JP4990692B2 - Immunochromatographic assay and kit - Google Patents

Description

  The present invention relates to an immunochromatography measurement method and a kit with improved measurement sensitivity, and particularly when a test sample containing a reaction inhibitor is used, such as detection of specific IgG in blood. The present invention relates to an immunochromatographic measurement method and kit capable of obtaining high measurement sensitivity even when using a test sample containing a concentration specimen.

  A second carrier capable of reacting immunologically to the specimen is prepared by preparing a membrane carrier having a capture site formed by previously immobilizing a first substance immunologically reactive with the specimen in a predetermined position. Immunochromatography in which a mixed solution of the substance and test sample is chromatographed on the membrane carrier toward the capture site, and a complex of the specimen and the second substance in the mixture is captured at the capture site The measuring method is publicly known (refer to Japanese translation of PCT publication No. 01-503174). Such a measurement method is widely used not only for clinical use but also for home use because the measurement is completed by a one-step operation of injecting the mixture into a membrane carrier and a result can be obtained quickly.

  However, the known immunochromatographic measurement method has a problem that the measurement sensitivity is low when detecting a test sample containing a reaction inhibitor, for example, specific IgG in blood. This is because, in the measurement of IgG, an antigen is used as the first substance and an anti-IgG antibody is used as the second substance. However, in the blood, other IgGs other than the specific IgG to be measured are used. This is because the second substance is bound to these other IgGs and consumed.

  In addition, even when the test sample contains a high-concentration sample, the ratio of the second antibody to the sample is low, so that a so-called prozone phenomenon in which the reaction appears to be weak appears, resulting in a decrease in measurement sensitivity.

  In order to improve the above-mentioned drawbacks, Japanese Patent Application Laid-Open No. 2001-337091 discloses a longitudinal portion provided with a capture site formed by previously fixing a first substance immunologically reactive with a specimen in a predetermined position. A test substance that penetrates into the membrane carrier from the side of the capture site through the dry porous material, a second substance that is immunologically reactive with the specimen, and the test sample An immunochromatographic measurement method comprising the step of: chromatographically developing the mixed solution with the membrane carrier in the longitudinal direction toward the capture site and binding the second substance to the sample captured at the capture site. Proposed.

  In this two-step method, although the disadvantage that the second antibody is consumed by substances other than the specimen and the prozone phenomenon are avoided for the time being, further improvement in measurement sensitivity has been desired.

Japanese Translation of National Publication No. 01-503174 JP 2001-337091 A

  Therefore, the present invention provides high measurement sensitivity even when a test sample containing a reaction inhibitor or a test sample containing a high concentration specimen is used, such as detection of specific IgG in blood. It is an object of the present invention to provide an immunochromatographic measurement method and kit capable of obtaining

  As a result of examining in detail the behavior when the test sample is chromatographed on the membrane carrier, the present inventors have made the test sample chromatographically developed and passed through the membrane carrier toward the capture site, The present inventors have found that the measurement sensitivity is significantly improved by developing the developing solvent with the membrane carrier in the direction opposite to the test sample in the direction opposite to the capture site, thereby completing the present invention.

  That is, according to one aspect of the present invention, a membrane carrier including a capture site formed by fixing a first substance immunologically reactive with a specimen in advance at a predetermined position is prepared. A developing solvent containing a second substance that is immunologically reactive with the specimen after being applied to the membrane carrier directly toward the capture site and chromatographically developed to pass through the capture site To the capture site in the direction opposite to the chromatographic development direction of the test sample, the first substance and the second substance at the capture site And an immunochromatographic measurement method characterized in that detection is performed by sandwiching between the two.

In the measurement method of the present invention, the developing solvent is previously mixed with the second substance in a container separate from the membrane carrier, and injected into the membrane carrier as a mixed solution containing the second substance. It may be chromatographed. Alternatively, the second substance is placed in advance on the membrane carrier, and when the developing solvent is injected into the membrane carrier and chromatographed, the second substance and the developing solvent are mixed together so that the developing solvent becomes the first solvent. Chromatographic development may be performed in a state containing the two substances.
In the measurement method of the present invention, the test sample is first applied directly to the membrane carrier and chromatographed on the membrane carrier toward the capture site, but the test sample passes from the capture site to the end of the membrane carrier. Although it does not prevent permeation, it is desirable that the membrane carrier is permeated slightly beyond the capture site so as not to impede the permeation of the membrane carrier of the next developing solvent.

Furthermore, according to another aspect of the present invention, as a kit suitable for carrying out the method of the present invention, there is provided an immunochromatography measurement kit comprising at least an immunochromatographic test strip and a housing containing the immunochromatographic test strip. And
The immunochromatographic test strip includes at least a first substance that can immunologically react with a specimen and a membrane carrier, and the first substance is previously fixed at a first position of the membrane carrier. Form a capture site,
Further, the immunochromatography measurement kit includes a second substance that is immunologically reactive with the specimen and is labeled, and the second substance is separated from the second position separated from the capture site. It is prepared so that it can be chromatographed on the membrane carrier toward the first position,
The housing includes a first opening and a second opening provided adjacent to each other so as to sandwich the first position and the second position, and the first opening Means for receiving and applying directly to the membrane carrier for chromatographic development towards the first position, the second opening receiving the developing solvent and containing it with the second substance An immunochromatography measurement kit is provided, which comprises means for performing chromatographic development on the membrane carrier toward the first position in a state where

The kit of the present invention may further include a container for preparing a developing solvent containing the second substance and injecting the developing solvent into the second opening. Alternatively, the second substance may be preliminarily placed on the membrane carrier and mixed with the second substance when the developing solvent is injected into the second opening.
In addition, as described above, in the present invention, the test sample is first chromatographed on the membrane carrier toward the capture site, but the capture site is not disturbed so as not to prevent permeation of the next developing solvent in the membrane carrier. It is desirable to allow the membrane carrier to permeate slightly above. Therefore, the kit of the present invention may include means for allowing the test sample to be received in the first opening by an amount that reaches the first position but does not reach the second position. . As such means, for example, a container capable of accommodating or weighing the test sample by the above amount is attached to the kit, or the first opening has a structure that cannot accommodate the test sample of the above amount or more. It is done.

  According to the present invention, a test sample is directly applied to a membrane carrier and chromatographed toward a capture site formed of a first substance that can immunologically react with a sample, and the capture site is After passing the sample, the developing solvent containing the second substance that can immunologically react with the specimen is chromatographed on the membrane carrier toward the capture site in the direction opposite to the chromatographic development direction of the test sample. Since the test sample passes through the capture site twice, the specimen in the test sample is given sufficient opportunity to come into contact with the first substance. Since the amount of integration increases, measurement sensitivity can be improved.

  In the present specification, the test sample means a sample subjected to immunoassay, and the specimen means a target substance (analyte) to be measured by immunoassay.

  Hereinafter, specific examples of the immunochromatography measurement kit of the present invention and the immunochromatography measurement method of the present invention using the same will be described in detail with reference to FIG.

  As shown in FIG. 1, the immunochromatography measurement kit includes a housing 10 and at least an immunochromatographic test strip accommodated in the housing 10.

  In the immunochromatographic test strip, the membrane carrier 3 is stuck on an adhesive sheet 4 longer than that. The membrane carrier 3 has an end on the chromatographic development start side (that is, the right side of the figure, hereinafter referred to as “upstream side”, and the opposite side, ie, the left side of the figure, hereinafter referred to as “downstream side”). The downstream end of the impregnation member 5 is placed on the adhesive sheet 4, and the upstream portion of the impregnation member 5 is attached to the adhesive sheet 4. Further, a developing solvent addition member 7 is placed on the impregnating member 5 so as to cover the entire surface of the impregnating member 5 and the upstream end of the adhesive sheet 4. Further, the absorbing member 6 is connected with the upstream end thereof placed on the downstream end of the membrane carrier 3, and the other part of the absorbing member 6 is adhered to the adhesive sheet 4. ing.

  The membrane carrier 3 is provided with a capture site 31 formed by immobilizing a first substance that can immunologically react with the specimen. Further, the impregnated member 5 is impregnated with a labeled second substance that can immunologically react with the specimen so that the membrane carrier 3 can be chromatographed. Further, the membrane carrier 3 is provided with a control portion 32 for capturing the second substance at a predetermined distance downstream from the capture portion 31 so that the completion of the reaction can be confirmed.

  The housing 10 includes openings T and C above the capture site 31 and the control site 32, respectively, so that the accumulation of the specimen or the second substance in the capture site 31 and the control site 32 can be determined by the naked eye. The housing 10 includes a first opening 1 for injecting a test sample at a position between the openings T and C, and a developing solvent above the developing solvent addition member 7 upstream of the impregnation member 5. A second opening 2 for injection is provided. As shown in FIG. 1, in the housing 10, the first opening 1 is located in the center in the longitudinal direction of the housing 10, and the openings T and C, the capture part 31, and the control part 32 are symmetrical to this. Therefore, it is convenient to quantify the coloration of the capture site 31 and the control site 32 with an immunochromatographic reader.

The membrane carrier 3 uses a nitrocellulose membrane filter, but the first substance that can chromatograph the specimen contained in the directly applied test sample and forms the capture site 31 Any cellulosic membrane, nylon membrane, glass fiber membrane, or the like can be used as long as it can fix.
The pressure-sensitive adhesive sheet 4 is not particularly limited as long as it does not change characteristics such as permeability of the membrane carrier 3, and a known one can be used.

  As the impregnating member 5, a band-shaped glass fiber nonwoven fabric is used, but is not limited thereto. For example, cellulose plastic cloth (filter paper, nitrocellulose membrane, etc.), porous plastic cloth such as polyethylene, polypropylene, etc. Etc. can also be used. In the present invention, the impregnating member 5 is not always necessary, and the second substance may be arranged directly on the membrane carrier 3 so that the second substance can be chromatographed. In the case where the second substance and the developing solvent are mixed in advance in a container separate from the immunochromatographic test strip, and the developing solvent is injected into the membrane carrier 3 containing the second substance, the impregnating member 5 is not essential.

As the developing solvent addition member 7, for example, a sheet or film of porous synthetic resin such as porous polyethylene and porous polypropylene, and paper or woven or non-woven fabric made of cellulose such as filter paper and cotton cloth are used. Can be used.
The absorbent member 6 may be made of any material that can quickly absorb and hold liquid, and examples thereof include cotton cloth, filter paper, and porous plastic nonwoven fabric made of polyethylene, polypropylene, etc., and filter paper is particularly suitable. It is.

  When the specimen is an antibody such as IgG, an antigen is usually used as the first substance immobilized on the capture site 31, and an antibody having specific reactivity with respect to the specimen as the second substance. Substances are used. When the specimen is an antigen, antibodies are usually used as the first substance and the second substance, and each antibody may independently be a polyclonal antibody or a monoclonal antibody, but at least one of them may be used. One is preferably a monoclonal antibody. Usually, the first substance (antibody) and the second substance (antibody) are used in a “hetero” combination, that is, a first substance that recognizes each antigenic determinant having a different position and structure on the antigen. One substance (antibody) and a second substance (antibody) are used in combination. However, the first antigenic determinant and the second antigenic determinant may be structurally the same as long as the positions on the antigen are different. In that case, the first substance (antibody) and the second substance The (antibody) may be a “homo” combination monoclonal antibody, ie the same monoclonal antibody can be used for both the first substance (antibody) and the second substance (antibody).

  The second substance is preferably labeled with a labeling substance in advance. Such a labeling substance may be any substance that can be used, and examples thereof include a color labeling substance, an enzyme labeling substance, and a radiation labeling substance. Among these, it is preferable to use a color labeling substance from the viewpoint that the color change at the capturing site 31 can be determined quickly and easily by observing with the naked eye. Examples of the color labeling substance include metal colloids such as gold colloid and platinum colloid, synthetic latex such as polystyrene latex colored with respective pigments such as red and blue, and latex such as natural rubber latex. Of these, metal colloids such as gold colloid are particularly preferred.

The labeled second substance is impregnated in the impregnating member 5 so that the developing solvent is chromatographed together when the developing solvent passes through the impregnating member 5 by impregnating the suspension in the impregnating member 5 and drying. Can be retained.
The substance that forms the control site 32 is not particularly limited as long as it has binding properties with the second substance, but is usually a substance that can immunologically react with the second substance, such as an antibody. Is used.

  The developing solvent is not particularly limited as long as it can be used in the immunochromatography measurement method, and an aqueous solvent such as water, physiological saline, buffer solution and the like can be used. Examples of the buffer include Tris buffer, phosphate buffer, HEPES (2-hydroxypiperazine-N′-2-ethanesulfonic acid) buffer, and the like. The developing solvent may contain various additives such as a pH adjuster, a surfactant, a preservative, an inorganic salt, and a polymer described in JP-A-2003-344406, if necessary. As a pH adjuster, hydrochloric acid, sodium hydroxide, etc. can be used, for example.

The test sample is not particularly limited as long as the membrane carrier can be chromatographed, and blood (whole blood, serum, or plasma), saliva, urine, organ emulsion, cloacaswab, tracheal swab, Biological samples such as feces, nasal aspirate, nasal swab, and throat swab are included. These biological samples may be appropriately diluted so that the membrane carrier can be chromatographed. For dilution, the same one as the developing solvent can be used.
The specimen is not particularly limited as long as it can be detected by an immunoassay. In addition to various antibodies such as IgG and biopolymers, pathogenic bacteria such as tuberculosis and O-157, viruses such as influenza virus and adenovirus, Examples include trace substances such as antigens such as C-reactive protein contained in blood and environmental hormone-like substances present in the environment.

Thus, after the sample collected from the living body is adjusted to a concentration capable of chromatographic development as necessary, it is injected as a test sample into the first opening 1 of the immunochromatographic test strip shown in FIG. 1 (FIG. 1). (b) Refer to vertical thin arrows), and the test sample is directly contacted with the membrane carrier 3 and chromatographed, and passes through the capture site 31 (see FIG. 1 (b) horizontal thin arrows). At this time, if a sample is present in the test sample, the sample is captured and accumulated in the first substance at the capture site 31 by the antigen-antibody reaction. At the same time, the test sample is chromatographed even toward the control unit 32.
At this time, the test sample only needs to slightly exceed the capture site 31 rather than penetrate through the capture site 31 of the membrane carrier 3 to the end. Therefore, the injection amount of the test sample 3 may be an amount sufficient to slightly exceed the capture site 31. This injection amount varies depending on the viscosity of the test sample, the type and size of the membrane carrier, the position of the capture site 31, and the like, and can be set as appropriate in consideration of these. In order to be able to measure or meter an appropriate injection volume, the kit of the present invention may be provided with a means capable of measuring or metering the volume of the test sample, and the dimensions of the first opening 1 may be adjusted to an appropriate volume. It is also possible to create a size that can accommodate only the test sample.

Thereafter, when the developing solvent is injected into the second opening 2 of the immunochromatographic test strip shown in FIG. 1 (see FIG. 1 (b) vertical thick arrow), after passing through the developing solvent addition member 7, the impregnation member 5, where the membrane carrier 3 together with the second substance is chromatographed toward the capture site 31. At this time, in order to push back the test sample that has passed through the capture site 31, the test sample is chromatographed again toward the capture site 31 (see the thick arrow in the horizontal direction in FIG. 1 (b)). At this time, if a specimen is present in the test sample, a complex of the second substance and the specimen is formed by the antigen-antibody reaction, and when the specimen reaches the capture site 31, it becomes a first substance at the capture site 31. The second substance that has been captured and accumulated while not forming a complex binds to the analyte already captured at the capture site 31, thus sandwiching the analyte with the first substance and the second substance. Can be captured. In this way, since the test sample passes through the capture site 31 twice, an opportunity for more specimens to be captured at the capture site 31 is provided, so that good measurement sensitivity can be obtained.
At this time, if a colored labeling substance such as colloidal gold is used as the labeling substance, the capture site 31 is colored due to the accumulation of the colored labeling substance. Therefore, the sample should be measured qualitatively or quantitatively immediately. Can do.

  In the specific example of FIG. 1, when the impregnation member 5 is not impregnated with the second substance, instead of the developing solvent, the second substance is previously separated from the developing solvent in a container separate from the immunochromatographic test strip. By mixing and injecting the mixed solution from the second opening 2, the same effect as described above can be obtained.

  Since the second substance that has moved the membrane carrier 3 together with the developing solvent to the downstream side is captured there when it reaches the control site 32, it can be confirmed that the measurement has been completed normally. In addition, the surplus developing solvent is absorbed by the absorbing member 6 to prevent backflow. The casing 10 is provided with an air vent hole 11 at a position above the absorbing member 6 to promote absorption of the developing solvent.

  When whole blood is used as a test sample, particularly when a colored labeling substance such as gold colloid is used as the labeling substance, a blood cell capturing membrane member is disposed on the membrane carrier 3 facing the first opening 1. May be. This prevents red blood cells from being developed on the membrane carrier 3, so that it is easy to confirm the accumulation of the colored label at the capture site 31 of the membrane carrier 3. As the blood cell capturing membrane member, a carboxymethylcellulose membrane is used. Specifically, ion exchange filter paper CM (trade name) sold by Advantech Toyo Co., Ltd. or ion exchange cellulose sold by Whatman Japan Co., Ltd. Paper or the like can be used.

  The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to these examples.

Example 1 (Preparation of a kit for immunochromatography measurement)
(1) Preparation of gold colloid solution Add 1 ml of 1% (v / w) chloroauric acid aqueous solution to 99 ml of ultrapure water boiled by heating, and 1 minute later, 1% (v / w) sodium citrate After 1.5 ml of an aqueous solution was added and heated to boil for 5 minutes, it was allowed to cool to room temperature. Subsequently, 200 mM potassium carbonate aqueous solution was added to this solution to adjust to pH 9.0, and ultrapure water was added thereto to make a total volume of 100 ml to obtain a gold colloid solution.

(2) Preparation of colloidal gold labeled anti-mouse IgG antibody solution The anti-mouse IgG antibody was labeled with colloidal gold according to the following procedure.
A protein equivalent weight of 1 μg of an anti-mouse IgG antibody (rabbit) (hereinafter, when the protein equivalent weight of an antibody is indicated, it is simply indicated by a weight value by gravimetric analysis of the purified protein) and 1 ml of a colloidal gold solution of (1) After mixing and allowing to stand for 2 minutes at room temperature to bind all of these antibodies to the gold colloid particle surface, 10% bovine serum albumin (hereinafter referred to as “BSA”) so that the final concentration in the gold colloid solution is 1%. An aqueous solution was added, and all the remaining surfaces of the colloidal gold particles were blocked with the BSA to prepare a colloidal gold labeled anti-mouse IgG antibody (rabbit) (hereinafter referred to as “gold colloid labeled antibody”) solution. . This solution was centrifuged (5600 × G, 30 minutes) to precipitate colloidal gold labeled antibody, and the supernatant was removed to obtain colloidal gold labeled antibody. The colloidal gold labeled antibody was suspended in a 50 mM Tris-HCl buffer (pH 7.4) containing 10% sucrose, 1% BSA, 0.5% Triton-X100 to obtain a colloidal gold labeled antibody solution.

(3) Preparation of immunochromatographic test strip The immunochromatographic test strip shown in FIG. 1 was prepared by the following procedure.

(3-1) Formation of capture site An elongated strip-shaped nitrocellulose membrane having a width of 5 mm and a length of 36 mm was prepared as a membrane carrier 3 for chromatographic development of a chromatographic medium.
0.5 μL of a solution containing 1.0 mg / ml of influenza virus A antigen was applied in a line at a position 7.5 mm from the upstream end of the membrane carrier for chromatographic development, dried at room temperature, and then captured 31.
(3-2) Formation of control line 0.5 μl of a solution containing anti-rabbit IgG antibody was applied onto the line at a position of 24.0 mm from the end of the chromatographic starting point side of the membrane carrier for chromatographic development. Dried to form a control site 32 that captures the colloidal gold labeled anti-mouse IgG antibody.

(3-3) Colloidal gold labeled antibody impregnated member
A 5 mm × 10 mm strip-shaped glass fiber nonwoven fabric was impregnated with 45 μl of a colloidal gold labeled antibody solution, which was dried at room temperature to obtain a colloidal gold labeled antibody impregnated member 5.

(3-4) Preparation of immunochromatographic test strip In addition to the chromatographic development membrane carrier 3 and the labeled antibody-impregnated member 5, a cotton cloth as a developing solvent addition member 7 and a filter paper as an absorbing member 6 were prepared. Then, in the same manner as in FIG. 1, these members were arranged on the adhesive sheet 4 to prepare an immunochromatographic test strip.
The immunochromatographic test strip was accommodated in the housing 10 shown in FIG. 1 to prepare an immunochromatographic measurement kit.

Example 2 (Detection of anti-influenza virus type A antibody from mouse serum)
Serum obtained from a mouse inoculated with influenza virus type A antigen was diluted 40-fold with a phosphate buffer (PBS) and prepared as a test sample. As a control, normal mouse serum was diluted 40-fold with PBS and used as a test sample. Only PBS was used as a blank.

Then, 34 μl of this test sample was dropped onto the first opening 1 of the kit prepared in Example 1 with a micropipette (see the vertical thin arrow in FIG. 1 (b)), and then PBS was added from the second opening 2. 100 μL was dropped (see the vertical thick arrow in FIG. 1 (b)). After standing at room temperature for 15 minutes, the capture amount of the complex of anti-influenza virus type A (mouse) IgG antibody captured at the capture site 31 and colloidal gold-labeled antibody was observed with the naked eye. The results are shown in Table 1. From Table 1, it can be seen that high measurement sensitivity can be obtained according to the present invention.

  The amount of captured color was observed with the naked eye in the degree of reddish purple coloration that increased or decreased in proportion to the amount. Then, the determination was made by classifying into four levels (w indicates weak color):-(no coloration), ± (weak coloration), + (clear coloration), and ++ (significant coloration).

Comparative Example 1
An elongated band-shaped nitrocellulose membrane having a width of 5 mm and a length of 36 mm contains 1.0 mg / ml of influenza virus A antigen at a position 6.0 mm from the upstream end of the chromatographic development membrane carrier 3 of the chromatographic medium. 0.5 μL of the solution was applied in a line shape, and this was dried at room temperature to obtain a capture site 31.
Further, 45 μl of a colloidal gold labeled antibody solution was impregnated into a 5 mm × 15 mm strip-shaped glass fiber nonwoven fabric, and this was dried at room temperature to obtain a colloidal gold labeled antibody impregnated member 5.
In addition, 0.5 μl of a solution containing an anti-rabbit IgG antibody was applied on the line at a position 14.5 mm from the end of the chromatographic starting membrane side of the membrane carrier for chromatographic development, dried at room temperature, A control site 32 that captures the mouse IgG antibody was formed.
Other than that was carried out similarly to Example 1, and created the immunochromatography test strip shown by FIG.2 (b).

As shown in FIG. 2A, the obtained immunochromatographic test strip has an opening CT that opens over the capture site 31 and the control site 32, and is used for adding a developing solvent upstream of the impregnation member 5. A housing 10 having an opening 1 located above the member 7 was prepared.
The immunochromatographic test strip was accommodated in the housing 10 to prepare an immunochromatography measurement kit.

  100 μL of the same test sample, control or blank as in Example 2 was dropped into the first opening 1 with a micropipette (see the thick arrow in FIG. 2 (b)). After standing at room temperature for 15 minutes, the capture amount of the complex of anti-influenza virus type A (mouse) IgG antibody captured at the capture site 31 and colloidal gold-labeled antibody was observed with the naked eye. The results are shown in Table 1. The amount captured was evaluated in the same manner as in Example 2. In this example, which is a one-step method, other IgG antibodies not targeted in the test sample react with gold colloid-labeled antibodies, and as shown in Table 1, almost no reaction was confirmed. The measurement sensitivity was significantly inferior to 2.

Comparative Example 2
An elongated band-like nitrocellulose membrane having a width of 5 mm and a length of 36 mm is composed of 1.0 mg / ml influenza virus A antigen at a position 16.0 mm from the upstream end of the chromatographic development membrane carrier 3 of the chromatographic medium. 0.5 μL of the solution was applied in a line shape, and this was dried at room temperature to obtain a capture site 31.
In addition, 0.5 μl of a solution containing an anti-rabbit IgG antibody was applied on the line at a position 23.0 mm from the end of the chromatographic starting membrane side of the membrane carrier for chromatographic development, dried at room temperature, A control site 32 that captures the mouse IgG antibody was formed.
Other than that was carried out similarly to Example 1, and created the immunochromatography test strip shown by FIG.3 (b).

As shown in FIG. 3 (a), the housing 10 (FIG. 2 (a)) of Comparative Example 1 is formed except that the opening 1 is formed at a position above the membrane carrier 3 on the downstream side of the impregnation member 5. A housing 10 having the same configuration was prepared.
The immunochromatographic test strip was accommodated in the housing 10 to prepare an immunochromatography measurement kit.

  After dropping 34 μl of the same test sample, control or blank as in Example 2 into the opening 1 with a micropipette (see the vertical thin arrow in FIG. 3 (b)), 100 μL of the developing solvent was added from the second opening 2. It was dripped (refer to FIG. 3 (b) vertical thick arrow). After standing at room temperature for 15 minutes, the capture amount of the complex of anti-influenza virus type A (mouse) IgG antibody captured at the capture site 31 and colloidal gold-labeled antibody was observed with the naked eye. The results are shown in Table 2 together with the results of Example 2. The amount captured was evaluated in the same manner as in Example 2. As shown in Table 2, this example is a two-step method. However, since the test sample dropping port, that is, the opening 1 is upstream of the capture site, the test sample passes through the capture site only once, and sensitivity is obtained. It was thought that it was not possible.

  The present invention enables highly sensitive immunochromatographic measurement, and is useful for quickly and easily detecting various antigens and antibodies using various biological samples and diagnosing related diseases. .

FIG. 1 is a schematic view showing a specific example of the immunochromatography measurement kit of the present invention, in which a is a plan view of the housing and b is a longitudinal sectional view of an immunochromatographic test strip accommodated in the housing. FIG. 2 is a schematic view showing the structure of a known immunochromatography measurement kit used in Comparative Example 1, wherein a is a plan view of the housing, and b is a longitudinal sectional view of an immunochromatographic test strip accommodated in the housing. . FIG. 3 is a schematic view showing the configuration of the immunochromatography measurement kit used in Comparative Example 2, wherein a is a plan view of the housing and b is a longitudinal sectional view of an immunochromatographic test strip accommodated in the housing.

Explanation of symbols

1, 2, C, T, CT Opening 3 Membrane carrier 31 Capture site 32 Control site 4 Adhesive sheet 5 Impregnation member 6 Absorbing member 7 Member for developing solvent addition 10 Housing 11 Air vent hole

Claims (12)

A membrane carrier having a capture site formed by previously immobilizing a first substance immunologically reactive with a specimen in a predetermined position is prepared, and a test sample is directed toward the capture site on the membrane carrier. The development solvent containing the second substance that can be immunologically reacted with the specimen after the direct application and chromatographic development to pass through the capture site is the chromatographic development direction of the test sample. by chromatographic development in the membrane carrier toward the capturing zone in the opposite direction, Louis be detected by sandwiched between said analyte and said first material in said capturing zone and the second material In the munochromatography measurement method , the membrane carrier includes a control site for capturing the second substance, and the test sample is directly applied to the membrane carrier between the capture site and the control site, in front Analyte is an IgG antibody, immunochromatographic assay, wherein the first substance is an antigen against the IgG antibody. The immunochromatography measurement method according to claim 1, wherein a developing solvent containing the second substance is prepared in a container separate from the membrane carrier, and then this is subjected to chromatographic development. When the second substance is placed in advance on the membrane carrier and the developing solvent is chromatographed, the second substance and the developing solvent are mixed and the developing solvent contains the second substance in the chromatographic development. The immunochromatographic measurement method according to claim 1 , which is adapted to be performed. The test sample, wherein at reaching the capture site, the the chromatographic development start point of the developing solvent is applied only to the film carrier amount is not reached, immunochromatographic assay according to claim 1. The immunochromatographic assay method according to claim 1, wherein the second substance is labeled with a metal colloid or latex. The immunochromatographic assay method according to claim 5, wherein the membrane carrier is a nitrocellulose membrane. An immunochromatographic test strip, and an immunochromatographic measurement kit comprising at least a housing containing the immunostrip test strip,
The immunochromatographic test strip includes at least a first substance that can immunologically react with a specimen and a membrane carrier, and the first substance is previously fixed at a first position of the membrane carrier. Form a capture site,
Further, the immunochromatography measurement kit includes a second substance that is immunologically reactive with the specimen and is labeled, and the second substance is separated from the second position separated from the capture site. It is prepared so that it can be chromatographed on the membrane carrier toward the first position,
Furthermore, the immunochromatographic test strip includes a control site formed on the membrane carrier so as to capture the second substance chromatographed from the second position through the first position,
The housing is provided so as to sandwich the first opening disposed between the first position and the control part, and the first position and the second position between the first opening. and a second and a opening, the first opening constitute the means for chromatographic development toward the directly applied to the first position to the film carrier to accept the test sample, wherein The second opening constitutes a means for receiving the developing solvent and chromatographically developing it on the membrane carrier toward the first position and the control site in a state containing the second substance. a such Louis Takeno chromatography measurement kit, wherein the analyte is an IgG antibody, immunochromatographic assay kit wherein the first material is characterized in that an antigen against the IgG antibody. The kit according to claim 7, further comprising a container for preparing a developing solvent containing the second substance and injecting the developing solvent into the second opening. The kit according to claim 7, wherein the second substance is arranged in advance on a membrane carrier and is mixed with the second substance when the developing solvent is injected into the second opening. The kit according to claim 7, further comprising means for causing the first opening to receive the test sample in an amount that reaches the first position but does not reach the second position. The kit according to claim 7, wherein the second substance is labeled with a metal colloid or latex. The kit according to claim 11, wherein the membrane carrier is a nitrocellulose membrane.

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