the upstream side, but the projecting length is long. Since the time difference can be adjusted by adjusting the above time difference, the time difference is not limited to the configuration shown in FIG. 2 (A), and other configurations can be adopted as long as the time difference is achieved.

The sample addition section B shown in FIG. 2B is provided with a liquid permeable sheet 6 that covers the liquid permeable sheet 5, so that the liquid sample is added onto the liquid permeable sheet 6. This is different from the sample addition section B shown in FIG. 2 (A). As shown in FIG. 2B, the liquid permeable sheet 6 has a length longer than that of the liquid permeable sheet 3, and its upstream end coincides with the upstream end of the liquid permeable sheet 3. The downstream end portion of the membrane carrier 2 is covered with the upstream end portion of the membrane carrier 2. As a result, the sample addition portion B of FIG. 2B is from the first flow path (see the triangular arrow) from the downstream side of the liquid permeable sheet 6 to the membrane carrier 2 and the upstream side of the liquid permeable sheet 6. It is provided with a second flow path (see arrowhead arrow) leading to the membrane carrier 2 via the liquid permeable sheet 3. Since the distance of the first flow path to the membrane carrier 2 is shorter than that of the second flow path, the liquid sample added to the sample addition part B is similar to the sample addition part B of FIG. 2 (A). It is automatically divided into two fractions and is configured to reach the capture site 21 with a time lag. Since the liquid sample is added to the liquid permeable sheet 6 in the sample addition portion B of FIG. 2B, it is possible to prevent the liquid sample from scattering at the time of addition. In the aspect of FIG. 2B, the liquid permeable sheet 6 corresponds to the “reception site to which the liquid sample is added” in the present invention.

The sample addition part B shown in FIG. 2C shows an application example of the sample addition part B shown in FIG. 2B. The sample addition part B of FIG. 2C is impregnated with an arbitrary reagent inside the liquid permeable sheet 6 in the middle of the first flow path (see the triangular arrow) of the sample addition part B of FIG. 2B. The impregnation pad 32 that can be made to be applied is arranged. In the case of the sample addition portion B of FIG. 2C, for example, the impregnated member 32 is impregnated with the enzyme-labeled antibody so that it can be chromatographed, and the impregnated member 31 is impregnated with the enzyme-labeled color former so as to be chromatographically developed. Can be made to. In this case, when the liquid sample is added to the liquid permeable sheet 6, the fraction of the liquid sample that has passed through the first flow path (see the triangular arrow) becomes the subject and the enzyme-labeled antibody when passing through the impregnating member 32. The complex is formed and penetrates into the membrane carrier 2, and the complex accumulates at the capture site 21. Then, the fraction of the liquid sample that has passed through the second flow path (see the arrowhead arrow) dissolves the color-developing substrate when passing through the impregnating member 31, penetrates into the membrane carrier 2, and passes through the first flow path. The capture site 21 can be reached later than the fraction, and the substrate can be colored by the labeling enzyme of the complex accumulated therein. Therefore, enzyme-colored immunochromatographic measurement using an enzyme-labeled antibody can be performed in one step.

Further, in the sample addition portion B of FIG. 2C, the antibody against the subject can be labeled with the color labeling substance and impregnated into both the impregnating member 31 and the impregnating member 32. In this method, more labeled antibodies can be developed when one impregnated member cannot contain many labeled antibodies or when the concentration of the solution impregnated in the impregnated member cannot be increased. It is effective for increasing the sensitivity. Further, by using an antibody that recognizes different epitopes of the subject as the labeled antibody to be impregnated in the impregnated member 31 and the impregnated member 32, steric hindrance due to the labeling antibody binding to the subject can be avoided, and the steric hindrance between the labeled antibodies can be avoided. Since competition can be prevented, high sensitivity can be achieved.

FIG. 2D shows a sample addition section B provided with three flow paths. In the sample addition section B of FIG. 2 (D), the same liquid permeable sheet 7 as the liquid opaque sheet 5 is placed on the liquid permeable sheet 6 of the sample addition section B of FIG. 2 (C). , The liquid permeable sheet 8 that covers the upper surface of the liquid permeable sheet 7 is laminated. Since the liquid permeable sheet 7 is arranged between both ends of the liquid permeable sheet 6, both ends of the liquid permeable sheet 6 project from the corresponding ends of the liquid permeable sheet 7. Since the liquid permeable sheet 8 has the same dimensions as the liquid permeable sheet 6, both ends of the liquid permeable sheet 8 coincide with the corresponding ends of the liquid permeable sheet 6. The liquid impermeable sheet 7 is arranged on the downstream side of the liquid impermeable sheet 5. The impregnation pad 32 is arranged in the center of the liquid impermeable sheet 5.

In the case of the sample addition part B of FIG. 2 (D), when the liquid sample is added onto the liquid permeable sheet 8, the liquid sample branches in both the upstream side and the downstream side as shown in FIG. 2 (D). And it will flow. The fraction of the liquid sample flowing to the downstream side is the membrane carrier 2 from the downstream end of the liquid permeable sheet 7 and the liquid permeable sheet 6 protruding from the downstream end to the downstream side. The membrane carrier 2 is chromatographed and the subject contained therein is accumulated at the capture site 21. On the other hand, a part of the fraction of the liquid sample flowing to the upstream side permeates the membrane carrier 2 from the upstream end of the liquid impermeable sheet 7 through the liquid permeable sheet 6 as in FIG. 2C. Then, the rest of the fraction permeates the membrane carrier 2 from the upstream end of the liquid impermeable sheet 5 through the liquid permeable sheet 3 in the same manner as in FIG. 2C. Since the liquid permeable sheet 7 is arranged on the downstream side of the liquid permeable sheet 5, the fraction permeated into the membrane carrier 2 via the liquid permeable sheet 6 passes through the liquid permeable sheet 3. The capture site 21 is reached earlier than the fraction that has penetrated the membrane carrier 2. Therefore, after the enzyme-labeled antibody contained in the impregnated member 32 binds to the subject accumulated in the capture site 21, the color-developing substrate contained in the impregnated member 31 reaches the capture site 21 and accumulates in the composite. The substrate can be colored by the body's labeling enzyme. Therefore, enzyme-colored immunochromatographic measurement using an enzyme-labeled antibody can be performed in one step. Similarly, by stacking the liquid impermeable sheet and the liquid permeable sheet in multiple layers, the sample addition portion B having four or more flow paths can be formed.

In the apparatus shown in FIG. 1, as in the liquid absorbing member 4, the downstream end portion of the liquid permeable sheet 3 may be laminated on the upstream end portion of the membrane carrier 2 to connect the two. Similarly, as in the liquid permeable sheet 3 shown in FIG. 1, the upstream end of the liquid absorbing member 4 may be abutted against the downstream end of the membrane carrier 2 to connect the two.

In the present invention, as the material constituting the pressure-sensitive adhesive sheet, the membrane carrier, the liquid absorbing member and the impregnating member, those conventionally used for immunochromatographic strips can be used.

The pressure-sensitive adhesive sheet is not particularly limited as long as the film carrier can be attached on the pressure-sensitive adhesive surface.

The membrane carrier is not particularly limited as long as it can chromatographically develop a liquid sample by a capillary phenomenon, and examples thereof include a nitrocellulose membrane filter and other cellulose membranes, nylon membranes, and glass fiber membranes. Be done.

The liquid absorbing member is not particularly limited as long as it is made of a material capable of quickly absorbing and holding a liquid, and examples thereof include cotton cloth, filter paper, and porous plastic non-woven fabric made of polyethylene, polypropylene, etc., and particularly filter paper. Is the best.

The impregnating member is not particularly limited as long as it can be chromatographically impregnated with a reagent such as a labeled antibody or an enzyme-labeled color former, and is not particularly limited. For example, a glass fiber non-woven fabric or a cellulose cloth (filter paper, nitrocellulose film). Etc.), porous plastic cloths such as polyethylene and polypropylene, and the like.

As the liquid permeable sheet, for example, a cellulosic paper or woven cloth or non-woven fabric such as filter paper and cotton cloth, and a sheet or film of a porous synthetic resin such as porous polyethylene and porous polypropylene are used. Can be done. In the present invention, the "sheet" includes a "film".

As the liquid impermeable sheet, for example, a plastic film such as a vinyl chloride tape, a cellophane film, an acetate film, a polyethylene film, or a polypropylene film can be used. Since the liquid permeable sheet is arranged on the upper surface of the liquid permeable sheet in the embodiment shown in FIG. 2 (A), a plastic tape coated with an adhesive or an adhesive on one side is placed on the upper surface of the liquid permeable sheet. It may be formed by bonding, or in the embodiment shown in FIGS. 2 (B) to 2 (D), since it is laminated between two adjacent liquid permeable sheets, an adhesive or an adhesive is applied to one side or both sides. The applied plastic tape may be adhered to the upper surface and / or the lower surface of the liquid permeable sheet and laminated between the two liquid permeable sheets.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these contents.

Example 1 (Detection of serum-specific IgA antibody in patients with nontuberculous mycobacteriosis)
An immunochromatography measuring device having the same configuration as that of FIG. 1 except that the sample addition portion of FIG. 2B was provided was produced. As the membrane carrier 2, an elongated strip-shaped nitrocellulose membrane filter having a width of 4 mm and a length of 35 mm was used and attached to the middle of the pressure-sensitive adhesive sheet 1 having the same width. The membrane carrier 2 has a Glycopeptidolipid core antigen (hereinafter, GPL-), which is a Mycobacterium avium complex (hereinafter, referred to as MAC) -specific antigen of nontuberculous mycobacteria, located 6.5 mm downstream from the upstream end. (referred to as core antigen) was immobilized to form a capture site 21. A control line 22 was provided at a position of 14.5 mm from the upstream end to the downstream side of the membrane carrier 2. The control line 22 was formed by immobilizing an anti-rabbit polyclonal antibody (a product of DAKO) on the membrane carrier 2. As the liquid permeable sheet 3, a cellulose non-woven fabric having a width of 4 mm and a length of 30 mm was used and attached to the adhesive sheet 1. At that time, an impregnated member 31 (width 4 mm, length 10 mm) impregnated with a platinum-gold colloid-labeled anti-human IgA rabbit antibody was sandwiched between the pressure-sensitive adhesive sheet 1 and the liquid-permeable sheet 3. The impregnating member 31 was arranged so that the end on the upstream side was located 10 mm downstream from the end on the upstream side of the pressure-sensitive adhesive sheet 1. Then, as the liquid permeable sheet 5, a cellophane tape having a width of 4 mm and a length of 25 mm was used, and the downstream end was arranged so as to coincide with the downstream end of the liquid permeable sheet 3. Further, as shown in FIG. 2B, a cellulose non-woven fabric having a width of 4 mm and a length of 33 mm is used as the liquid permeable sheet 6, and the upstream end coincides with the upstream end of the liquid permeable sheet 3. The liquid-impermeable sheet 5 was coated with a portion extending from the downstream end to the upstream side up to 3 mm so as to overlap the upstream end of the membrane carrier 2.

Using a positive sample priced by Capilia MAC antibody ELISA (trade name: a product of Towns Co., Ltd.) prepared to the concentration shown in Table 1 using physiological saline as a liquid sample, the above-mentioned immunochromatography measuring apparatus. It was added to the sample addition part (the part indicated by the white arrow in FIG. 2B), and after 30 minutes, it was judged visually and by an immunochromatographic reader (ICR). The evaluation criteria for visual judgment were divided into five stages:-(no coloring), ± (weak coloring), + (clear coloring), ++ (significant coloring), and +++ (particularly remarkable coloring). .. The results are shown in Table 1.

The result of comparing the result by ICR of Table 1 with the measured value of the above-mentioned ELISA method is shown in FIG. From FIG. 3, a good correlation was observed between the two for positive samples having a concentration of 7.10 U / ml or more. Therefore, it was confirmed that the immunochromatography measuring apparatus of the present invention can detect the IgA antibody in blood much more easily than the ELISA method.

Example 2 (Detection of influenza virus type A antigen)
An immunochromatography measuring device having the same configuration as that of FIG. 1 except that the sample addition portion of FIG. 2C was provided was produced. As the membrane carrier 2, an elongated strip-shaped nitrocellulose membrane filter having a width of 4 mm and a length of 35 mm was used and attached to the middle of the pressure-sensitive adhesive sheet 1 having the same width. On the membrane carrier 2, an anti-influenza virus type A antibody was immobilized at a position 6.5 mm from the upstream end to the downstream end to form a capture site 21. A control line 22 was provided at a position of 14.5 mm from the upstream end to the downstream side of the membrane carrier 2. The control line 22 was formed by immobilizing an anti-mouse polyclonal antibody (a product of DAKO) on the membrane carrier 2. An impregnated member 31 impregnated with a first labeled antibody consisting of a platinum-gold colloid-labeled anti-influenza virus type A antibody having a width of 4 mm and a length of 10 mm is impregnated with an impregnated member 31 whose upstream end is from the upstream end of the adhesive sheet 1. It was arranged so as to be located 10.0 mm downstream. Further, as the liquid permeable sheet 3, a cellulose non-woven fabric having a width of 4 mm and a length of 30 mm was used, laminated on the impregnated member 31, and attached to the adhesive sheet 1. Then, as the liquid permeable sheet 5, a cellophane tape having a width of 4 mm and a length of 25 mm was used, and the downstream end of the cellophane tape was arranged so as to coincide with the downstream end of the liquid permeable sheet 3. Further, as shown in FIG. 2C, an impregnated member 32 impregnated with a second labeled antibody composed of a platinum-gold colloid-labeled anti-influenza virus type A antibody having a width of 4 mm and a length of 10 mm is placed upstream thereof. The side end was arranged so as to be located 5.0 mm downstream from the upstream end of the liquid impermeable sheet 5. Further, as shown in FIG. 2C, a cellulose non-woven fabric having a width of 4 mm and a length of 33 mm is used as the liquid permeable sheet 6, and the upstream end coincides with the upstream end of the liquid permeable sheet 3. The liquid-impermeable sheet 5 was coated by arranging the portion from the downstream end to the upstream side up to 3 mm so as to overlap the upstream end of the membrane carrier 2. At that time, the impregnating member 32 was sandwiched between the liquid permeable sheet 5 and the liquid permeable sheet 6. The first labeled antibody and the second labeled antibody are monoclonal antibodies that recognize different epitopes of the same influenza virus type A.

Influenza virus type A antigen isolated and cultured from a clinical sample, using a sample extract (a product of Towns), and a sample prepared at 1 × 10 ³ PFU / ml as a test sample, the above immunochromatography measuring device Was added to the sample addition section (the part indicated by the white arrow in FIG. 2C), and determined by an immunochromatographic reader (ICR) 5 minutes, 8 minutes, 15 minutes, 20 minutes and 40 minutes after the addition. As a control, Immuno Ace Flu (trade name: a product of Towns Co., Ltd.) was used, the same test sample was added, and the determination was made under the same conditions. The results are shown in Table 2 and FIG. From the results of Table 2 and FIG. 4, it was found that the immunochromatographic measuring apparatus of the present invention can obtain higher sensitivity than the control product, although the measuring time is long.

Example 3 (Detection of influenza virus type A antigen)
An immunochromatography measuring device similar to that of Example 2 was produced except that the position of the upstream end of the impregnating member 31 was 10.0 mm, 13.0 mm or 20.0 mm from the upstream end of the pressure-sensitive adhesive sheet 1. The above-mentioned immunochromatography measuring device uses a sample prepared in 1 × 10 ⁴ PFU / ml using a sample extract (a product of Towns) using an influenza virus type A antigen isolated and cultured from a clinical sample as a test sample. Was added to the sample addition section (the part indicated by the white arrow in FIG. 2C), and determined by an immunochromatographic reader (ICR) 3 minutes, 5 minutes, 8 minutes, 15 minutes, 20 minutes, and 30 minutes after the addition. did. As a control, Immuno Ace Flu (trade name: a product of Towns Co., Ltd.) was used, the same test sample was added, and the determination was made under the same conditions. The results are shown in Table 3 and FIG.

From the results of Table 3 and FIG. 5, it was clarified that the detection sensitivity changes depending on the arrangement position of the impregnating member 31. It was clarified that the sensitivity increases as the position of the impregnating member 31 is closer to the upstream side, that is, the distance from the membrane carrier is increased.

Claims (11)

A chromatographic development unit having a membrane carrier having a capture site formed by immobilizing a substance that can be immunologically bound to a subject, and a liquid sample being supplied to the membrane carrier and directed toward the capture site. An immunochromatography measuring apparatus including at least a sample addition portion communicating with the membrane carrier so as to chromatolate the inside of the membrane carrier, wherein the sample addition portion includes a receiving site to which the liquid sample is added and the receiving portion. The plurality of flow paths including a plurality of flow paths communicating with the site and a discharge site communicating with the plurality of flow paths so as to supply the liquid sample to the membrane carrier, the plurality of flow paths include the liquid passing through different flow paths. An immunochromatography measuring device configured such that the samples reach the capture site at different times. The immunochromatography measuring apparatus according to claim 1, wherein the plurality of channels have different distances from the receiving site to the capturing site. The sample addition section includes a liquid impermeable sheet arranged at the receiving site, and a first flow path that guides the added liquid sample from one end portion of the liquid impermeable sheet to the membrane carrier. The immunochromatography measuring apparatus according to claim 2, wherein the liquid impermeable sheet and a second flow path leading from the other end portion of the liquid permeable sheet to the membrane carrier are formed. The liquid permeable sheet is arranged on the upper surface of the first liquid permeable sheet connected to one end of the film carrier, and the added liquid sample is applied to the film carrier of the liquid permeable sheet. A first flow path leading from the end on the side to the membrane carrier, and a first channel for guiding the added liquid sample from the end opposite to the end of the liquid impermeable sheet to the liquid permeable sheet. The immunochromatography measuring apparatus according to claim 3, which forms the second flow path. The immunochromatography measuring apparatus according to claim 4, wherein the sample addition section further includes a second liquid permeable sheet that coats the liquid permeable sheet. The immunochromatography according to any one of claims 1 to 5, wherein the subject is an antibody contained in the liquid sample, and the immobilized substance is an antigen to which the antibody specifically binds. measuring device. The immunochromatography measuring apparatus according to claim 6, wherein an antibody that specifically binds to the antibody accumulated at the capture site is arranged in the second flow path so that it can be chromatographically developed. The invention according to any one of claims 1 to 5, wherein the subject is an antigen contained in the liquid sample, and the immobilized substance is a first antibody that specifically binds to the antigen. Immunochromatography measuring device. The immunochromatography measuring apparatus according to claim 8, wherein a second antibody that specifically binds to the antigen accumulated at the capture site is arranged in the second flow path so that it can be chromatographically developed. The immunochromatography measuring device according to claim 9, wherein the second antibody is pre-labeled with a color labeling substance. An enzyme-labeled antibody that specifically binds to the antigen accumulated at the capture site is arranged in the first flow path so as to be chromatographically developed, and a coloring substrate that develops the color of the enzyme-labeled antibody is used as the second color-developing substrate. The immunochromatography measuring apparatus according to claim 8, which is arranged so as to be chromatographically developed in the flow path of the above.

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