JP7337371B2 - Immunochromatography measurement device - Google Patents

Description

TECHNICAL FIELD The present invention relates to an immunochromatographic measurement device capable of high-sensitivity detection in one step.

A conventional immunochromatographic measurement device includes a chromatographic development unit provided with a membrane carrier having a capture site formed by immobilizing a substance that can bind immunologically to a subject, and a liquid sample on the membrane carrier. a sample application unit communicating with the membrane carrier so as to supply and chromatographically develop the inside of the membrane carrier toward the capture site, and specifically bind to the analyte between the sample application unit and the chromatographic development unit An impregnated member impregnated with a possible labeled antibody is arranged. Then, the liquid sample is added to the sample addition part, and after the liquid sample passes through the impregnated member, a complex between the analyte and the antibody is formed, and then the complex is captured by the capture site, The analyte has been detected by coloring the capture site (see Patent Document 1).

WO2007/074812

However, conventional immunochromatography measurement devices have the problem that sufficient detection sensitivity cannot be obtained when a large amount of analyte that reacts with a labeled antibody is present in a liquid sample. Specifically, when the subject is a specific IgG antibody in serum, the labeled antibody is consumed in the reaction with an IgG antibody other than the subject contained in the serum, and the specific IgG antibody that is the subject The amount of capture at the capture site of the complex of and labeled antibody is reduced, resulting in insufficient sensitivity and, in some cases, even failure to detect. In addition, even if the analyte is an antigen, when it is present in a large amount in a liquid sample, the antigen labeling rate with the labeled antibody decreases, and sufficient sensitivity cannot be obtained.

In order to solve the above problems, it is conceivable to first develop only the liquid sample on the membrane carrier to capture the analyte at the capture site, and then develop the labeled antibody on the membrane carrier. Since a two-step process of addition and addition of the labeled antibody is required, the operation becomes complicated, resulting in the problem of impairing the simplicity of the operation.

The present invention has been made in view of the above circumstances, and it is an object of the present invention to provide an immunochromatographic measurement apparatus that can be operated in one step by adding a liquid sample to a sample addition part and can detect with high sensitivity. do.

As a result of intensive research, the present inventors provided a plurality of flow paths leading to a membrane carrier in the sample addition section of an immunochromatography measurement device, and the liquid sample added to the sample addition section was captured at a time lag from the plurality of flow paths. The inventors have found that the above object is achieved by achieving the above, and have completed the present invention.

That is, one aspect of the present invention provides a chromatographic development section comprising a membrane carrier having a capture site formed by immobilizing a substance that can bind immunologically to a subject; an immunochromatographic measurement device that includes at least a sample addition unit that communicates with the membrane carrier so as to supply it to the carrier and chromatographically develop the inside of the membrane carrier toward the capture site, wherein the sample addition unit includes the liquid sample a receiving site to which is added, a plurality of channels communicating with the receiving site, and an outlet site communicating with the plurality of channels to supply the liquid sample to the membrane carrier, wherein the plurality of channels provides an immunochromatographic measurement device configured such that the liquid samples that have passed through different channels reach the capture sites at different times.

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

According to another preferred embodiment, the sample application section comprises a liquid-impermeable sheet arranged at the receiving site, and the added liquid sample is transferred from one end of the liquid-impermeable sheet to the A first channel leading to the membrane carrier and a second channel leading from the other end of the liquid impermeable sheet to the membrane carrier are formed.

According to another preferred embodiment, said liquid-impermeable sheet is arranged on top of a first liquid-permeable sheet connected to one end of said membrane carrier, and said liquid sample added to said a first channel leading from the end of the liquid-impermeable sheet on the side of the membrane carrier to the membrane carrier; A second channel is formed leading to the liquid permeable sheet.

According to another preferred embodiment, the sample application section further comprises a second liquid permeable sheet covering the liquid impermeable sheet.

According to another preferred embodiment, the analyte is an antibody contained in the liquid sample, the immobilized substance is an antigen to which the antibody specifically binds, and preferably the capture site An antibody that specifically binds to the antibody accumulated in the second channel is arranged so as to be capable of being chromatographically developed.

According to another preferred embodiment, the analyte 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 in the capture site is arranged in the second channel so as to be chromatographically developable. The second antibody is preferably pre-labeled with a color labeling substance. According to another preferred embodiment, an enzyme-labeled antibody that specifically binds to the antigen accumulated at the capture site is arranged in the first channel so as to be chromatographically developable, and the enzyme-labeled antibody is A chromogenic substrate for color development is arranged in the second channel so as to be chromatographically developable.

According to the present invention, the sample addition part of the immunochromatographic measurement device is provided with a plurality of flow paths from a receiving site where a liquid sample is added to a membrane carrier, and the plurality of flow paths are used for liquids that have passed through different flow paths. Since the sample is configured to reach the ejection site at different times and the capture site at different times as well, the reaction that conventionally had to be performed in two steps can be performed in one step.

FIG. 1 is a longitudinal cross-sectional view showing an example of the immunochromatographic measuring device of the present invention. FIG. 2(A) is a longitudinal cross-sectional view showing a part of the sample addition section and the chromatographic development section connected thereto of the immunochromatographic measurement device of FIG. 1, and FIG. 2(B) is FIG. 2(A). FIG. 2C is a similar view showing a modification of the sample addition part of FIG. 2B, and FIG. 2D is a view similar to FIG. 2(C) is a similar view showing a modification of the sample addition portion; FIG. FIG. 3 is a diagram showing the sensitivity correlation between the immunochromatographic measurement device obtained in Example 1 and a conventional product. FIG. 4 is a graph showing the results of Example 2, where the solid line shows the results of the immunochromatographic measurement device of the present invention and the dashed line shows the results of the control product. FIG. 5 is a graph showing the results of Example 3. The solid line, dotted line and broken line indicate that the upstream end of the impregnated member 31 is 10 mm, 13 mm and 20 mm downstream from the upstream end of the pressure-sensitive adhesive sheet 1, respectively. The results are shown for the case where it is arranged so as to be located at the point, and the two-dot chain line shows the results for the control product.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In this specification, chromatographic development is performed from the right side to the left side of the drawing, and the chromatographic starting point side, that is, the right side of FIG. is referred to as "downstream side".

FIG. 1 shows one embodiment of the immunochromatographic measuring device of the present invention. In the immunochromatographic measurement device 10 of FIG. 1, a long and thin strip-shaped membrane carrier 2 is attached to the middle of a long and thin strip-shaped adhesive sheet 1, and a liquid-permeable sheet 3 is attached to the upstream margin of the adhesive sheet 1. A liquid absorbing member 4 is adhered to the downstream side of the adhesive sheet 1 . The downstream end of the liquid permeable sheet 3 is placed in abutment with the upstream end of the membrane carrier 2, as shown in FIG. It is connected to the carrier 2 so that it can be supplied with liquid. 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 laminated on the adhesive sheet 1. The liquid absorbing member 4 is adhered on the downstream margin so that the liquid absorbing member 4 is connected so as to be able to absorb the liquid contained on the downstream side of the membrane carrier 2 .

As is well known, the membrane carrier 2 has a trapping site 21 formed by immobilizing a substance immunologically capable of binding to the subject on the membrane carrier 2 . Between the liquid-permeable sheet 3 and the adhesive sheet 1 is an impregnated member 31 for preparing a reagent such as a labeled antibody that reacts with the analyte accumulated in the capture site 21 so as to be capable of chromatographic development. are placed. Further, a control line 22 is provided at a position downstream of the capture site 21 of the membrane carrier 2, as is well known, for confirming that the reaction has ended regardless of the presence or absence of the analyte. This control line 22 is provided as necessary, but generally, it may be formed of a substance that reacts with the reagent contained in the impregnated member 31. As the reagent, a labeled antibody that specifically reacts with the subject 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 immunochromatographic measurement apparatus of FIG. 1 is characterized in that a liquid impermeable sheet 5 is placed on the upper surface of the liquid permeable sheet 3, and a liquid sample is added onto the liquid impermeable sheet 5. and The length of the liquid-impermeable sheet 5 is slightly shorter than the length of the liquid-permeable sheet 3, and the downstream end of the liquid-impermeable sheet 5, as shown in FIG. is aligned with the downstream end of the Accordingly, the liquid-permeable sheet 3 has its upstream end projecting upstream from the upstream end of the liquid-impermeable sheet 5, exposing its upper surface. In FIG. 1, the membrane carrier 2 and the liquid absorbing member 4 constitute the chromatographic developing section A of the present invention, and the liquid permeable sheet 3 and the liquid impermeable sheet 5 positioned upstream therefrom are the chromatographic developing section A of the present invention. A sample addition section B is configured. Note that the impregnating member 31 can be provided at any position in the flow channel through which the liquid sample passes according to the purpose, and the position at which it is arranged is not limited to the position shown in FIG.

In the case of the immunochromatographic measurement device of FIG. 1, the liquid sample is added onto the liquid-impermeable sheet 5, so that the liquid sample flows in both the upstream and downstream directions, as shown in FIG. 2(A). It will branch and flow. The fraction of the liquid sample that has flowed downstream immediately permeates the membrane carrier 2 from the downstream end of the liquid-impermeable sheet 5 and into the membrane carrier 2, as indicated by the triangular arrow in FIG. is chromatographically developed, and the analyte contained therein accumulates at the capture site 21 . On the other hand, the fraction of the liquid sample that has flowed upstream passes from the upstream end of the liquid-impermeable sheet 5 through the liquid-permeable sheet 3, as indicated by the arrowhead in FIG. The membrane carrier 2 is likewise permeated. That is, the sample application section B of the apparatus of FIG. It has a second flow path leading to the membrane carrier 2 via the . Since the first channel has a shorter distance to the membrane carrier 2 than the second channel, the liquid sample that has passed through the first channel first permeates the membrane carrier 2 and reaches the capture site 21. After that, the liquid that has passed through the second channel permeates the membrane carrier 2 with a delay and reaches the trapping site 21 . In this manner, the liquid sample added to the sample addition portion B of the apparatus of FIG. 1 is automatically divided into two fractions and is configured to reach the trapping site 21 with a time lag.

In the apparatus of FIG. 1, the position indicated by the downward white arrow above the liquid-impermeable sheet 5 corresponds to the "receiving site to which the liquid sample is added" in the present invention. and the second path correspond to the "plurality of flow paths communicating with the receiving site" in the present invention, and the downstream ends of the liquid-impermeable sheet 5 and the liquid-permeable sheet 3 respectively correspond to the corresponds to the "excretion site". In the device of FIG. 1, the first flow path and the second path differ from each other in the distance from the receiving site to the capture site 21 or the discharge site, so that the fraction of the liquid sample passing through each path is captured at different times. It reaches site 21 or the ejection site.

In the apparatus of FIG. 1, in order to facilitate the reception of the liquid sample, the upper surface of the liquid-impermeable sheet 5 is provided with grooves directed toward both ends, or the surface of the liquid-impermeable sheet 5 is added with It is preferable to apply a treatment to prevent the liquid sample from scattering. In a preferred embodiment, for example, the upper surface of the liquid impermeable sheet 5 is covered with the structure shown in FIG. Covering with a liquid permeable sheet 6 may be mentioned.

In the immunochromatographic measurement apparatus of FIG. 1, when a specific IgG antibody in serum is used as an analyte, the capture site 21 is formed by immobilizing the antigen recognized by the specific IgG antibody, which is the analyte, on the membrane carrier 2. The impregnated member 31 may be impregnated with a labeled antibody formed by labeling an antibody that binds to the constant region of a specific IgG antibody, which is the specimen, with an appropriate labeling substance. In this case, the fraction of the liquid sample that has passed through the first channel first reaches the capture site 21 and the unlabeled analyte accumulates at the capture site 21, and then the liquid that has passed through the second channel A fraction of the sample reaches the capture site 21 together with the labeled antibody and binds to the analyte accumulated there, so that the rate of analyte labeling at the capture site 21 increases, enabling highly sensitive detection.

In the immunochromatography measurement apparatus of FIG. 1, when antigens such as microorganisms such as viruses and bacteria or proteins are used as test samples, an antibody that recognizes the first epitope of the antigen that is the test sample is immobilized on the membrane carrier 2. The capture site 21 is formed by the transformation, and the impregnated member 31 is impregnated with a labeled antibody, which is an antibody that recognizes the second epitope of the antigen and is labeled with an appropriate labeling substance, so as to be chromatographically developable. In this case, the fraction of the liquid sample that has passed through the first channel first reaches the capture site 21, where the unlabeled antigen accumulates, and then the fraction of the liquid sample that has passed through the second channel. Since the fraction reaches the capture site 21 together with the labeled antibody and binds to the analyte accumulated there, the rate of labeling of the subject at the capture site 21 increases, enabling highly sensitive detection.

Any usable labeling substance may be used as the labeling substance, but a color labeling substance is preferably used because it can be measured in one step using the apparatus shown in FIG. Examples of color labeling substances include metal colloids such as colloidal gold and colloidal platinum, synthetic latexes such as polystyrene latex colored with red and blue pigments, and latexes such as natural rubber latex. Among them, metal colloids such as gold colloids are particularly preferable.

In the present invention, the time difference between the fraction of the liquid sample that has passed through the first channel of the sample addition part B and the fraction of the liquid sample that has passed through the second channel of the sample addition part B reaching the capture site 21 is 5 times the detection time. In the case of an immunochromatographic measurement device set to ~30 minutes, it is preferably about 3 to 15 minutes. This time lag is adjusted by appropriately changing the length of the liquid permeable sheet 3, the length and arrangement of the liquid impermeable sheet 5, and the physical properties of the liquid permeable sheet 3, such as the average pore size, density, and basis weight. can do. In FIG. 2A, the downstream end of the liquid-impermeable sheet 5 coincides with the downstream end of the liquid-permeable sheet 3, that is, the upstream end of the membrane carrier 2, but this is not the only option. is not required and other configurations may be employed so long as the above time difference is achieved. In FIG. 2A, the upstream end of the liquid-permeable sheet 3 protrudes upstream from the upstream end of the liquid-impermeable sheet 5. Since the time difference can be adjusted by adjusting the time difference, the configuration is not limited to that of FIG.

The sample application section B shown in FIG. 2B is provided with a liquid permeable sheet 6 covering the liquid impermeable sheet 5, and the liquid sample is added onto the liquid permeable sheet 6. This is different from the sample addition portion B shown in FIG. As shown in FIG. 2B, the liquid permeable sheet 6 is longer than the liquid permeable sheet 3, and its upstream end coincides with the upstream end of the liquid permeable sheet 3. so that its downstream end covers the upstream end of the membrane carrier 2 . As a result, the sample application section B in FIG. It has a second channel (see arrowheads) leading to the membrane carrier 2 via the liquid permeable sheet 3 . Since the first channel has a shorter distance to the membrane carrier 2 than the second channel, the liquid sample added to the sample addition part B is It is configured so that it is automatically divided into two fractions and reaches the capture site 21 with a time difference. Since the liquid sample is added to the liquid permeable sheet 6 in the sample adding section B of FIG. 2B, the liquid sample can be prevented from scattering during the addition. In addition, in the embodiment of FIG. 2(B), the liquid permeable sheet 6 corresponds to the "receiving part to which the liquid sample is added" according to the present invention.

The sample application portion B shown in FIG. 2(C) shows an application example of the sample application portion B shown in FIG. 2(B). In the sample application section B of FIG. 2(C), the inside of the liquid permeable sheet 6 is impregnated with an arbitrary reagent in the middle of the first channel (see triangular arrow) of the sample application section B of FIG. 2(B). It is an arrangement of impregnated pads 32 that can be In the case of the sample application portion B in FIG. 2(C), for example, the impregnation member 32 is impregnated with an enzyme-labeled antibody so as to enable chromatographic development, and the impregnation member 31 is impregnated with the enzyme-labeled coloring agent so as to enable chromatographic development. can be made In this case, when the liquid sample is applied to the liquid-permeable sheet 6, the fraction of the liquid sample that has passed through the first channel (see the triangular arrow) forms an analyte and an enzyme-labeled antibody when passing through the impregnation member 32. and permeates 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 channel (see the arrowhead arrow) dissolves the chromogenic substrate when passing through the impregnation member 31, permeates the membrane carrier 2, and passes through the first channel. The fraction reaches the capture site 21 later than the fraction collected, and the substrate can be colored by the labeling enzyme of the complex accumulated there. Therefore, enzymatic chromogenic immunochromatography measurement using an enzyme-labeled antibody can be performed in one step.

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

FIG. 2(D) shows a sample application section B having three channels. In the sample application section B of FIG. 2(D), a liquid-impermeable sheet 7 identical to the liquid-impermeable sheet 5 is placed on the liquid-permeable sheet 6 of the sample application section B of FIG. 2(C). , a liquid-permeable sheet 8 covering the upper surface of the liquid-impermeable sheet 7 is laminated. The liquid-impermeable sheet 7 is arranged between both ends of the liquid-permeable sheet 6 so that both ends of the liquid-permeable sheet 6 overhang the corresponding ends of the liquid-impermeable 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 are aligned with corresponding ends of the liquid permeable sheet 6 . The liquid impermeable sheet 7 is arranged downstream of the liquid impermeable sheet 5 . Note that the impregnated pad 32 is arranged in the center of the liquid impermeable sheet 5 .

In the case of the sample application portion B in FIG. 2(D), when the liquid sample is applied onto the liquid permeable sheet 8, the liquid sample branches in both the upstream and downstream directions, as shown in FIG. 2(D). and flow. The fraction of the liquid sample that has flowed downstream is the liquid permeable sheet 8 projecting downstream from the downstream end of the liquid impermeable sheet 7 and the membrane carrier 2 from the downstream end of the liquid permeable sheet 6 . permeates into the membrane carrier 2 and undergoes chromatographic development in the membrane carrier 2 , and the analyte contained therein accumulates in the capture site 21 . On the other hand, part of the liquid sample fraction that has flowed upstream permeates the membrane carrier 2 from the upstream end of the liquid-impermeable sheet 7 through the liquid-permeable sheet 6 as in FIG. 2(C). 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 as in FIG. 2(C). Since the liquid-impermeable sheet 7 is arranged on the downstream side of the liquid-impermeable sheet 5, the fraction that permeates the membrane carrier 2 via the liquid-permeable sheet 6 passes through the liquid-permeable sheet 3. It reaches the trapping site 21 earlier than the fraction that has permeated the membrane carrier 2 by the force. Therefore, after the enzyme-labeled antibody contained in the impregnated member 32 binds to the analyte accumulated at the capture site 21, the chromogenic substrate contained in the impregnated member 31 reaches the capture site 21 and the complex accumulated there. The substrate can be developed by the body's labeling enzyme. Therefore, enzymatic chromogenic immunochromatography measurement using an enzyme-labeled antibody can be performed in one step. Similarly, by stacking multiple layers of liquid-impermeable sheets and liquid-permeable sheets, it is possible to construct a sample application section B having four or more channels.

In the apparatus shown in FIG. 1, like the liquid absorbent member 4, the downstream end of the liquid permeable sheet 3 may be laminated on the upstream end of the membrane carrier 2 to connect them. Similarly, like the liquid permeable sheet 3 shown in FIG. 1, the upstream end of the liquid absorbent member 4 may be butted against the downstream end of the membrane carrier 2 to connect them.

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

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

The membrane carrier is not particularly limited as long as it can chromatographically develop a liquid sample by capillary action. Examples thereof include nitrocellulose membrane filters 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 that can quickly absorb and retain liquid, and examples thereof include cotton cloth, filter paper, and porous plastic nonwoven fabrics made of polyethylene, polypropylene, etc., particularly filter paper. is optimal.

The impregnation member is not particularly limited as long as it can be impregnated with a reagent such as a labeled antibody or an enzyme-labeled coloring agent together with a liquid sample so as to allow chromatographic development. etc.), and porous plastic cloths such as polyethylene and polypropylene.

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

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

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

Example 1 (Detection of specific IgA antibody in serum of non-tuberculous mycobacterial disease patient)
An immunochromatographic measurement device having the same configuration as that shown in FIG. 1 except that the sample addition part shown in FIG. 2(B) was provided was prepared. As the membrane carrier 2, a 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 adhesive sheet 1 having the same width. On the membrane carrier 2, a Glycopeptidolipid core antigen (hereinafter, GPL- core antigen) was immobilized to form a capture site 21 . A control line 22 was provided at a position 14.5 mm downstream from the upstream end of the membrane carrier 2 . A control line 22 was formed by immobilizing an anti-rabbit polyclonal antibody (manufactured by DAKO) on the membrane carrier 2 . A cellulose non-woven fabric having a width of 4 mm and a length of 30 mm was used as the liquid-permeable sheet 3 and attached to the pressure-sensitive adhesive sheet 1 . At that time, an impregnated member 31 (width 4 mm, length 10 mm) impregnated with platinum-gold colloid-labeled anti-human IgA rabbit antibody was sandwiched between the adhesive sheet 1 and the liquid-permeable sheet 3 . The impregnated member 31 was arranged such that the upstream end of the adhesive sheet 1 was positioned 10 mm downstream from the upstream end of the pressure-sensitive adhesive sheet 1 . A cellophane tape having a width of 4 mm and a length of 25 mm was used as the liquid-impermeable sheet 5 and arranged so that the downstream end thereof coincided with the downstream end of the liquid-permeable sheet 3 . Further, as shown in FIG. 2(B), a cellulose non-woven fabric having a width of 4 mm and a length of 33 mm was used as the liquid permeable sheet 6, and the upstream end was aligned with the upstream end of the liquid permeable sheet 3. , and a portion extending from the downstream end to the upstream end by 3 mm overlaps the upstream end of the membrane carrier 2 , and is covered with the liquid impermeable sheet 5 .

A positive specimen evaluated by Capillary MAC antibody ELISA (trade name: product of Towns Inc.) was prepared with physiological saline to the concentration shown in Table 1. Using a positive specimen as a liquid sample, the immunochromatographic measurement device It was added to the sample addition part (the part indicated by the white arrow in FIG. 2(B)), and after 30 minutes had passed, it was evaluated visually and by an immunochromatographic reader (ICR). The evaluation criteria for visual judgment were divided into 5 stages of - (no coloring), ± (weak coloring), + (clear coloring), ++ (remarkable coloring), and +++ (particularly remarkable coloring). . Table 1 shows the results.

FIG. 3 shows the results of comparing the ICR results in Table 1 with the measured values of the ELISA method. From FIG. 3, a good correlation was observed between the two positive samples with a concentration of 7.10 U/ml or more. Therefore, it was confirmed that the immunochromatographic measuring device of the present invention can detect blood IgA antibodies very simply compared to the ELISA method.

Example 2 (Detection of influenza virus type A antigen)
An immunochromatographic measurement device having the same configuration as that shown in FIG. 1 was prepared except that the sample addition part shown in FIG. 2(C) was provided. As the membrane carrier 2, a 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 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 downstream from the upstream end to form a capture site 21. A control line 22 was provided at a position 14.5 mm downstream from the upstream end of the membrane carrier 2 . A control line 22 was formed by immobilizing an anti-mouse polyclonal antibody (product of DAKO) on the membrane carrier 2 . An impregnated member 31 impregnated with a first labeled antibody consisting of an anti-influenza virus type A antibody labeled with colloidal platinum-gold and having a width of 4 mm and a length of 10 mm is extended from the upstream end of the adhesive sheet 1 at its upstream end. It was arranged so as to be positioned 10.0 mm downstream. Further, as the liquid permeable sheet 3 , a cellulose nonwoven 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 . A cellophane tape having a width of 4 mm and a length of 25 mm was used as the liquid-impermeable sheet 5 , and its downstream end was arranged so as to coincide with the downstream end of the liquid-permeable sheet 3 . Furthermore, as shown in FIG. 2(C), an impregnated member 32 impregnated with a second labeled antibody consisting of an anti-influenza virus type A antibody labeled with colloidal platinum-gold and having a width of 4 mm and a length of 10 mm is placed upstream of it. The side end was positioned 5.0 mm downstream from the upstream end of the liquid-impermeable sheet 5 . Further, as shown in FIG. 2(C), a cellulose nonwoven fabric having a width of 4 mm and a length of 33 mm was used as the liquid permeable sheet 6, and the upstream end of the liquid permeable sheet 3 was aligned with the upstream end of the liquid permeable sheet 3. , and a portion extending from the downstream end to the upstream end by 3 mm overlapped with the upstream end of the membrane carrier 2 , and was covered with the liquid impermeable sheet 5 . At that time, the impregnated member 32 was sandwiched between the liquid-impermeable 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 specimen was adjusted to 1×10 ³ PFU/ml using specimen extract (a product of Townes Inc.). (point indicated by the white arrow in FIG. 2(C)), and evaluated by an immunochromatographic reader (ICR) 5, 8, 15, 20 and 40 minutes after the addition. As a control, Immunoace Flu (trade name: product of Townes) was used, the same test sample was added, and determination was made under the same conditions. The results are shown in Table 2 and FIG. From the results shown in Table 2 and FIG. 4, it was found that the immunochromatography measurement device of the present invention provided higher sensitivity than the control product, although the measurement time was long.

Example 3 (Detection of influenza virus type A antigen)
An immunochromatographic measuring device similar to that of Example 2 was produced, except that the arrangement position of the upstream end of the impregnated member 31 was 10.0 mm, 13.0 mm or 20.0 mm from the upstream end of the adhesive sheet 1 . Influenza virus type A antigen isolated and cultured from a clinical specimen was adjusted to 1×10 ⁴ PFU/ml using a specimen extract (a product of Townes Co.). 3 minutes, 5 minutes, 8 minutes, 15 minutes, 20 minutes, and 30 minutes after the addition of the sample addition part (point indicated by the white arrow in FIG. 2 (C)) Determination by immunochromatographic reader (ICR) did. As a control, Immunoace Flu (trade name: product of Townes) was used, the same test sample was added, and 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 has become clear that the detection sensitivity changes depending on the arrangement position of the impregnated member 31 . It has been clarified that the sensitivity increases as the position of the impregnating member 31 is closer to the upstream side, that is, as the distance from the membrane carrier increases.

Claims (11)

A chromatographic development unit comprising a membrane carrier having a capture site formed by immobilizing a substance that can bind immunologically to a subject; and supplying a liquid sample to the membrane carrier and directing it to the capture site. an immunochromatographic measurement device including at least a sample addition unit communicating with the membrane carrier so as to chromatographically develop the membrane carrier, wherein the sample addition unit includes a receiving portion to which the liquid sample is added; a plurality of channels in communication with the sites; and a discharge site in communication with the plurality of channels so as to supply the liquid sample to the membrane carrier, wherein the plurality of channels communicate with the liquid that has passed through different channels. configured such that the sample reaches the capture site at different times ;
The sample application section includes a liquid-impermeable sheet disposed at the receiving site, and a first flow channel that guides the added liquid sample from one end of the liquid-impermeable sheet to the membrane carrier. and a second channel leading from the other end of the liquid-impermeable sheet to the membrane carrier . 2. The immunochromatographic measuring device according to claim 1, wherein said receiving portion is formed on the upper surface side of said liquid-impermeable sheet and between said two end portions. The liquid-impermeable sheet is arranged on the upper surface of a first liquid-permeable sheet connected to one end of the membrane carrier, and the liquid sample added to the liquid-impermeable sheet is placed on the membrane carrier of the liquid-impermeable sheet. a first channel leading from one end of the membrane carrier to the membrane carrier; The immunochromatographic measurement device according to claim 1 , which forms two channels. 4. The immunochromatographic measurement device according to claim 3, wherein said sample application section further comprises a second liquid permeable sheet covering said liquid impermeable sheet. 5. The immunochromatographic measuring device according to any one of claims 1 to 4, wherein said membrane carrier is a single membrane carrier. The immunochromatography according to any one of claims 1 to 5, wherein the analyte is an antibody contained in the liquid sample, and the immobilized substance is an antigen to which the antibody specifically binds. measuring device. 7. The immunochromatographic measuring device according to claim 6, wherein an antibody that specifically binds to the antibody accumulated in the capture site is arranged in the second channel so as to be chromatographically developable. 6. The subject according to any one of claims 1 to 5, wherein the analyte is an antigen contained in the liquid sample, and the immobilized substance is a first antibody that specifically binds to the antigen. immunochromatographic measurement device. 9. The immunochromatographic measurement device according to claim 8, wherein a second antibody that specifically binds to the antigen accumulated in the capture site is arranged in the second channel so as to be chromatographically developable. 10. The immunochromatographic 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 channel so as to be capable of being chromatographically developed, and a chromogenic substrate that causes the enzyme-labeled antibody to develop a color is added to the second channel. 9. The immunochromatographic measurement device according to claim 8, arranged so as to be capable of chromatographic development in the channel of the.

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