Competitive immunochromatographic analysis and immunochromatographic sensor

Abstract

The present invention provides a competitive immunochromatographic analysis method that can intuitively display the molecular concentration of a substance to be measured and that allows easy visual judgment, and an immunochromatographic sensor that uses the analysis method. [Solution] The competitive immunochromatographic analysis method of the present invention is characterized in that a determination section is composed of a membrane carrier to which a labeled antibody that reacts specifically with the substance to be measured and a text antibody consisting of an antibody that reacts specifically with the substance to be measured are immobilized in the form of letters or figures, and a mask antigen consisting of the same antigen as the substance to be measured is immobilized on the membrane carrier so as to surround the immobilized text antibody in the form of letters or figures, and a mixture of a test solution containing the substance to be measured and the labeled antibody is supplied to the determination section, and the labeled antibody is caused to competitively bind to the substance to be measured, the mask antigen, and the text antibody in the determination section, thereby determining the concentration of the substance to be measured based on the signal from the labeled antibody bound to the text antibody and the mask antigen. [Selected Figure] Figure 2

Description

Technical Field

[0001] The present invention relates to an improved competitive immunochromatographic analysis method capable of semi-quantifying the molecular concentration of a substance to be measured in a test solution visually, and an immunochromatographic sensor using the analysis method.

Background Art

[0002] In recent years, with the diversification of clinical tests, point-of-care testing (POCT) at the clinical site has been attracting attention. As one of the simple test methods for realizing POCT, a sensor using a competitive immunochromatographic method has already been proposed (Patent Document 1). The immunochromatographic sensor proposed in Patent Document 1 is a sensor for measuring bilirubin, and the sensor includes an impregnated member impregnated with a labeled substance obtained by labeling a conjugate of bilirubin and a polymer compound with a labeling substance, a membrane carrier provided with a capture site where an anti-bilirubin antibody is immobilized, and is configured such that in the impregnated member, the labeled substance is mixed in the test solution, and the mixed solution of the test solution and the labeled substance is chromatographically developed on the membrane carrier toward the capture site, so that at the capture site, the labeled substance and bilirubin in the test solution competitively react with the anti-bilirubin antibody, and the labeled substance and bilirubin in the test solution are competitively captured at the capture site. With the above-described configuration, if bilirubin does not exist in the test solution, the labeled substance reacts with the anti-bilirubin antibody immobilized at the capture site by an antibody-antigen reaction and is captured, so that the capture site strongly develops color. If bilirubin exists in the test solution, bilirubin and the labeled substance are competitively captured at the capture site, so that the color development of the capture site becomes weak. Thereby, it becomes possible to visually determine the presence or absence of bilirubin in the test solution based on the color of the capture site.

Prior Art Documents

Patent Documents

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2007-218593 [Summary of the Invention] [Problems to be Solved by the Invention]

[0004] According to the above-described conventional competitive immunochromatographic sensor, since the result can be visually determined only by adding a test solution to the sensor, it is useful as a sensor for point-of-care testing. However, in the above-described conventional competitive immunochromatographic sensor, since the signal (i.e., color) of the test line (i.e., the capture site) shows a negative correlation with the molecular concentration of the substance to be measured in the test solution, the displayed result is not intuitive, and there is a problem that the user may misrecognize the determination of the result. Specifically, in the sensor of Patent Document 1, the more the amount of biopyrine in the test solution, the lighter the color of the capture site, and the less the amount of biopyrine, the darker the color of the capture site. Therefore, there is a possibility that the user may misunderstand that the amount of biopyrine is large because the color of the capture site is dark. Further, according to the conventional competitive immunochromatographic method, the molecular concentration of the substance to be measured must be determined by the darkness of the color of the test line. However, it is very difficult for a human to determine the concentration based on the darkness of the color, it is difficult to determine the cut-off value, and there is a problem that quantification in a narrow concentration range is impossible. An object of the present invention is to solve the above-described conventional problems, and to provide a competitive immunochromatographic analysis method capable of intuitively displaying the molecular concentration of a substance to be measured and an immunochromatographic sensor using the analysis method, which is easy to visually determine. [Means for Solving the Problems]

[0005] To achieve the above object, the competitive immunochromatographic analysis method according to the present invention fixes a text antibody composed of an antibody that specifically reacts with a labeled antibody that specifically reacts with a substance to be measured in the form of characters or figures on a membrane carrier, and fixes a mask antigen composed of the same antigen as the substance to be measured on the membrane carrier so as to surround the text antibody fixed in the form of the characters or figures. A mixed solution obtained by mixing a test solution containing the substance to be measured and the labeled antibody is supplied to a determination unit. In the determination unit, the labeled antibody is competitively bound to the substance to be measured, the mask antigen, and the text antibody, and the concentration of the substance to be measured is determined based on the signal from the labeled antibody bound to the text antibody and the mask antigen. In the above analysis method, a plurality of determination units can be provided, and the ratio of the amount of the text antibody disposed in each determination unit to the amount of the mask antigen disposed can also be changed. In the above analysis method, a plurality of determination units can be provided on the membrane carrier along the flow direction of the test solution, and the ratio of the amount of the mask antigen disposed relative to the text antibody in the downstream determination unit can be made lower than the ratio of the amount of the mask antigen disposed relative to the text antibody in the upstream determination unit. Conversely, it is also possible to make the ratio of the amount of the mask antigen disposed relative to the text antibody in the downstream determination unit higher than the ratio of the amount of the mask antigen disposed relative to the text antibody in the upstream determination unit. Preferably, the text antibody and the mask antigen can be inkjet printed on the membrane carrier. When printing, depending on the types of the text antibody and the mask antigen used, for example, a method of first printing the text antibody in the form of characters or figures on the membrane carrier and then printing the mask antigen so as to cover the text antibody, and a method of first printing the mask antigen on the membrane carrier and then printing the text antibody in the form of characters or figures on the mask antigen can be selected. In either case, the mask antigen surrounds the periphery of the text antibody. In addition, the competitive immunochromatographic sensor according to the present invention comprises a text antibody composed of an antibody that specifically reacts with a labeled antibody that specifically reacts with a substance to be measured, which is fixed to a membrane carrier in the form of characters or a figure. A mask antigen composed of the same antigen as the substance to be measured is fixed to the membrane carrier so as to surround the text antibody fixed in the form of the characters or figure, and has a determination unit formed thereby. Furthermore, the competitive immunochromatographic sensor according to the present invention comprises a sample introduction unit for introducing a sample solution containing a substance to be measured, a labeled antibody-containing unit disposed downstream of the sample introduction unit and containing a labeled antibody that specifically reacts with the substance to be measured, at least one determination unit provided downstream of the labeled antibody-containing unit, and a suction unit disposed downstream of the determination unit, which are arranged on a membrane carrier. The sample solution introduced into the sample introduction unit is configured to pass through the labeled antibody-containing unit and the determination unit in sequence and flow to the suction unit. The determination unit fixes a text antibody composed of an antibody that specifically reacts with the labeled antibody to the membrane carrier in the form of characters or a figure, and fixes a mask antigen composed of the same antigen as the substance to be measured to the membrane carrier so as to surround the text antibody fixed in the form of the characters or figure. Also, a plurality of the determination units may be provided, and the ratio of the amount of the text antibody disposed in each determination unit to the amount of the mask antigen disposed therein may be configured to be changed. Furthermore, a plurality of the determination units may be provided along the flow direction of the sample solution, and the ratio of the amount of the mask antigen to the text antibody in the downstream determination unit may be configured to be lower than the ratio of the amount of the mask antigen to the text antibody in the upstream determination unit. Additionally, the ratio of the amount of the mask antigen to the text antibody in the downstream determination unit may be configured to be higher than the ratio of the amount of the mask antigen to the text antibody in the upstream determination unit. Preferably, the text antibody and the mask antigen can be inkjet printed on the determination unit. In this case, the amounts of the text antibody and the mask antigen to be arranged can be adjusted by the number of printing times. When printing, depending on the types of the text antibody and the mask antigen to be used, for example, after first printing the text antibody in a character or figure shape on the membrane carrier, a method of printing the mask antigen so as to cover the text antibody, and a method of first printing the mask antigen on the membrane carrier and then printing the text antibody in a character or figure shape on the mask antigen can be selected. In any case, the mask antigen surrounds the text antibody.

Advantages of the Invention

[0006] The competitive immunochromatographic analysis method according to the present invention fixes a text antibody composed of an antibody that specifically reacts with a labeled antibody that specifically reacts with a substance to be measured in a character or figure shape on a membrane carrier, and fixes a mask antigen composed of the same antigen as the substance to be measured on the membrane carrier so as to surround the text antibody fixed in the character or figure shape. A mixed solution of a test solution containing the substance to be measured and the labeled antibody is supplied to the determination unit, and in the determination unit, the labeled antibody is competitively bound to the substance to be measured, the mask antigen, and the text antibody, and based on the signals of the labeled antibody bound to the text antibody and the mask antigen, the concentration of the substance to be measured is determined. Here, the labeled antibody specifically reacts with the substance to be measured, the mask antigen, and the text antibody, respectively, but the substance to be measured and the text antibody do not specifically react with each other. Therefore, when a mixed solution of the labeled antibody and the test solution is supplied to the determination unit, the labeled antibody competitively reacts with the substance to be measured contained in the test solution, the mask antigen fixed to the determination unit, and the text antibody. Here, the labeled antibody bound to the substance to be measured cannot bind to the mask antigen but can bind to the text antibody. Also, the labeled antibody not bound to the substance to be measured can bind to both the mask antigen and the text antibody. Then, in the determination unit, the text antibody is laid out to represent characters or figures, and the mask antigen is laid out to surround the text antibody. When the amount of the substance to be measured contained in the test solution is small, the ratio of the labeled antibody that binds to the text antibody remains unchanged, but the amount of the labeled antibody that binds to the substance to be measured becomes relatively small. As a result, the amount of the labeled antibody that binds to the mask antigen becomes relatively large. In addition to the labeled antibody that has not bound to the substance to be measured, the labeled antibody that has bound to the substance to be measured binds to the text antibody. However, since the amount of the labeled antibody that binds to the substance to be measured is relatively small and the amount of the labeled antibody that binds to the mask antigen is relatively large, the entire determination unit develops color and the characters or figures drawn by the text antibody become invisible. When the amount of the substance to be measured contained in the test solution is large, the ratio of the labeled antibody that binds to the text antibody remains unchanged, but the amount of the labeled antibody that binds to the substance to be measured becomes relatively large. As a result, the amount of the labeled antibody that binds to the text antibody increases. In addition, since the labeled antibody that has bound to the substance to be measured cannot bind to the mask antigen, the amount of the labeled antibody that binds to the mask antigen becomes relatively small. In addition to the labeled antibody that has not bound to the substance to be measured, the labeled antibody that has bound to the substance to be measured binds to the text antibody. Since the amount of the labeled antibody that binds to the mask antigen is relatively small, the color development of the mask antigen part becomes weak, and the color development of the text antibody part becomes strong, and the characters or the like mimicked by the text antibody become visible. As described above, according to the competitive immunochromatographic analysis method of the present invention, the analysis result can be determined by the visibility of characters, figures, etc. in the determination unit, which appears by the contrast between the color of the text antibody part and the color of the mask antigen part, rather than the shade of the color of the determination unit. Thus, the determination becomes easy. In addition, it is also possible to control the cut-off by adjusting the arrangement amounts of the text antibody and the mask antigen fixed to the determination unit. In the conventional analysis method, when the test line shows color, it is determined as negative, and when it does not show color, it is determined as positive. However, in order for the test line not to show complete color when it is positive, the concentration of the substance to be measured (specimen) in the test solution must be relatively high, which has the drawback of poor sensitivity. According to the method of the present invention, since the determination can be made based on the contrast between the color of the text antibody part and the color of the mask antigen part, even if the mask antigen part (mask part) shows some color, it can be determined as positive when the text appears due to the color development of the text antibody part. Therefore, it becomes possible to lower the lower limit of the concentration of the substance to be measured that can be visually determined compared to the conventional analysis method. Also, as described above, according to the competitive immunochromatographic analysis method of the present invention, the more the amount of the substance to be measured in the test solution, the easier it is to see the characters or figures, and the less the amount of the substance to be measured in the test solution, the more difficult it is to see the characters or figures. If the test solution does not contain the substance to be measured, the characters or figures cannot be seen. Therefore, the determination result shows a positive correlation with the concentration of the substance to be measured, and the user will not misunderstand the determination result. In addition, by providing a plurality of the determination units and changing the ratio of the arrangement amounts of the text antibody and the mask antigen in each determination unit, it becomes possible to perform analysis on the same test solution with a plurality of determination units having different conditions, so that more accurate determination becomes possible. Also, by using the determination results of the plurality of determination units with different conditions, it becomes easier to set the cut-off. In this case, a plurality of determination units are provided on the membrane carrier along the flow direction of the test solution, and by making the ratio of the arrangement amount of the mask antigen to the text antibody in the downstream determination unit lower or higher than the ratio of the arrangement amount of the mask antigen to the text antibody in the upstream determination unit, it becomes possible to compare the appearance of the characters or figures in the upstream determination unit with the appearance of the characters or figures in the downstream determination unit and perform the determination, and it becomes possible to more easily perform a more accurate determination. In addition, by inkjet printing the text antibody and the mask antigen on a membrane carrier, the manufacturing becomes extremely easy. By using inkjet printing, it becomes possible to freely and easily select characters and figures drawn by the text antibody, and also to easily adjust the amount of the text antibody and the mask antigen to be arranged. Specifically, for example, by increasing the number of overprinting times, the amount of the text antibody and the mask antigen to be fixed increases, so that the density of the text antibody and the mask antigen can be adjusted according to the number of printing times. The competitive multi-immunochromatographic sensor according to the present invention has a determination unit formed by fixing a text antibody composed of an antibody that specifically reacts with a labeled antibody that specifically reacts with a measurement target substance in a character or figure shape on a membrane carrier, and fixing a mask antigen composed of the same antigen as the measurement target substance on the membrane carrier so as to surround the text antibody fixed in the character or figure shape. Therefore, by simply dropping a test solution onto the determination unit, it becomes possible to easily know the concentration of the measurement target substance. Another competitive multi-immunochromatographic sensor according to the present invention has a test solution introduction unit for introducing a test solution containing a measurement target substance, a labeled antibody-containing unit arranged downstream of the test solution introduction unit and containing a labeled antibody that specifically reacts with the measurement target substance, at least one determination unit provided downstream of the labeled antibody-containing unit, and a suction unit arranged downstream of the determination unit on a membrane carrier. The test solution introduced into the test solution introduction unit is configured to pass through the labeled antibody-containing unit and the determination unit in sequence and flow to the suction unit. The determination unit is formed by fixing a text antibody composed of an antibody that specifically reacts with the labeled antibody in a character or figure shape on a membrane carrier, and fixing a mask antigen composed of the same antigen as the measurement target substance on the membrane carrier so as to surround the text antibody fixed in the character or figure shape. Therefore, by simply introducing a test solution from the test solution introduction unit, it becomes possible to easily know the concentration of the measurement target substance.

Brief Description of the Drawings

[0007]

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Embodiments for Carrying Out the Invention

[0008] Hereinafter, with reference to one embodiment shown in the accompanying drawings, embodiments of the competitive immunochromatographic analysis method and immunochromatographic sensor according to the present invention will be described.

[0009] First, the competitive immunochromatographic analysis method according to the present invention will be described. FIG. 1(a) is a conceptual diagram showing the relationship between the substance to be measured, mask antigen, and text antibody used in the competitive immunochromatographic analysis method according to the present invention, and FIG. 1(b) is a conceptual diagram of the determination unit of the immunochromatographic sensor for performing this analysis method. As shown in the drawings, the competitive immunochromatographic analysis method is used to semi-quantify the concentration of the substance to be measured A contained in the test solution. In FIG. 1(b), reference symbol B indicates a labeled antibody that specifically reacts with the substance to be measured A, and reference symbol C indicates an antibody that specifically reacts with the labeled antibody B (hereinafter, referred to as a text antibody in this specification). A text antibody C that does not react with the substance to be measured A is selected. As shown in FIG. 1(b), in the determination unit, the text antibody C is fixed in the form of characters or figures (in this example, the number "1"). And the same antigen D as the substance to be measured A is fixed so as to surround the text antibody C in the determination unit (hereinafter, referred to as a mask antigen D in this specification). Preferably, these text antibodies C and mask antigens D can be inkjet-printed. Specifically, for example, after first printing the text antibody C in any form (the number "1" in this example) on a membrane carrier, a method of printing the mask antigen D so as to cover the text antibody C, and a method of first printing the mask antigen D on the membrane carrier and then printing the text antibody C in any form (the number "1" in this example) on the mask antigen D can be selected. Which method to select can be determined according to the types of the text antibody and mask antigen used, but in any case, the mask antigen surrounds the periphery of the text antibody. Also, by using inkjet printing to fix the text antibody C and the mask antigen D, it becomes easy to fixedly arrange the text antibody C in the form of any characters, figures, etc. Further, by increasing the number of overprinting times, the amount of the text antibody and mask antigen to be fixed increases, so the density of the text antibody and mask antigen can be adjusted by the number of printing times. Before determining the concentration of the measurement target substance A using the above-described determination unit, a mixed solution in which the labeled antibody B is previously mixed with the test solution is prepared in advance. When this mixed solution is supplied to the determination unit, the labeled antibody B competitively reacts and binds with the measurement target substance A contained in the test solution, the mask antigen D fixed to the determination unit, and the text antibody C. Here, the labeled antibody B not bound to the measurement target substance A can bind to both the mask antigen D and the text antibody C, but the labeled antibody B bound to the measurement target substance A cannot bind to the mask antigen D and can only bind to the text antibody C. When the mixed solution of the above-described test solution and the labeled antibody is dropped onto the determination unit, the following phenomenon occurs. Figure 2 is a diagram schematically showing the behavior of the labeled antibody in the determination unit. 1. When the amount of the measurement target substance A contained in the test solution is small The proportion of labeled antibody B that binds to text antibody C remains unchanged. However, since the amount of labeled antibody B that binds to the substance A to be measured is relatively small, the amount of labeled antibody B that binds to the mask antigen D becomes relatively large. In addition to the labeled antibody B that is not bound to the substance A to be measured, the labeled antibody B that is bound to the substance A to be measured binds to the text antibody C. However, since the amount of labeled antibody B that binds to the substance A to be measured is relatively small and the amount of labeled antibody B that binds to the mask antigen D is relatively large, the entire determination unit develops color, and the characters (in this example, "1") drawn by the text antibody C cannot be seen (see Fig. 2(a)). 2. When there is a large amount of substance A to be measured in the test solution The proportion of labeled antibody B that binds to text antibody C remains unchanged. However, since the amount of labeled antibody B that binds to the substance A to be measured is relatively large, as a result, the amount of labeled antibody B that binds to the text antibody C increases. In addition, since the labeled antibody B that is bound to the substance A to be measured cannot bind to the mask antigen D, the amount of labeled antibody B that binds to the mask antigen D becomes relatively small. In addition to the labeled antibody B that is not bound to the substance A to be measured, the labeled antibody B that is bound to the substance A to be measured binds to the text antibody C, and since the amount of labeled antibody B that binds to the mask antigen D is relatively small, the color development in the part of the mask antigen B becomes weak, the color development in the part of the text antibody C becomes strong, and the characters or the like mimicked by the text antibody C can be seen (see Fig. 2(b)). As described above, according to the competitive immunochromatographic analysis method according to this example, the analysis result can be determined by the appearance of characters, figures, etc. in the determination unit that appear by the contrast between the color of the part of the text antibody C and the color of the part of the mask antigen D, rather than by the presence or absence of color development in the determination unit, so the determination becomes easier. In addition, the more the amount of the substance A to be measured in the test solution, the easier it is to see the characters or figures. The less the amount of the substance A to be measured in the test solution, the harder it is to see the characters or figures. If the substance A to be measured is not contained in the test solution, the characters or figures cannot be seen. Therefore, the determination result shows a positive correlation with the concentration of the substance A to be measured, and the user will not misunderstand the determination result.

[0010] Next, a second embodiment in which the determination unit configured as described above is applied to a lateral flow immunoassay sensor will be described. FIG. 3 is a schematic top view of a second embodiment of the immunoassay sensor according to the present invention, and FIG. 4 is a schematic side view of the immunoassay sensor shown in FIG. 3. This multi - immunoassay sensor has a single strip - shaped membrane carrier 5, and a sample introduction part 1 for introducing a sample is provided at the upstream end of the membrane carrier 5. Downstream of the sample introduction part 1, specifically, a labeled antibody - containing part 2 is provided so as to partially overlap the sample introduction part 1. And further downstream of the labeled antibody - containing part 2, a determination part 3 is arranged, and a sample suction pad 4 is provided downstream of the determination part 3. The membrane carrier 5 is made of, for example, filter paper (specifically, nitrocellulose). When a sample is dropped into the sample introduction part 1, the sample enters the labeled antibody - containing part 2 by capillary action, and then passes through the determination part 3 and reaches the sample suction pad 4. Although not shown, a confirmation part in which an antibody capable of capturing labeled antibody B is fixed can be provided downstream of the determination part 3. This confirmation part can be used to confirm whether the sample has passed through the determination part 3 normally.

[0011] The labeled antibody - containing part 2 is impregnated with a labeled antibody B that specifically reacts with the substance A to be measured, and this labeled antibody B is labeled with an arbitrary labeling substance such as colloidal gold or latex. The determination part 3 has the same configuration as the determination part shown in the above - described embodiment, that is, a text antibody C that specifically reacts with the labeled antibody B is fixed in the form of characters or figures, and a mask antigen D composed of the same antigen as the substance A to be measured is fixed so as to surround the text antibody C. According to the competitive immunoassay sensor configured as described above, When a sample is dropped from the sample introduction part 1, the sample containing the substance A to be measured enters the labeled antibody - containing part 2, where the labeled antibody B is mixed with the sample. The mixture obtained by mixing the labeled antibody B and the test solution enters the determination unit 3, where the labeled antibody B competes with the measurement target substance A, mask antigen D, and text antibody C contained in the test solution. When the amount of the measurement target substance A contained in the test solution is small, the proportion of the labeled antibody B that binds to the text antibody C remains unchanged, but the amount of the labeled antibody B that binds to the measurement target substance A is relatively small. Therefore, the amount of the labeled antibody B that binds to the mask antigen D becomes relatively large. In addition to the labeled antibody B that is not bound to the measurement target substance A, the labeled antibody B that is bound to the measurement target substance A binds to the text antibody C. However, since the amount of the labeled antibody B that binds to the measurement target substance A is relatively small and the amount of the labeled antibody B that binds to the mask antigen D is relatively large, the entire determination unit 3 develops color, and the characters or figures drawn by the text antibody C become invisible. When the amount of the measurement target substance A contained in the test solution is large, the proportion of the labeled antibody B that binds to the text antibody C remains unchanged, but the amount of the labeled antibody B that binds to the measurement target substance A is relatively large. As a result, the amount of the labeled antibody B that binds to the text antibody C increases. In addition, since the labeled antibody B that is bound to the measurement target substance A cannot bind to the mask antigen D, the amount of the labeled antibody B that binds to the mask antigen D becomes relatively small. In addition to the labeled antibody B that is not bound to the measurement target substance A, the labeled antibody B that is bound to the measurement target substance A binds to the text antibody C, and since the amount of the labeled antibody B that binds to the mask antigen D is relatively small, the color development of the mask antigen D part becomes weak, and the color development of the text antibody C part becomes strong, and the characters or the like mimicked by the text antibody C can be seen. In addition, although not shown, the competitive immunochromatographic sensor configured as described above is housed in a suitable housing. Preferably, a window portion is formed in the housing at a position corresponding to the determination unit 3 so that the reaction result in the determination unit 3 can be viewed through the window portion.

[0012] According to the competitive immunochromatographic sensor according to this embodiment configured as described above, it is possible to semi-quantify the measurement target substance in the test solution only by dropping the test solution onto the test solution supply unit.

[0013] Next, using the competitive immunochromatographic sensor configured as described above, the results of actually measuring the substance to be measured are shown in FIGS. 5 to 7. These measurement results are the results of measuring five samples of each of nine types of immunochromatographic sensors with different amounts of the text antibody and the mask antigen arranged in the determination unit, and measuring samples of five concentrations. In this example, after printing the text antibody C first, the mask antigen D was printed so as to cover the text antibody C. Here, anti-goat IgG was used as the text antibody C, human IgG was used as the mask antigen, and anti-human IgG labeled with gold colloid was used as the labeled antibody, and human IgG in sample solutions of five concentrations (0 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, and 50 μg / mL) was measured. In addition, in each immunochromatographic sensor, the labeled antibody-containing part 2 used a conjugate pad prepared by the following method. 1 mL of gold nanoparticles (753610, ALDRICH) was placed in a 1.5 mL tube, and 100 μL of a phosphate buffer PB (pH 7.4, 10 mM) containing 100 μg / mL anti-human IgG (I3382, SIGMA) was added thereto and allowed to stand for 15 minutes. Then, 100 μL of a phosphate buffer PB (pH 7.4, 10 mM) containing 10% (w / v) bovine serum albumin BSA (010-25783, Wako) and 100 μL of a phosphate buffer PB (pH 7.4, 10 mM) containing 1% polyethylene glycol PEG20000 (168-11285, Wako) were added and allowed to stand for 15 minutes. This was centrifuged (15000 rpm, 4 °C, 30 min) to remove the supernatant, and 50 μL of a conjugate pad buffer (phosphate buffer PB (pH 7.4, 10 mM) containing 2% (w / v) bovine serum albumin BSA and 10% (w / v) sucrose) was added. This was redissolved using an ultrasonic cleaner filled with ice water, 5 μL was added per one conjugate pad (5×8 mm), and then it was dried at 37 °C for 2 hours using a dryer. Figure 5(a) shows the state of the determination unit 3 of five immunochromatographic sensors. Each sensor is created by printing text antibody C once in the form of the letter "D" using inkjet printing with an antibody concentration of 0.1 mg / mL per print, and printing the mask antigen once using inkjet printing with an antigen concentration of 0.1 mg / mL per print. The samples with five different concentrations (0 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, and 50 μg / mL) are measured with each immunochromatographic sensor. Figure 5(b) shows the state of the determination unit 3 of five immunochromatographic sensors. Each sensor is created by printing text antibody C once in the form of the letter "D" using inkjet printing with an antibody concentration of 0.1 mg / mL per print, and printing the mask antigen five times using inkjet printing with an antigen concentration of 0.1 mg / mL per print. The samples with five different concentrations (0 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, and 50 μg / mL) are measured with each immunochromatographic sensor. Figure 5(c) shows the state of the determination unit 3 of five immunochromatographic sensors. Each sensor is created by printing text antibody C once in the form of the letter "D" using inkjet printing with an antibody concentration of 0.1 mg / mL per print, and printing the mask antigen ten times using inkjet printing with an antigen concentration of 0.1 mg / mL per print. The samples with five different concentrations (0 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, and 50 μg / mL) are measured with each immunochromatographic sensor. Figure 6(a) shows the state of the determination unit 3 of five immunochromatographic sensors. Each sensor is created by printing text antibody C three times in the form of the letter "D" using inkjet printing with an antibody concentration of 0.1 mg / mL per print, and printing the mask antigen once using inkjet printing with an antigen concentration of 0.1 mg / mL per print. The samples with five different concentrations (0 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, and 50 μg / mL) are measured with each immunochromatographic sensor. Figure 6(b) shows the state of the determination unit 3 of five immunochromatographic sensors each created by printing text antibody C three times in the form of the letter "D" using inkjet printing in which an antibody with a concentration of 0.1 mg / mL is fixed in one printing, and printing the mask antigen five times using inkjet printing in which an antigen with a concentration of 0.1 mg / mL is fixed in one printing, and measuring sample solutions of five concentrations (0 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, and 50 μg / mL) with each immunochromatographic sensor. Figure 6(c) shows the state of the determination unit 3 of five immunochromatographic sensors each created by printing text antibody C three times in the form of the letter "D" using inkjet printing in which an antibody with a concentration of 0.1 mg / mL is fixed in one printing, and printing the mask antigen ten times using inkjet printing in which an antigen with a concentration of 0.1 mg / mL is fixed in one printing, and measuring sample solutions of five concentrations (0 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, and 50 μg / mL) with each immunochromatographic sensor. Figure 7(a) shows the state of the determination unit 3 of five immunochromatographic sensors each created by printing text antibody C five times in the form of the letter "D" using inkjet printing in which an antibody with a concentration of 0.1 mg / mL is fixed in one printing, and printing the mask antigen once using inkjet printing in which an antigen with a concentration of 0.1 mg / mL is fixed in one printing, and measuring sample solutions of five concentrations (0 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, and 50 μg / mL) with each immunochromatographic sensor. FIG. 7(b) shows the state of the determination unit 3 of five immunochromatographic sensors prepared by printing the text antibody C five times in the form of the letter "D" using inkjet printing in which an antibody with a concentration of 0.1 mg / mL is fixed in one printing, and printing the mask antigen five times using inkjet printing in which an antigen with a concentration of 0.1 mg / mL is fixed in one printing, and measuring sample solutions with five concentrations (0 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, and 50 μg / mL) with each immunochromatographic sensor. FIG. 7(c) shows the state of the determination unit 3 of five immunochromatographic sensors prepared by printing the text antibody C five times in the form of the letter "D" using inkjet printing in which an antibody with a concentration of 0.1 mg / mL is fixed in one printing, and printing the mask antigen ten times using inkjet printing in which an antigen with a concentration of 0.1 mg / mL is fixed in one printing, and measuring sample solutions with five concentrations (0 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, and 50 μg / mL) with each immunochromatographic sensor. From the results shown in FIGS. 5 to 7, it was found that there is a correlation between the amounts of the text antibody and the mask antigen fixed to the determination unit and the concentration of the substance to be measured in the sample, and it was confirmed that the cut-off can be determined by the amounts of the text antibody and the mask antigen.

[0014] Next, a third embodiment in which the determination unit configured as described above is applied to a competitive immunochromatographic sensor having a lateral flow structure will be described. FIG. 8 is a schematic top view of a third embodiment of the immunochromatographic sensor according to the present invention, and FIG. 9 is a schematic side view of the immunochromatographic sensor shown in FIG. 8. In this embodiment, the configuration is the same as that of the immunochromatographic sensor of the second embodiment except that three determination units 3a to 3c are provided in series, so overlapping explanations will be omitted. The layouts of the three determination units 3a to 3c of the text antibody C are different. In determination unit 3a, the text antibody C is laid out in the form of the number "3", in determination unit 3b, in the form of the number "2", and in determination unit 3c, in the form of the number "1". Also, the arranged amounts of the fixed mask antigen D of the three determination units are changed. That is, in determination unit 3a, the mask antigen D is, for example, printed 10 times in overlay, in determination unit 3b, the mask antigen D is, for example, printed 5 times in overlay, and further, in determination unit 3c, the mask antigen D is only printed, for example, 1 time. Assume that the text antibody C is printed the same number of times only with different forms. Thereby, as going downstream, the arranged amount of the mask antigen D decreases, and as a result, it becomes possible to change the way of representing numbers with respect to the content of the measurement target substance A in the test solution among the three determination units 3a to 3c. That is, when configured as described above, in determination unit 3a, no number appears unless the content of the measurement target substance A is a certain amount or more, but in determination unit 3b, a number appears even with a smaller content, and in determination unit 3c, a number appears even with an even smaller content. By adjusting the content of the measurement target substance at which a number appears in each of the determination units 3a to 3c with the arranged amounts of the text antibody C and the mask antigen D, it becomes possible to semi-quantify the content of the measurement target substance. In the above-described embodiment, the ratio of the arranged amount of the mask antigen D with respect to the text antibody C in the downstream determination unit is made lower than the ratio of the arranged amount of the mask antigen D with respect to the text antibody C in the upstream determination unit, but this is not limited to this embodiment, and the ratio of the arranged amount of the mask antigen D with respect to the text antibody C in the downstream determination unit may be made higher than the ratio of the arranged amount of the mask antigen D with respect to the text antibody C in the upstream determination unit. Even when configured in this way, it becomes possible to change the way of representing numbers with respect to the content of the measurement target substance A in the test solution among the three determination units 3a to 3c, and by adjusting with the arranged amounts of the text antibody C and the mask antigen D, it becomes possible to semi-quantify the content of the measurement target substance.

[0015] Figures 10 to 13 show the results of measuring samples with different concentrations using five immunochromatographic sensors each having three determination units with different amounts of the text antibody C and the mask antigen D arranged. In the examples of Figures 10, 11, 12, and 13, the ratios of the amounts of the text antibody C and the mask antigen D arranged are different from each other. Here, anti-goat IgG was used as the text antibody C, human IgG was used as the mask antigen, anti-human IgG labeled with gold colloid was used as the labeled antibody, and experiments were conducted by changing the concentration of human IgG in the sample solution. In this example, after printing the text antibody C first, the mask antigen D was printed so as to cover the text antibody C. The conjugate pad constituting the labeled antibody-containing portion 2 used the same conjugate pad as that in the examples shown in Figures 5 to 7. The amounts of the text antibody C and the mask antigen D fixed to the determination unit 3 were adjusted by changing the number of overprinting times of inkjet printing. The text antibody C was printed in the forms of "1", "2", and "3" using inkjet printing in which an antibody with a concentration of 0.1 mg / mL was fixed in one printing. The mask antigen D used inkjet printing in which an antigen with a concentration of 0.1 mg / mL was fixed in one printing. Samples with a concentration of 0, 1 μg / mL, 5 μg / mL, 10 μg / mL, and 50 μg / mL of human IgG as the substance to be measured were used. Figure 10 shows the results of measuring samples with a concentration of 0, 1 μg / mL, 5 μg / mL, 10 μg / mL, and 50 μg / mL using five immunochromatographic sensors each having a determination unit in which the text antibody was printed once for all the determination units 3a to 3c, the mask antigen was overprinted 10 times for the determination unit 3a at the most upstream, the mask antigen was overprinted 5 times for the second determination unit 3b, and the mask antigen was printed once for the determination unit 3c at the most downstream. FIG. 11 shows the results of measuring samples with a concentration of 0, 1 μg / mL, 5 μg / mL, 10 μg / mL, and 50 μg / mL using five immunochromatographic sensors each having a determination unit in which a text antibody was printed three times for all the determination units 3a to 3c, a mask antigen was overprinted 10 times for the most upstream determination unit 3a, a mask antigen was overprinted 5 times for the second determination unit 3b, and a mask antigen was printed once for the most downstream determination unit 3c. FIG. 12 shows the results of flowing samples with a concentration of 0, 1 μg / mL, 5 μg / mL, 10 μg / mL, and 50 μg / mL using five immunochromatographic sensors each having a determination unit in which a text antibody was printed five times for all the determination units 3a to 3c, a mask antigen was overprinted 10 times for the most upstream determination unit 3a, a mask antigen was overprinted 5 times for the second determination unit 3b, and a mask antigen was printed once for the most downstream determination unit 3c. FIG. 13 shows the results of measuring samples with a concentration of 0, 1 μg / mL, 5 μg / mL, 10 μg / mL, and 50 μg / mL using five immunochromatographic sensors each having a determination unit in which a text antibody was printed once for the determination unit 3a and a mask antigen was overprinted 10 times, a text antibody was overprinted three times and a mask antigen was overprinted 5 times for the determination unit 3b, and a text antibody was overprinted five times and a mask antigen was printed once for the determination unit 3c.

[0016] As shown in FIGS. 10 to 13, it was confirmed that by adjusting the amounts of the text antibody and the mask antigen disposed, it became possible to identify and discriminate test solutions having different concentrations of the measurement target substance A. Thereby, it was confirmed that semi - quantification of the concentration of the measurement target substance A became possible.

[0017] In the above description, human IgG is described as the substance to be measured. However, the substance to be measured is not limited to this example and can be any target substance. For example, it goes without saying that 8-OHdG can be measured. When measuring 8-OHdG, anti-8-OHdG (derived from rat) is used as the labeled antibody, and anti-rat IgG, for example, is used as the text antibody, and an 8-OHdG / BSA conjugate can be used as the mask antigen. Also, when creating a determination unit for measuring 8-OHdG, the mask antigen can be printed first, and then the text antibody can be printed on top of it.

[0018] Next, a fourth embodiment in which a determination unit configured to measure 8-OHdG is applied to a lateral flow competitive immunochromatographic sensor will be described. FIG. 14 is a schematic top view of a fourth embodiment of the immunochromatographic sensor according to the present invention, FIG. 15 is a schematic side view of the immunochromatographic sensor shown in FIG. 14, and FIG. 16 is a diagram showing the amounts of the text antibody C and the mask antigen D arranged in each determination unit represented by the number of printing times. In this embodiment, except for providing a confirmation unit 6 downstream of the determination units 3a to 3c arranged in series, the basic structure is the same as that of the immunochromatographic sensor of the third embodiment, so duplicate explanations will be omitted. In this embodiment, after printing the mask antigen D first, the text antibody C was printed on top of the mask antigen D. Here, anti-rat IgG was used as the text antibody C, an 8-OHdG / BSA conjugate was used as the mask antigen, and anti-8-OHdG labeled with gold colloid was used as the labeled antibody, and 8-OHdG in sample solutions of four concentrations (0 ng / mL, 5 ng / mL, 10 ng / mL, and 25 ng / mL) was measured. Also, the labeled antibody-containing unit 2 used a conjugate pad created by the following method. Place 1 mL of gold nanoparticles (753637, Sigma - Aldrich) into a 2 mL tube, add 100 μL of 40 μg / mL anti - 8 - OHdG thereto, and shake for 30 minutes. Then add 100 μL of 10% (w / v) bovine serum albumin BSA (010 - 25783, Wako) and 50 μL of 1% (w / v) polyethylene glycol PEG20000 (168 - 11285, Wako), and shake for 10 minutes. Add 450 μL of the storage buffer for conjugate pad (Tris - HCl buffer (pH 8.2, 20 mM) containing 0.05% (w / v) polyethylene glycol PEG20000, 1% (w / v) BSA, 0.1% (w / v) sodium azide, and 150 mM sodium chloride) thereto. Centrifuge this (2500 g, 4 °C, 30 min), remove the supernatant, add 75 μL of the coating buffer for conjugate pad (Tris - HCl buffer (pH 8.2, 20 mM) containing 0.05% (w / v) polyethylene glycol PEG20000, 5% (w / v) sucrose, and 150 mM sodium chloride) and 33 μL of ultrapure water. Redissolve this using a vortex mixer, add 25 μL per one conjugate pad (5×8 mm), and then dry at 37 °C for 2 hours using a dry heater. The layouts of the text antibody C are different in the three determination units 3a to 3c and the one confirmation unit 6. In the determination unit 3a, the text antibody C is laid out in the form of "Lo", in the determination unit 3b in the form of "Md", in the determination unit 3c in the form of "Hi", and in the confirmation unit 6 in the form of "Tr". Also, in the three determination units and the one confirmation unit, the amounts of the fixed text antibody C disposed are changed. That is, in the determination unit 3a, the text antibody C is, for example, printed 14 times in overlay, in the determination unit 3b the text antibody C is, for example, printed 10 times in overlay, in the determination unit 3c the text antibody C is, for example, printed 6 times, and further, in the confirmation unit 6, the text antibody C is, for example, printed 20 times. In addition, the amounts of the fixed mask antigen D arranged by the three determination units 3a to 3c are different. That is, in determination unit 3a, the mask antigen D is, for example, printed 10 times in overlay, in determination unit 3b, the mask antigen D is, for example, printed 15 times in overlay, and further, in determination unit 3c, the mask antigen D is, for example, printed 20 times. As a result, as going downstream, the amount of the text antibody C arranged for the mask antigen D is small, and as a result, it becomes possible to change the way the text appears for the content of the measurement target substance A in the test solution by the three determination units 3a to 3c. That is, when configured as described above, in determination unit 3c, text does not appear unless the content of the measurement target substance A is a certain amount or more, but in determination unit 3b, text appears even with a smaller content, and in determination unit 3a, text appears even with an even smaller content. On the other hand, in confirmation unit 6, text appears regardless of the content of the measurement target substance A. By adjusting the content of the measurement target substance at which text appears in each of the determination units 3a to 3c with the amounts of the text antibody C and the mask antigen D arranged, it becomes possible to semi-quantify the content of the measurement target substance.

[0019] FIG. 17 shows the results of measuring samples with different concentrations by creating four immunochromatographic sensors each having three determination units 3a to 3c and one confirmation unit 6 with different amounts of the text antibody C and the mask antigen D arranged. Since the basic configuration of the immunochromatographic sensor is the same as the configuration described in FIGS. 14 to 16, only the parts corresponding to the three determination units 3a to 3c and one confirmation unit 6 are shown in FIG. 17. Here, anti-rat IgG was used as the text antibody C, 8-OHdG / BSA conjugate was used as the mask antigen D, anti-8-OHdG labeled with gold colloid was used as the labeled antibody, and experiments were conducted by changing the concentration of 8-OHdG in the sample solution. In this example, after printing the mask antigen D first, the text antibody C was printed on the mask antigen D. The conjugate pad constituting the labeled antibody containing unit 2 used the same one as the conjugate pad of the example shown in FIGS. 14 to 16. The amounts of the text antibody C and the mask antigen D fixed to the determination unit 3 were adjusted by changing the number of overprinting times in inkjet printing. The text antibody C was printed in the forms of "Tr", "Hi", "Md", and "Lo" using inkjet printing in which an antibody with a concentration of 0.1 mg / mL was fixed in one printing. The mask antigen D was printed using inkjet printing in which an antigen with a concentration of 0.1 mg / mL was fixed in one printing. Samples with a concentration of 8-OHdG of 0, 5 ng / mL, 10 ng / mL, and 25 ng / mL as the substance to be measured were used. For the determination unit 3a, the text antibody was overprinted 14 times and the mask antigen was overprinted 10 times. For the determination unit 3b, the text antibody was overprinted 10 times and the mask antigen was overprinted 15 times. For the determination unit 3c, the text antibody was overprinted 6 times and the mask antigen was overprinted 20 times. Furthermore, four immunochromatographic sensors having a determination unit in which the text antibody was overprinted 20 times were prepared for the confirmation unit 6, and the results of measuring samples with a concentration of 0, 5 ng / mL, 10 ng / mL, and 25 ng / mL using these sensors are shown. As shown in FIG. 17, it was confirmed that by adjusting the amounts of the text antibody and the mask antigen arranged, it became possible to identify and discriminate test solutions with different concentrations of 8-OHdG as the substance to be measured A. Thereby, it was confirmed that semi-quantification of the concentration of 8-OHdG as the substance to be measured A became possible.

Explanation of symbols

[0020] A Substance to be measured B Labeled antibody C Text antibody D Mask antigen 1 Test solution introduction part 2 Labeled antibody-containing part 3 Determination unit 4 Suction part 5 Membrane carrier 6 Confirmation part

Claims

1. a determination section including a membrane carrier having a text antibody C, which is composed of a labeled antibody B that specifically reacts with a substance A to be measured and an antibody that specifically reacts with the labeled antibody B, immobilized in the form of letters or graphics, and a mask antigen D, which is composed of the same antigen as the substance A to be measured, immobilized on the membrane carrier so as to surround the text antibody C immobilized in the form of letters or graphics; supplying a mixture of a test solution containing a substance A to be measured and the labeled antibody B; In the determination unit, The labeled antibody B is allowed to competitively bind to the analyte A, the mask antigen D, and the text antibody C, The concentration of the measured substance A is determined based on the signal from the labeled antibody bound to the text antibody C and the mask antigen D. A competitive immunochromatographic analysis method comprising the steps of:

2. A plurality of the determination units are provided, The ratio of the amount of the text antibody C and the amount of the mask antigen D in each test section is changed.

2. The method of claim 1 .

3. A plurality of determination sections are provided on the membrane carrier along the flow direction of the test solution, The ratio of the amount of the mask antigen D to the amount of the text antibody C in the downstream determination section is set lower than the ratio of the amount of the mask antigen D to the amount of the text antibody C in the upstream determination section.

3. The method of claim 2.

4. A plurality of determination sections are provided on the membrane carrier along the flow direction of the test solution, The ratio of the amount of the mask antigen D to the amount of the text antibody C in the downstream determination section is made higher than the ratio of the amount of the mask antigen D to the amount of the text antibody C in the upstream determination section.

3. The method of claim 2.

5. The text antibody C and mask antigen D are inkjet printed onto a membrane carrier. The method according to any one of claims 1 to 4.

6. The method has a determination section in which a text antibody C consisting of a labeled antibody B that specifically reacts with a substance A to be measured and an antibody that specifically reacts with the labeled antibody B is immobilized on a membrane carrier in the form of letters or graphics, and a mask antigen D consisting of the same antigen as the substance A to be measured is immobilized on the membrane carrier so as to surround the text antibody C immobilized in the form of letters or graphics. A competitive immunochromatographic sensor comprising:

7. A test solution introduction section 1 for introducing a test solution containing a measurement target substance A; A labeled antibody-containing section 2 is disposed downstream of the sample liquid inlet section 1 and contains a labeled antibody B that specifically reacts with a substance A to be measured; At least one determination section 3 provided downstream of the labeled antibody-containing section 2; a suction unit 4 disposed downstream of the determination unit 3; is placed on the membrane carrier 5, A competitive immunochromatographic sensor configured such that a test solution introduced into a test solution introduction section 1 flows into a suction section 4 after passing through a labeled antibody-containing section 2 and a determination section 3 in this order, The determination unit 3 is configured by immobilizing a text antibody C, which is an antibody that specifically reacts with the labeled antibody B, on a membrane carrier in the form of letters or graphics, and immobilizing a mask antigen D, which is the same antigen as the measurement target substance A, on the membrane carrier so as to surround the text antibody C immobilized in the form of letters or graphics. A competitive immunochromatographic sensor comprising:

8. The determination unit 3 is provided in plurality, The ratio of the amount of the text antibody C to the amount of the mask antigen D in each determination section 3 is different.

8. The sensor according to claim 7.

9. The determination unit 3 is provided in a plurality of locations along the flow direction of the test solution, The ratio of the amount of mask antigen D to the amount of text antibody C in the downstream test zone is lower than the ratio of the amount of mask antigen D to the amount of text antibody C in the upstream test zone.

9. The sensor according to claim 8.

10. The determination unit 3 is provided in a plurality of locations along the flow direction of the test solution, The ratio of the amount of mask antigen D to the amount of text antibody C in the downstream test zone is higher than the ratio of the amount of mask antigen D to the amount of text antibody C in the upstream test zone.

9. The sensor according to claim 8.

11. The text antibody C and mask antigen D are ink-jet printed on the test section 3. The sensor according to any one of claims 7 to 10.

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