Test kit and test strip

Abstract

To provide a test kit and a test strip capable of shortening a time from the application of a specimen extraction liquid to a sample pad to the development of a reaction result in a detection region as compared with a conventional time.SOLUTION: Since the test strip 1 is equipped with a development pad DP in which a compound having a phosphorylcholine group is held in a dry state as a sensitizing agent, the time from the application of the specimen extraction liquid to the sample pad SP to the development of a line on a first test line TL1 or the like can be shortened as compared with the related art.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to test kits and test strips. [Background technology]

[0002] Point of care testing (POCT) is widely used in tests for infectious diseases such as influenza virus and pregnancy tests. For example, a test kit using the principle of immunoassay such as immunochromatography is known as POCT. Immunochromatography is a measurement method in which, when a specimen extract from which a specimen has been extracted moves on a membrane by capillary action, a complex is formed by an antigen as a specimen in the specimen extract and a labeled antibody bound to colored particles as a labeling substance, and the complex is captured by a capture antibody as a capture substance applied to the membrane, and the accumulation of the complex is visually confirmed.

[0003] Such a test kit utilizing immunochromatography includes a test strip for immunochromatography on which the labeled antibody is held in a wet, releasable state and on which an antigen capture antibody and a labeled capture antibody are immobilized, and a case having an opening for dripping a sample extract onto the test strip and an opening through which the accumulation of immune complexes on the test strip can be confirmed.

[0004] 1, a test strip is configured by arranging a sample pad SP, a conjugate pad CP, a membrane M, and an absorbent pad 111 in this order along the spreading direction x on a belt-shaped soft backing sheet 11. When a specimen extract is dropped onto the sample pad SP of the test strip 110, the specimen extract is spread by capillary action onto the conjugate pad CP, the membrane M, and the absorbent pad 111 in that order.

[0005] The conjugate pad CP holds, in a wet and releasable state, for example, a labeled antibody A1 that specifically reacts with a predetermined antigen and has colored particles bound thereto, and a labeled antibody B1 that specifically reacts with a different type of antigen from the antigen and has colored particles bound thereto of a different color from the labeled antibody A1. The membrane M is provided with a detection region in which a first test line TL1, a second test line TL2, and a control line CL are arranged in this order from the conjugate pad CP toward the absorbent pad 111. In the test strip 110, the specimen extract that has passed through the conjugate pad CP can pass through the first test line TL1, the second test line TL2, and the control line CL in this order by capillary action.

[0006] Immobilized on the first test line TL1 is an antigen capture antibody A2 that reacts with the same antigen as the labeled antibody A1 but has a different epitope from that of the labeled antibody A1. Immobilized on the second test line TL2 is an antigen capture antibody B2 that reacts with the same antigen as the labeled antibody B1 but has a different epitope from that of the labeled antibody B1. Immobilized on the control line CL is a labeled capture antibody r that specifically reacts with the labeled antibodies A1 and B1.

[0007] In a test using a test kit, first, the test strip 110 is placed in a case and placed horizontally on a flat surface. Then, a specimen extract from an extracted specimen is dropped onto the sample pad SP of the test strip 110 from the opening of the case, and the specimen extract is absorbed by the sample pad SP. The specimen extract is then spread onto the conjugate pad CP adjacent to the sample pad SP. At this time, as shown in 1002 in FIG. 1, if antigen G (which specifically reacts with the labeled antibody A1) is contained in the specimen extract, the antigen G is also spread onto the conjugate pad CP together with the specimen extract.

[0008] In the conjugate pad CP, the specimen extract flowing in from the sample pad SP is developed by capillary action, and the retained labeled antibody A1 and labeled antibody B1 are released into the specimen extract. As a result, the labeled antibody A1 reacts specifically with the antigen G in the specimen extract, and a complex C is generated in the specimen extract. The labeled antibody B1 in the specimen extract does not react with the antigen G. Therefore, the labeled antibody B1 and complex C are developed onto the membrane M together with the specimen extract.

[0009] In the membrane M, the specimen extract flowing in from the conjugate pad CP is developed by capillary action and reaches the detection area. In the first test line TL1 in the detection area, the antigen capture antibody A2 reacts specifically with the antigen G of the complex C, the complex C is captured by the antigen capture antibody A2, and the complex C accumulates in the first test line TL1. In the first test line TL1, a line of the same color as the colored particles of the labeled antibody A1 of the complex C appears due to the colored particles. On the other hand, in the second test line TL2, the antigen capture antibody B2 that does not react specifically with the antigen G is immobilized, so the complex C is not captured and no line appears. In this way, the user of the test kit can visually confirm the accumulation of the complex C and can determine whether or not the antigen G is contained in the specimen extract from the presence or absence of the appearance of a line (for example, see non-patent literature).

[0010] In addition, the control line CL has a labeled capture antibody r immobilized thereon that specifically reacts with the labeled antibodies A1 and B1, so that the labeled antibodies A1 and B1 are captured and accumulated on the control line CL, and a line of a color that is a mixture of the color of the colored particles of the labeled antibody A1 and the color of the colored particles of the labeled antibody B1 appears. The user can determine whether the test was performed properly based on the appearance of a line on the control line CL. The specimen extract that has passed through the detection area reaches the absorbent pad 111 and is absorbed by the absorbent pad 111.

[0011] In such tests using immunochromatography, it takes a certain amount of time for a line to appear after the specimen extract is dropped, so a judgment time within a specified time range within which the line should be confirmed is predetermined. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Nichirei Biosciences Corporation, "Influenza Virus Kit ImmunofineTM FLUII", [online], searched on October 2, 2023, Internet (URL: https: / / nichireibiosciences.co.jp / wp-content / themes / nichirei / pdf / %E3%80%90Web%E7%94%A8%E3%80%91IF%20FLU%E2%85%A1%20%E6%93%8D%E4%BD%9C%E6%96%B9%E6%B3%95%E3%82%AB%E3%83%BC%E3%83%88%E3%82%99_2204.pdf) Summary of the Invention [Problem to be solved by the invention]

[0013] However, if the time required for the line to appear after the specimen extract is dropped is long, the user of the test kit may forget the judgment time, and the presence or absence of the line may not be determined within the specified judgment time. If the test strip is left for longer than the judgment time, the detection area dries out, and the specimen extract once absorbed by the absorbent pad flows back into the detection area, or a secondary release of the specimen extract remaining in the conjugate pad occurs, causing the labeled substance in the specimen extract to remain in the detection area. If the labeled substance remains in the detection area, there are problems that lead to erroneous judgment, such as a decrease in the visibility of the detection area, the labeled substance remaining in the detection area appearing as a line, or the appearance of a non-specific reaction. Therefore, it is desired to shorten the time from the application of the specimen extract to the sample pad until the line appears (the reaction result appears in the detection area) more than before.

[0014] Therefore, the present invention has been made in consideration of the above points, and aims to provide a test kit and test strip that can shorten the time from when a specimen extract is applied to the sample pad to when a reaction result is produced in the detection area compared to conventional methods. [Means for solving the problem]

[0015] The test kit of the present invention is a test kit having a test strip placed in a case, the test strip comprising: a sample pad to which a specimen extract containing a specimen is applied; a conjugate pad in which a labeling substance that reacts specifically with the specimen developed from the sample pad by capillary action to form a complex is held in a wet, releasable state; a spreading pad in which a sensitizer that can be contained in the specimen extract developed from the conjugate pad by capillary action is held; and a membrane having a detection region in which a capture substance that reacts specifically with the complex developed from the spreading pad by capillary action is immobilized, and the spreading pad holds a compound having a phosphorylcholine group in a dry state as the sensitizer.

[0016] The test strip also comprises a sample pad to which a specimen extract containing a specimen is applied, a conjugate pad in which a labeling substance that reacts specifically with the specimen developed from the sample pad by capillary action to form a complex is held in a wet, releasable state, a spreading pad in which a sensitizer that can be contained in the specimen extract developed from the conjugate pad by capillary action is held, and a membrane having a detection region in which a capture substance that reacts specifically with the complex developed from the spreading pad by capillary action is immobilized, and the spreading pad holds a compound having a phosphorylcholine group in a dry state as the sensitizer. Effect of the Invention

[0017] By providing a development pad that holds a compound having a phosphorylcholine group in a dry state as a sensitizer, the present invention can shorten the time from when a specimen extract is applied to the sample pad to when a reaction result is expressed in the detection area compared to the conventional method. [Brief description of the drawings]

[0018] [Figure 1] Reference numeral 1001 is a schematic diagram showing the configuration of a test strip used in a conventional test kit, and reference numeral 1002 is a schematic diagram showing the appearance of a specimen extract containing an antigen when the test strip is developed. [Diagram 2] FIG. 2 is a top perspective view showing the configuration of the test kit according to the present embodiment. [Diagram 3] 1 is a perspective view showing a configuration of a test strip according to an embodiment of the present invention. [Figure 4] FIG. 1 is a side view showing the configuration of a test strip according to an embodiment of the present invention. [Diagram 5] 1 is a table showing the results of a verification test in which the performance of the test kit according to the present embodiment was compared with that of an existing product. [Figure 6] This is a table showing the results of a validation test in which performance was compared by changing the configuration of the test kit. [Figure 7] 13 is a table showing the results of a verification test in which performance was compared by changing the presence or absence of a spreading pad, the application position of the sensitizer, and the amount of the sensitizer used. [Figure 8] 13 is a table showing the results of a verification test in which performance was compared by changing the distance L2 of the deployment pad. [Figure 9] 13 is a table showing the results of a verification test in which performance was compared by changing the length of the deployment pad. [Figure 10] 13 is a table showing the results of a verification test in which performance was compared by changing the distance L0 from the downstream end of the development pad to the inspection area. [Figure 11] 1 is a table showing the results of a verification test in which performance was compared by changing the amount of a compound having a phosphorylcholine group with a hydrophilic side chain used. [Figure 12]1 is a table showing the results of a verification test in which performance was compared by changing the amount of a compound having a phosphorylcholine group with a hydrophobic side chain used. [Figure 13] 1 is a table showing the results of a verification test in which performance was compared by changing the amount of a compound having a phosphorylcholine group with an anionic side chain used. [Figure 14] 13 is a table showing the results of a verification test in which performance was compared using compounds having phosphorylcholine groups with other side chains. [Figure 15] 1011 is a side view showing the configuration of a test strip in which the development pad has a laminated portion overlapping both the conjugate pad and the membrane, and 1012 is a side view showing the configuration of a test strip in which the development pad has a laminated portion overlapping both the sample pad and the conjugate pad. [Figure 16] 1 is a table showing the results of validation testing comparing the performance of test strips in which a spreading pad overlies both a conjugate pad and a membrane. [Figure 17] 1 is a table showing the results of validation testing in which performance comparisons were performed on dipstick-type test strips. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will now be described in detail with reference to the accompanying drawings. In the following description, the same components are designated by the same reference numerals, and duplicated description will be omitted.

[0020] First Embodiment (1) Configuration of the test kit according to this embodiment As shown in Fig. 2, the test kit 100 of this embodiment, like conventional test kits, is composed of a test strip (described later) and a case 101 that stores the test strip during testing. Fig. 2 is a top perspective view showing the configuration of the test kit 100 of this embodiment in a state in which the test strip is stored in the case 101. The case 101 includes a lid 102 and a base 103, and is configured so that the lid 102 and the base 103 can be fitted together. The test strip is stored in the internal space surrounded by the lid 102 and the base 103 in the case 101.

[0021] In this embodiment, the longitudinal direction of case 101 is the x-direction, the lateral direction of case 101 is the y-direction, and the height direction in which lid 102 and base 103 are stacked in case 101 is the z-direction. The x-direction, which is the longitudinal direction of case 101, is the longitudinal direction of a test strip described below, and is the direction in which a specimen extract develops in the test strip. The y-direction, which is the lateral direction of case 101, is the width direction of the test strip.

[0022] The upper surface of the cover 102 is provided with a specimen dropping opening 105 having a peripheral portion recessed from the upper surface, and a determination opening 106 having a peripheral portion recessed from the upper surface of the cover 102. In the test kit 100, a sample pad of a test strip described later is disposed below the specimen dropping opening 105, and a detection area provided on a membrane of the test strip is disposed below the determination opening 106. Therefore, in the test kit 100, with the test strip stored in the case 101, a specimen extract obtained by extracting a specimen collected from a test subject is dropped onto the sample pad exposed to the outside from the specimen dropping opening 105 to perform a test, and the reaction result appearing in the detection area from the determination opening 106 can be visually confirmed by the user.

[0023] (2) Composition of the test strip Next, the test strip 1 of this embodiment will be described with reference to Figures 3 and 4. As shown in Figure 3, the test strip 1 has a sample pad SP, a conjugate pad CP, a spreading pad DP, a membrane M, and an absorbent pad AbP arranged in this order on a backing sheet 11.

[0024] As an example of test strip 1, for example, the length of test strip 1 is about 77.0 [mm], the width is about 4.0 [mm], and the height is about 0.4 [mm].

[0025] When a specimen extract is dropped onto the sample pad SP of the test strip 1, the specimen extract is absorbed into the sample pad SP. In the test strip 1, the specimen extract absorbed into the sample pad SP spreads in the x direction by capillary action through the conjugate pad CP, spreading pad DP, membrane M, and absorbent pad AbP in that order. In this embodiment, in the spreading direction in which the specimen extract spreads in the test strip 1, the sample pad SP side is referred to as the upstream side, and the absorbent pad AbP side is referred to as the downstream side.

[0026] The sample pad SP is provided with a specimen extract containing an antigen as a specimen. The sample pad SP is, for example, a filter paper made of glass fiber, and can absorb the dropped specimen extract and spread the specimen extract by capillary action. In this embodiment, the sample pad SP has a laminated portion 3a in which the end of the sample pad SP is laminated on the end of the conjugate pad CP, and the end of the sample pad SP is raised by the film thickness of the conjugate pad CP.

[0027] The conjugate pad CP is, for example, a filter paper made of glass fiber, and can develop a specimen extract by capillary action. The conjugate pad CP specifically reacts with a predetermined antigen developed from the sample pad SP, and holds at least one type of labeled antibody (not shown) bound to colored particles in a wet, releasable state. The labeled antibody as the labeling substance varies depending on the test subject. In this embodiment, the downstream end of the conjugate pad CP on the opposite side to the sample pad SP has a laminated portion 3b laminated on the end of the adjacent development pad DP, and the end of the conjugate pad CP is raised by the film thickness of the development pad DP.

[0028] In the conjugate pad CP, when the specimen extract is spread from the sample pad SP through the laminated portion 3a of the sample pad SP and the conjugate pad CP, the specimen extract is spread to the conjugate pad CP by capillary action, and the retained labeled antibody is released into the specimen extract and reacts specifically with the antigen in the specimen extract, resulting in the formation of a complex between the antigen and the labeled antibody.

[0029] In this embodiment, the sample pad SP and the conjugate pad CP are made of glass fiber as described above. The glass fiber sample pad SP and the conjugate pad CP can hold a relatively small amount of water, so a smaller amount of sample liquid needs to be dripped than when using a sample pad SP or a conjugate pad CP made of cellulose fiber, which can hold a large amount of water. Therefore, the glass fiber sample pad SP and the conjugate pad CP are preferable because they are less likely to become clogged due to components derived from the sample and allow the sample extract to spread smoothly.

[0030] Thus, the sample pad SP and the conjugate pad CP are preferably made of glass fiber, but may be made of other materials such as cellulose fiber, pulp fiber, cotton, etc. In the present embodiment, nonwoven fabric is used for the sample pad SP and the conjugate pad CP, but there is no particular limitation as long as the material generates capillary action, and woven fabric, filter paper, or other paper materials may also be used.

[0031] In addition to this configuration, in this embodiment, a spreading pad DP is provided between the conjugate pad CP and the membrane M. The spreading pad DP is, for example, a filter paper made of glass fiber, and can spread the specimen extract by capillary action. In this embodiment, the downstream end of the spreading pad DP opposite the conjugate pad CP has a laminated portion 3c laminated on the end of the adjacent membrane M, and the end of the spreading pad DP is raised by the thickness of the membrane M.

[0032] The spreading pad DP holds in a dry state a sensitizer that can be contained in the specimen extract developed from the conjugate pad CP by capillary action. When the spreading pad DP becomes wet with the specimen extract developed from the conjugate pad CP, the sensitizer is contained in the specimen extract. When the spreading pad DP becomes wet, the specimen extract is spread from the conjugate pad CP through the laminated portion 3b of the conjugate pad CP and the spreading pad DP by capillary action, and the spreading pad DP becomes wet, the held sensitizer is contained in the specimen extract.

[0033] The spreading pad DP according to the present embodiment is made of glass fiber, and therefore can hold a relatively small amount of water, so that the amount of sample to be dripped is smaller than that required when using a spreading pad made of cellulose fiber, which can hold a large amount of water. Therefore, the spreading pad DP made of glass fiber is less likely to become clogged due to sample-derived components, and the sample extract can be spread smoothly.

[0034] Thus, the expansion pad DP is preferably made of glass fiber, but may be made of other materials such as cellulose fiber, pulp fiber, cotton, etc. In addition, in this embodiment, a nonwoven fabric is used for the expansion pad DP, but there is no particular limitation as long as the material generates capillary action, and the expansion pad DP may be made of woven fabric, filter paper, or other paper materials.

[0035] The width of the development pad DP is preferably about the same as that of the test strip 1, and is preferably 2.0 mm or more and 8.0 mm or less, from the viewpoint of appropriately controlling the flow rate of the specimen extract and from the viewpoint of the manufacturing method and stability of the test strip 1. The thickness of the development pad DP in the height direction is preferably 0.1 mm or more and 2.0 mm or less, from the viewpoint of appropriately controlling the flow rate of the specimen extract. The length of the development pad DP will be described later.

[0036] The sensitizer to be held in the development pad DP in a dry state contains a compound having a phosphorylcholine group. The compound having a phosphorylcholine group is a functional group derived from a living body, and is a polymer mainly composed of a monomer 2-methacryloyloxyphosphorylcholine (MPC) having a polar group of phospholipids, the "phosphorylcholine group". In addition, the compound having a phosphorylcholine group used as the sensitizer preferably has at least one side chain among a hydrophilic side chain, a hydrophobic side chain, an anionic side chain, a cationic side chain, and a hydrogen-bonding side chain. The sensitizer may contain a mixture of two or more compounds having a phosphorylcholine group with a hydrophilic side chain, a hydrophobic side chain, an anionic side chain, a cationic side chain, or a hydrogen-bonding side chain, such as a mixture of a compound having a phosphorylcholine group with a hydrophilic side chain and a compound having a phosphorylcholine group with a hydrophobic side chain.

[0037] As the sensitizer, for example, commercially available products such as Lipidure (registered trademark)-BL103 (manufactured by NOF Corporation), Lipidure (registered trademark)-BL203 (manufactured by NOF Corporation), Lipidure (registered trademark)-BL405 (manufactured by NOF Corporation), Lipidure (registered trademark)-BL502 (manufactured by NOF Corporation), Lipidure (registered trademark)-BL702 (manufactured by NOF Corporation), Lipidure (registered trademark)-BL1201 (manufactured by NOF Corporation), and Lipidure (registered trademark)-BL1301 (manufactured by NOF Corporation) can be used.

[0038] Lipidure (registered trademark)-BL103 is a compound having a phosphorylcholine group with a hydrophilic side chain. Lipidure (registered trademark)-BL405 is a compound having a phosphorylcholine group with an anionic side chain. Lipidure (registered trademark)-BL502 is a compound having a phosphorylcholine group with a cationic side chain. Lipidure (registered trademark)-BL702 is a compound having a phosphorylcholine group with a hydrogen-bonding side chain. Lipidure (registered trademark)-BL203, Lipidure (registered trademark)-BL1201, and Lipidure (registered trademark)-BL1301 are compounds having a phosphorylcholine group with a hydrophobic side chain.

[0039] Here, the development pad DP is manufactured as follows. For example, a strip-shaped pad material made of glass fiber or the like is prepared. A sensitizer-containing liquid is applied to the pad material, and the sensitizer-containing liquid is spread evenly over the pad material to absorb it. The development pad DP is then dried using a freeze dryer or the like to produce a development pad DP in which the sensitizer is dried and retained in the pad material. The concentration of the sensitizer in the development pad DP can be adjusted by the concentration of the sensitizer-containing liquid to be applied and the amount of application.

[0040] The membrane M is made of, for example, nitrocellulose, and is capable of developing a specimen extract by capillary action. The membrane M has a first test line TL1, a second test line TL2, and a control line CL provided in this order as detection regions from the developing pad DP toward the absorbent pad AbP.

[0041] The first test line TL1 has an antigen capture antibody immobilized as a capture substance that specifically reacts with a specific antigen, and captures a complex containing the antigen that is developed by capillary action together with the sample extract. Similarly, the second test line TL2 has an antigen capture antibody immobilized as a capture substance that specifically reacts with a specific antigen different from the antigen in the first test line TL1, and captures a complex containing the antigen that is developed by capillary action together with the sample extract. The control line CL has a labeled capture antibody immobilized as a capture substance that specifically reacts with multiple types of labeled antibodies, and captures the labeled antibodies in the sample extract and the labeled antibodies in the complex.

[0042] The membrane M according to this embodiment has a laminated portion 3d at the end on the absorbent pad AbP side where the ends of the adjacent absorbent pads AbP are laminated. At the laminated portion 3d where the membrane M and a part of the absorbent pad AbP are laminated, the absorbent pad AbP is raised by the thickness of the membrane M.

[0043] In such a membrane M, when the specimen extract is developed from the development pad DP through the laminated portion 3c of the development pad DP and the membrane M, the specimen extract is developed on the membrane M by capillary action, passes through the detection area, and reaches the laminated portion 3d. At this time, the complex in the specimen extract is captured on either the first test line TL1 or the second test line TL2. In the first test line TL1 and the second test line TL2, a line of the same color as the colored particles bound to the labeled antibody of the complex appears as a result of capturing the complex. In addition, the labeled antibody in the specimen extract is captured on the control line CL, and a line of a color that is a mixture of the colors of the colored particles bound to the two types of labeled antibodies appears on the control line CL.

[0044] In this case, in the membrane M, a sensitizer is contained in the sample extract by the development pad DP, and the sensitizer has a sensitizing effect, thereby improving the detection sensitivity at least at the first test line TL1 and the second test line TL2, and shortening the detection time.

[0045] In the absorbent pad AbP, the specimen extract is developed from the membrane M through the laminated portion 3d. The absorbent pad AbP of this embodiment is made of cellulose fiber. The absorbent pad AbP can absorb the specimen extract of the membrane M by capillary action, and excess specimen extract is removed from the membrane M.

[0046] 4, in this embodiment, the distance by which the spreading pad DP overlaps the membrane M in the longitudinal direction of the test strip 1 is defined as L1. Also, the distance by which the spreading pad DP overlaps neither the conjugate pad CP nor the membrane M in the longitudinal direction of the test strip 1 is defined as L2. Furthermore, the distance by which the spreading pad DP overlaps the conjugate pad CP in the longitudinal direction of the test strip 1 is defined as L3.

[0047] In this embodiment, the distance from the downstream end of the development pad DP to the first test line TL1 disposed downstream of the development pad DP in the longitudinal direction of the test strip 1 is defined as L0 1 In addition, the distance from the downstream end of the development pad DP to the second test line TL2 disposed downstream of the development pad DP in the longitudinal direction of the test strip 1 is defined as L0. 2 Furthermore, the distance from the downstream end of the development pad DP to the control line CL disposed downstream of the development pad DP in the longitudinal direction of the test strip 1 is defined as L0 3 Let us assume that.

[0048] In a conventional test kit, it generally takes about 5 minutes for the first test line TL1, the second test line TL2, and the control line CL to develop a line (called the line detection time or simply the detection time). In contrast, in the test kit 100 according to the present embodiment, the expected detection time is about 3 minutes, and the conditions such as the distance L1 and the distance L2 of the development pad DP are selected so that the determination result can be obtained in a shorter time than in the past.

[0049] Furthermore, in the test kit 100 of this embodiment, by selecting each condition such as the above-mentioned distance L1 and distance L2, high line intensity can be observed at the first test line TL1, the second test line TL2, and the control line CL, even if the detection time is shortened, and further, no phenomena such as non-specific reactions or white spots are observed, and the configuration is also good in terms of back bleed.

[0050] A non-specific reaction refers to a phenomenon in which a false positive or false negative occurs for some reason, resulting in a color reaction, etc. A whiteout refers to a phenomenon in which the first test line TL1, the second test line TL2, etc., become white during or after the development of the specimen extract, making it appear as if there are lines before and after it. A good backout refers to a state in which there is little labeled antibody of the complex remaining on the entire membrane M during or after the development of the specimen extract, making it easy to determine the presence or absence of a line.

[0051] (3) Performance comparison between the test kit according to this embodiment and existing products The existing product test kit "Immunofine TM A test sample was used, and a performance comparison was carried out between the test sample and the test kit 100 according to the present embodiment. Here, the test sample is referred to as Comparative Example 1, and the test kit 100 according to the present embodiment is referred to as Example 1.

[0052] In Example 1, the case 101, specimen extract, backing sheet 11, sample pad SP, conjugate pad CP, membrane M, and absorbent pad AbP were the same as those in Comparative Example 1. A buffer solution containing salts, surfactants, etc. was used as the specimen extract. The amount of the dripped liquid was 78 [μL]. The same capture antibody for membrane M was used in Example 1 and Comparative Example 1. Here, an antibody that binds to influenza A virus antigen (FluA) was fixed to the first test line TL1, and an antibody that binds to influenza B virus antigen (FluB) was fixed to the second test line TL2. A labeled capture antibody that specifically reacts with the labeled antibody was fixed to the control line CL.

[0053] In Comparative Example 1 and Example 1, influenza A virus antigen (FluA) and influenza B virus antigen (FluB) were used as positive samples. The influenza A virus antigen (FluA) and influenza B virus antigen (FluB) were used in amounts several tens of times greater than the antigen amount that results in the minimum detection sensitivity of the existing product, Comparative Example 1. The negative sample was only the sample extract.

[0054] Example 1 differs from Comparative Example 1 only in that a spreading pad DP was provided between the conjugate pad CP and the membrane M, and other aspects such as the length of the test strip were the same. The spreading pad DP in Example 1 was made of a glass fiber pad material in which Lipidure (registered trademark)-BL103 (manufactured by NOF Corporation) was dried and held as a sensitizer.

[0055] The deployment pad DP is 8.0 mm long, 4.0 mm wide, and 0.43 mm thick, with a volume of 13.76 mm 3 ]. The amount of sensitizer used and dried and retained on the development pad DP was 21 [μg]. The notation "μg" representing the amount of sensitizer used and dried and retained on the development pad DP is the theoretical value of the weight of the sensitizer after the sensitizer is applied to the development pad DP in a solution state and dried. Here, the concentration of Lipidure (registered trademark)-BL103 used is 0.11 [%], and the amount of sensitizer solution applied per test (amount of sensitizer solution applied to the development pad DP per test) is 18.67 [μl]. If the weight (amount used) of the sensitizer is X [μg] and the above amount of sensitizer used 21 [μg] is calculated as X, then X = (0.11 [%] / 100) × 18.67 [μl] × 1000 = 20.5 [μg] (≒ 21 [μg]).

[0056] In the test kits of Example 1 and Comparative Example 1, 78 [μL] of specimen extract was dropped into the specimen dropping opening 105, and the case 101 was opened at the timing when the predetermined line detection time came, the sample pad SP, the conjugate pad CP, the spreading pad DP, and the absorption pad AbP were peeled off from the backing sheet 11, and the remaining membrane M was dried for 30 minutes in a thermostatic oven at 37 degrees. After that, the intensity of each line was measured with an immunochromato reader (C10066-10, Hamamatsu Photonics). The occurrence of white spots and non-specific reactions, and the quality of backdrop were visually confirmed.

[0057] Since the line detection time (judgment time) of Comparative Example 1, which is an existing product, is 5 minutes, the line detection time of the first test line TL1, the second test line TL2, and the control line CL was set to 5 minutes in Comparative Example 1. In contrast, in Example 1, the line detection time of the first test line TL1, the second test line TL2, and the control line CL was set to 3 minutes, which is shorter than that of Comparative Example 1.

[0058] As a result, the results shown in Figure 5 were obtained. In Figure 5, the line intensity of the first test line TL1 is shown as "A line intensity", the line intensity of the second test line TL2 is shown as "B line intensity", and the line intensity of the control line CL is shown as "C line intensity". In addition, the column showing the occurrence or non-occurrence of a non-specific reaction is marked "non-specific", the column showing the occurrence or non-occurrence of a white gap is marked "white gap", and the column showing the quality of the back gap is marked "back gap".

[0059] When evaluating the line strength, the highest line strength value among the samples in the verification test (here, Example 1 and Comparative Example 1) was determined as a reference value for each of the A line strength, B line strength, and C line strength, and the ratio of the line strength value of the other samples was obtained from this reference value. Then, based on the obtained ratio, the line strength was evaluated according to the following Table 1. For example, in the A line strength column shown in FIG. 5, since the line strength of Example 1 was higher than that of Comparative Example 1, the A line strength value of Example 1 was set as the reference value. Then, the ratio of the A line strength value of Comparative Example 1 to the reference value was obtained. As a result, the ratio of the A line strength of Comparative Example 1 from the reference value was between 56[%] and 70[%], and the evaluation was "+". Similarly, the B line strength and C line strength were also evaluated. Note that in the B line strength column, since the line strength value of Example 1 was high, the B line strength value of Example 1 was set as the reference value. In the C line strength column, since the line strength value of Comparative Example 1 was high, the C line strength value of Comparative Example 1 was set as the reference value.

[0060] [Table 1]

[0061] From FIG. 5, it was confirmed that, although the detection time of Example 1 was shorter than that of Comparative Example 1, the A line intensity, B line intensity, and C line intensity had sufficient performance. In particular, a significant increase in the A line intensity and B line intensity was confirmed even with a short detection time. Furthermore, in Example 1, as in Comparative Example 1, no non-specific reaction or white spots that affect the performance of the product occurred, and the backing was also good. From the above, it was confirmed that, in Example 1, even if the detection time was shorter than that of Comparative Example 1, the basic performance as a test kit could be sufficiently guaranteed, and some line intensities could be enhanced.

[0062] (4) Comparison of performance when the composition of the test kit is changed Next, the distance L1 and the distance L2 of the deployment pad DP, and the distance L0 from the deployment pad DP to the first test line TL1 1, the distance L0 from the deployment pad DP to the second test line TL2 2 A verification test was conducted to change the length of the development pad DP itself (distance L3 + distance L2 + distance L1) and the amount of sensitizer used, and to check the A line intensity, B line intensity, C line intensity, the occurrence of non-specific reactions, the occurrence of white spots, and the quality of backing. The results shown in Figure 6 were obtained.

[0063] (4-1) Preparation of conjugate pad The conjugate pads CP used in Examples 11 to 14 and Comparative Examples 11 to 14 were prepared as follows.

[0064] (4-1-1) Preparation of colored latex particle-labeled antibodies A mixture was generated by mixing 30 μL of a 10 wt% aqueous dispersion of blue carboxyl latex particles (polystyrene, particle size 400 nm) with 270 μL of 50 mM MES buffer (pH 6.0). 60 μL of MES buffer containing 100 mg / mL WSC (water-soluble carbodiimide) was added to the mixture twice at 20-minute intervals to activate the particles at room temperature.

[0065] After centrifugation and washing, 150 μL of anti-type A influenza virus antibody adjusted to a concentration of 1 mg / mL was added and reacted at room temperature for one hour. Next, the mixture was blocked with monomethanolamine, centrifuged, washed, and then blocked with 1 wt% casein / PBS solution. Next, the mixture was centrifuged and suspended in 10 mL of Tris-HCl buffer containing 5 wt% sucrose, 1 wt% BSA, and 1 wt% casein, and then subjected to an ultrasonic dispersion device to obtain a blue particle-labeled anti-type A antibody dispersion.

[0066] Separately, a mixture was prepared by mixing 30 μL of a 10 wt% aqueous dispersion of red carboxyl latex particles (polystyrene, particle size 400 nm) with 270 μL of 50 mM MES buffer (pH 6.0). 60 μL of MES buffer containing 100 mg / mL WSC (water-soluble carbodiimide) was added to the mixture twice at 20-minute intervals for activation at room temperature. After centrifugation and washing, 150 μL of anti-type B influenza virus antibody adjusted to 1 mg / mL was added and reacted at room temperature for one hour. Next, the mixture was blocked with monomethanolamine, centrifuged, washed, and then blocked with 1 wt% casein / PBS solution. The mixture was then centrifuged and suspended in 10 mL of Tris-HCl buffer containing 5 wt% sucrose, 1 wt% BSA, and 1 wt% casein, and then subjected to an ultrasonic dispersion device to obtain a red particle-labeled anti-B antibody dispersion.

[0067] (4-1-2) Preparation of conjugate pad Next, the obtained blue particle-labeled anti-A antibody dispersion and the obtained red particle-labeled anti-B antibody dispersion were mixed to generate a colored particle mixed dispersion. The colored particle mixed dispersion was applied at a rate of 46.7 [μL / cm] to multiple glass nonwoven fabric pads (manufactured by Merck Millipore) arranged on a net, spread evenly, and then dried overnight in an oven to obtain a conjugate pad CP to be used in the verification test.

[0068] (4-2) Preparation of membrane The membrane M used in Examples 11 to 14 and Comparative Examples 11 to 14 was prepared as follows.

[0069] (4-2-1) Preparation of antibody-immobilized membrane sheet An anti-influenza A virus antibody with a different epitope from the above-mentioned colored particle-labeled anti-influenza A antibody, whose concentration was adjusted to 1.0 mg / mL with Tris-HCl buffer, was applied in a line 1 mm wide onto a nitrocellulose membrane sheet (Merck Millipore, 3.0 cm long, 45 cm wide) to create a first test line TL1 (also referred to as the A line).

[0070] In addition, downstream of line A on the nitrocellulose membrane sheet, a second test line TL2 (also referred to as line B) was created by applying a 1 mm-wide line of anti-influenza B virus antibody, which has an epitope different from that of the above-mentioned colored particle-labeled anti-influenza B antibody, with a concentration adjusted to 1.0 mg / mL with Tris-HCl buffer.

[0071] Furthermore, downstream of the B line on the nitrocellulose membrane sheet, a control line CL (also referred to as the R (reference) line) was created by applying a 1 mm-wide line of goat anti-mouse IgG antibody adjusted to 1.0 mg / mL with Tris-HCl buffer.

[0072] The A line was provided at a position 11 [mm] from the upstream end of the membrane sheet, the B line was provided at a position 14.5 [mm] from the upstream end of the membrane sheet, and the R line was provided at a position 18.0 [mm] from the upstream end of the membrane sheet. The anti-type influenza virus A antibody, the anti-type influenza virus B antibody, and the goat anti-mouse IgG antibody were applied using an immunochromatography dispenser (manufactured by Biodot Co., Ltd.) and set to 1 [μL / cm]. After the anti-type influenza virus A antibody, the anti-type influenza virus B antibody, and the goat anti-mouse IgG antibody were applied, the nitrocellulose membrane sheet was dried overnight at 37 [℃].

[0073] (4-2-2) Blocking of antibody-immobilized membrane sheet Next, the antibody-immobilized membrane sheet thus prepared was subjected to blocking according to the technique described in Japanese Patent No. 6530114. "Blocking" refers to coating the membrane sheet with an aqueous solution of protein and / or polymer in order to prevent nonspecific adsorption of the labeled antibody onto the membrane sheet in the test strip (immunochromatography).

[0074] Specifically, while an absorbent paper was placed over the entire downstream end of the antibody-immobilized membrane sheet, the sheet was immersed 5 mm below the liquid surface in a container filled with the primary blocking solution, and the primary blocking solution was absorbed.

[0075] In addition, while an absorbent paper was placed over the entire downstream end of the antibody-immobilized membrane, the antibody-immobilized membrane was immersed 5 mm below the liquid surface in a container filled with the secondary blocking solution, and the secondary blocking solution was absorbed.

[0076] Next, the entire antibody-immobilized membrane sheet was immersed in a container filled with the secondary blocking solution. Next, the antibody-immobilized membrane sheet soaked with the secondary blocking solution was turned upside down, and the upstream end of the antibody-immobilized membrane sheet was pressed against absorbent paper placed on the surface for 1 minute, allowing the soaked secondary blocking solution to be absorbed into the absorbent paper. Next, the blocked antibody-immobilized membrane sheet was dried overnight using a vacuum dryer to produce Membrane M.

[0077] (4-3) Preparation of expansion pad A glass nonwoven pad (Merck Millipore) was prepared as a pad material, and a predetermined amount of Lipidure (registered trademark)-BL103 (sensitizer-containing liquid, 0.11 [wt%]) was applied to a number of pad materials arranged on a net, spread evenly on the pad material, and then dried overnight in a freeze dryer. In this way, a pad material with a dried sensitizer was obtained as a spreading pad DP. The length (distance L3 + distance L2 + distance L1) and the amount of sensitizer used in the spreading pad DP used in Examples 11 to 14 and Comparative Examples 11 to 14 were the values ​​shown in FIG. 6. In FIG. 6, the sensitizer held in the spreading pad DP is expressed in "μg". The sensitizer is applied to the spreading pad DP in a solution state, and the amount of sensitizer used shown in FIG. 6 indicates the theoretical value of the weight of the sensitizer after drying.

[0078] (4-4) Preparation of test strips and test kits In Examples 11 to 14 and Comparative Examples 11 to 14, the prepared membrane M was attached to a backing sheet (manufactured by Lohmann) 11 serving as a support, and then a similarly prepared spreading pad DP was attached to overlap the membrane M by several millimeters. In addition, in Examples 11 to 14 and Comparative Examples 11 to 14, a conjugate pad CP was attached to the upstream side of the backing sheet 11 in the spreading direction so as to overlap the spreading pad DP by several millimeters.

[0079] Furthermore, in Examples 11 to 14 and Comparative Examples 11 to 14, a sample pad SP made of a glass nonwoven fabric (manufactured by Merck Millipore) was attached to the upstream side in the developing direction of the backing sheet 11 so as to overlap the conjugate pad CP by several millimeters. Then, in Examples 11 to 14 and Comparative Examples 11 to 14, an absorption pad AbP (manufactured by Merck Millipore) for receiving excess reagent was attached to the downstream side of the membrane M so as to overlap the membrane M by several millimeters.

[0080] Then, it was cut to a width of 4 [mm] to obtain band-shaped test strips (immunochromatography test pieces) of Examples 11 to 14 and Comparative Examples 11 to 14. The test strips obtained as the examples and comparative examples were each stored in a case 101 to prepare a test kit.

[0081] For each of Examples 11 to 14 and Comparative Examples 11 to 14, the distance L1 of the development pad DP, the distance L2, the distance L0 from the downstream end of the development pad DP to the first test line TL1, 1 , the distance L0 from the downstream end of the deployment pad DP to the second test line TL2 2 Test strips were prepared so that the values ​​shown in FIG. 6 were obtained.

[0082] (4-4) Verification test and considerations Using each of the test kits of Examples 11 to 14 and Comparative Examples 11 to 14, a performance comparison was made under the following conditions. Here, a positive test for influenza A virus antigen was performed using a self-controlled specimen positive for influenza A virus, a positive test for influenza B virus antigen was performed using a self-controlled specimen positive for influenza B virus, and a negative test for influenza A and B virus antigen was performed using a negative self-controlled specimen. The positive test was performed using the existing test kit "Immunofine" TM The amount of antigen was several tens of times the amount of antigen that provides the minimum detection sensitivity of the antibody "FLUII" (Nichirei Biosciences Corporation), and was therefore sufficient to perform a general test.

[0083] 78 [μL] of specimen extract was dropped into the specimen dropping opening 105 of each of the test kits of Examples 11 to 14 and Comparative Examples 11 to 14, and when the line detection time came, the case 101 was opened and each test strip was taken out of the case 101. Then, the sample pad SP, conjugate pad CP, spreading pad DP, and absorption pad AbP were peeled off from the backing sheet 11, and the remaining membrane M was dried in a thermostatic oven at 37 degrees for 30 minutes. Thereafter, the intensity of each line was measured with an immunochromatograph reader (C10066-10, Hamamatsu Photonics). The occurrence of white spots and non-specific reactions, and the quality of backdrop were visually confirmed. The line detection time (also referred to as judgment time or simply detection time) was measured using the existing product test kit "Immunofine". TM The detection time was set at 3 minutes, which is shorter than the 5 minutes required by the "Nichirei Biosciences" (FLUII) system.

[0084] In Example 12, the length of the expansion pad DP is 6 [mm], which is shorter than that of the other Examples 11, etc. In Example 13, the length of the expansion pad DP is 9 [mm], which is longer than that of the other Examples 11, etc., and the distance L2 of the expansion pad DP is 2 [mm], which is longer than that of the other Examples 11, etc. In Example 14, the distance L0 from the downstream end of the expansion pad DP to the first test line TL1 is 1 [mm]. 1 and the distance L0 from the downstream end of the deployment pad DP to the second test line TL2. 2and were set to 9 [mm] and 12.5 [mm], which are larger than those of the other examples such as Example 11.

[0085] In Comparative Example 11, a simple pad material that does not dry and retain sensitizer was provided as the development pad. In Comparative Example 12, the amount of sensitizer that is dried and retained in the development pad DP was set to 75 [μg], which is larger than in the other Examples 11 and the like. In Comparative Example 13, the distance L2 of the development pad DP was set to 7 [mm], which is larger than in the other Examples 11 and the like, and the length of the development pad DP itself was also set to 14 [mm], which is larger than in the other Examples 11 and the like. In Comparative Example 14, the distance L0 from the downstream end of the development pad DP to the first test line TL1 was set to 10 [mm], which is larger than in the other Examples 11 and the like. 1 and the distance L0 from the downstream end of the deployment pad DP to the second test line TL2. 2 and were set to 15 [mm] and 18.5 [mm], which are even larger than those in Example 14.

[0086] When evaluating the A line intensity, B line intensity, and C line intensity, the line intensity (A line intensity, B line intensity, and C line intensity) of each sample (Example 11 shown in FIG. 6, etc.) was measured using an immunochromatograph reader (C10066-10, Hamamatsu Photonics), and the highest line intensity value among the samples for each A line intensity, B line intensity, and C line intensity was determined as the reference value. Next, the ratio of the line intensity of the remaining samples to the reference value was calculated. Then, from the obtained ratio, the A line intensity, B line intensity, and C line intensity were evaluated based on Table 1 with "+++", "++", "+", and "±".

[0087] For example, in the A-line intensity column shown in FIG. 6, the line intensity value of Example 14 was the highest. Therefore, the A-line intensity value of Example 14 was set as the reference value. Then, the ratio of the A-line intensity values ​​of other Examples 11, 12, etc. to the reference value was obtained. As a result, in Example 11, the evaluation was "++" based on the ratio obtained for the A-line intensity. In the same manner, evaluations were obtained for other Examples 12, etc. In the B-line intensity column, the line intensity value of Example 14 was the highest, so the B-line intensity value of Example 14 was set as the reference value. In the C-line intensity column, the line intensity value of Example 11 was the highest, so the C-line intensity value of Example 11 was set as the reference value.

[0088] In this verification test, the sample had a spreading pad DP that kept the sensitizer dry, and even in 3 minutes, which is shorter than the conventional detection time, high line intensity was observed at least in the A line intensity and the B line intensity, and non-specific reaction and whiteout did not occur, and the backing out was also good. As shown in FIG. 6, in Comparative Example 11, the A line intensity was "±" and the B line intensity was "+". In Comparative Example 11, since there was no sensitizer, sufficient line intensity was not obtained in the detection time of 3 minutes. In Comparative Example 12, since the amount of sensitizer used was large, whiteout occurred strongly, and it was impossible to measure the A line intensity, B line intensity, and C line intensity. In Comparative Example 13, the A line intensity was "±" and the B line intensity and C line intensity were "+". In Comparative Example 13, the line intensity was weak, and the backing out was poor, which was at a level that affected visual judgment. In Comparative Example 14, the intensity of each line was weak.

[0089] On the other hand, in Examples 11 to 14, the A line intensity and B line intensity were "+++" or "++", and a significant increase in line intensity was observed. In addition, in Examples 11 to 13, the C line intensity was "+++", and a significant increase in line intensity was observed. Furthermore, in all of Examples 11 to 14, there was no non-specific reaction or white voids, the back voids were good, and the visibility of the lines was sufficiently ensured.

[0090] It was found that the line intensity decreased as the distance from the downstream end of the development pad DP to the first test line TL1, the second test line TL2, and the control line CL increased. This is thought to be because the sensitizing effect of the sensitizer was not sufficient in a detection time of 3 minutes.

[0091] From the above, the distance L2 of the deployment pad DP and the distance L0 from the deployment pad DP to the first test line TL1 are 1 , the distance L0 from the deployment pad DP to the second test line TL2 2 It was confirmed that the length of the spreading pad DP itself (distance L3 + distance L2 + distance L1) and the amount of sensitizer used affect the expression of A line intensity, B line intensity, and C line intensity, and also affect the occurrence of non-specific reactions and white spots, and the quality of backing.

[0092] (5) Comparison of performance when using or not using a spreading pad, when changing the application position of the sensitizer, and when changing the amount of sensitizer used Next, verification tests were conducted to confirm the A-line intensity, B-line intensity, the occurrence of non-specific reactions, the occurrence of white spots, and the quality of backing by changing the presence or absence of the spreading pad DP, the application position of the sensitizer, and the amount of sensitizer used. The results are shown in Figure 7.

[0093] The test strips of Example 21 and Comparative Examples 21 to 26 were prepared using the sample pad SP, conjugate pad CP, spreading pad DP, membrane M, and absorbent pad AbP used in the above-mentioned "(4) Performance comparison when the configuration of the test kit is changed". Example 21 has a configuration in which the sensitizer is dried and retained in the spreading pad DP. The amount of the sensitizer used that is dried and retained in the spreading pad DP was 41 [μg]. The amount of the sensitizer used is the theoretical value of the weight of the sensitizer after the sensitizer is applied to the spreading pad DP in a solution state and dried.

[0094] Comparative Examples 21 and 22 had the same configuration as the existing product, in which the development pad DP was not provided, and the conjugate pad CP was directly laminated on the end of the membrane M. Comparative Examples 21 and 22 were test strips of the same configuration, and were prepared to evaluate the A-line intensity, etc., by changing the line detection time to 3 minutes and 5 minutes.

[0095] In Comparative Example 23, a spreading pad DP in which the sensitizer was not dried and retained was provided. In Comparative Example 24, a spreading pad DP in which the sensitizer was not dried and retained was provided, and the sensitizer was dried and retained in a conjugate pad CP arranged upstream of the spreading pad DP (indicated as "Conjugate" in the sensitizer application position column), and the position where the sensitizer was applied was different from that of Example 21. The amount of the sensitizer used to be dried and retained in the conjugate pad CP was 41 [μg].

[0096] In Comparative Example 25, the test strip itself, including the development pad DP, was configured so that the sensitizer was not dried and retained, and the test strip had the same configuration as in Comparative Example 23. The sensitizer was then added to the specimen extract.

[0097] In Comparative Example 26, a development pad DP on which the sensitizer was not dried was provided, and the sensitizer was dried and retained in a sample pad SP arranged upstream of the development pad DP and the conjugate pad CP (indicated as "sample pad" in the "sensitizer application position" column), and the position at which the sensitizer was applied was different from that in Example 21. The amount of sensitizer used to be dried and retained in the sample pad SP was 72 [μg].

[0098] 78 [μL] of specimen extract was dropped into the specimen dropping opening 105 of each test kit of Example 21 and Comparative Examples 21 to 26, and the case 101 was opened at the timing when the line detection time came, the sample pad SP, the conjugate pad CP, the spreading pad DP, and the absorption pad AbP were peeled off from the backing sheet 11, and the remaining membrane M was dried for 30 minutes in a thermostatic oven at 37 degrees. After that, the intensity of each line was measured with an immunochromatograph reader (C10066-10, Hamamatsu Photonics). The occurrence of white spots and non-specific reactions, and the quality of backdrop were visually confirmed.

[0099] In Example 21 and Comparative Examples 22 to 26, the line detection time (judgment time) was compared with that of the existing test kit "Immunofine TM The line detection time was set to 3 minutes, which is shorter than the 5 minutes required by the existing test kit "Immunofine" (manufactured by Nichirei Biosciences Corporation). TM The line detection time was set to 5 minutes, similarly to the "FLUII" (manufactured by Nichirei Biosciences Corporation). When evaluating the A line intensity and the B line intensity, the line intensity of each sample (Example 21 shown in FIG. 7, etc.) was measured using an immunochromatograph (C10066-10, Hamamatsu Photonics) in the same manner as in the above-mentioned verification test, and the highest line intensity value among the samples was set as the reference value for each of the A line intensity and the B line intensity. Then, the ratio of the line intensity of the remaining samples to the reference value was calculated, and the evaluation was performed with "+++", "++", "+", and "±" based on Table 1. In addition, in the A line intensity column and the B line intensity column, since the line intensity value of Example 21 was the highest in both cases, the A line intensity value and the B line intensity value of Example 21 were set as the reference value, respectively.

[0100] In this verification test, the sample had a spreading pad DP that dried and retained the sensitizer, and even at 3 minutes, which is shorter than the conventional detection time, high line intensity was observed at least for the A line intensity and the B line intensity, and non-specific reaction and white voids did not occur, and the back void was also good. As shown in Figure 7, from the evaluation results obtained in Comparative Examples 21 and 22, it was confirmed that a significantly higher line intensity could be observed for the B line intensity when the line detection time was set to 5 minutes, which is longer than 3 minutes. This is because the antigen-antibody reaction progresses as the reaction time is longer.

[0101] When the A-line intensity and the B-line intensity of Example 21 and Comparative Examples 22 to 26, in which the line detection time was set to 3 minutes, were compared, the highest line intensity was observed for both the A-line intensity and the B-line intensity in Example 21, in which the sensitizer was dried and retained in the spreading pad DP. In Comparative Example 25, non-specific reactions and strong white spots occurred, making it impossible to measure the intensity of each line.

[0102] Moreover, in Example 21, the A-line intensity was observed to be about 1.73 times higher than that of Comparative Example 21, despite the line detection time being 3 minutes, which was shorter than that of Comparative Example 21. In Example 21, the A-line intensity and the B-line intensity were both observed to be about 2 times higher than that of Comparative Example 23. Furthermore, in Example 21, as in the above-mentioned verification test, no non-specific reaction or white spots occurred, and the back spots were also good.

[0103] From the above, it was confirmed that by providing a developing pad DP on the test strip and applying the sensitizer to the developing pad DP, it is possible to shorten the line detection time while increasing the A line intensity and B line intensity more than before. It was also confirmed that by applying the sensitizer to the developing pad DP, it is possible to provide a test strip that does not cause non-specific reactions or white spots and has good back spots.

[0104] (6) Distance L2 of the deployment pad (6-1) Verification test Next, the distance L2 of the spreading pad DP, where the spreading pad DP does not overlap with either the conjugate pad CP or the membrane M, will be described. Here, the length of the spreading pad DP itself is the same, and multiple test strips with different distances L2 and L3 of the spreading pad DP are prepared, and verification tests were performed to confirm the A line intensity, B line intensity, C line intensity, the occurrence of non-specific reactions, the occurrence of blank spots, and the quality of backing for each test strip. As a result, the results shown in Figure 8 were obtained.

[0105] The test strips of Examples 31 to 33 and Comparative Examples 31 and 32 were prepared using the sample pad SP, conjugate pad CP, spreading pad DP, membrane M, and absorbent pad AbP used in the above-mentioned "(4) Performance comparison when the configuration of the test kit is changed." The length of the sample pad SP in the longitudinal direction of the test strip was 14 [mm], the length of the conjugate pad CP in the longitudinal direction of the test strip was 10 [mm], and the length of the spreading pad DP in the longitudinal direction of the test strip was 8 [mm].

[0106] Distance L0 from the downstream end of the deployment pad DP to the first test line TL1 1 is 9 [mm], and the distance L0 from the downstream end of the deployment pad DP to the second test line TL2 is 2 was 12.5 [mm]. The spreading pad DP was configured to retain the sensitizer in a dried state, and the amount of sensitizer retained in the spreading pad DP was 21 [μg]. The amount of sensitizer used is the theoretical value of the weight of the sensitizer after applying the sensitizer in a solution state to the spreading pad DP and drying. The line detection time was set to 3 minutes, which is shorter than the conventional 5 minutes. In this verification test, as in the above-mentioned verification test, a positive test for influenza A virus antigen using a self-controlled specimen positive for influenza A virus, a positive test for influenza B virus antigen using a self-controlled specimen positive for influenza B virus, and a negative test for influenza A and B virus antigens using a negative self-controlled specimen were performed.

[0107] When evaluating the A line intensity, B line intensity, and C line intensity, the line intensity of each sample (Example 31 shown in FIG. 8, etc.) was measured using an immunochromatography reader (C10066-10, Hamamatsu Photonics) in the same manner as in the above-mentioned verification test, and the highest line intensity value among the measurement results was set as the reference value for each of the A line intensity, B line intensity, and C line intensity. Then, the ratio of the line intensity of the remaining samples to the reference value was obtained, and evaluation was performed using "+++", "++", "+", and "±" based on Table 1. In addition, in the A line intensity column, the B line intensity column, and the C line intensity column, the line intensity value of Example 33 was the highest, so the A line intensity value, the B line intensity value, and the C line intensity value of Example 33 were set as the reference value, respectively. The occurrence of whiteout and non-specific reaction, and the quality of backing out were visually confirmed.

[0108] In this verification test, the sample had a developing pad DP that kept the sensitizer dry, and even in 3 minutes, which is shorter than the conventional detection time, high line intensity was observed at least for the A line intensity and the B line intensity, and non-specific reaction and whiteout did not occur, and the backing was also good. The evaluation results of Examples 31 to 33 confirmed that by setting the distance L2 of the developing pad DP to 0 [mm] or more and 2 [mm] or less, the line intensity could be increased for each line intensity even if the line detection time was 3 minutes, and furthermore, non-specific reaction and whiteout did not occur, and the backing was also good. In addition, in Examples 31 to 33, the C line intensity was also sufficiently visible.

[0109] (6-2) Optimal range In view of the above, it is preferable that in the test strip 1, the development pad DP does not overlap either the conjugate pad CP or the membrane M in the longitudinal direction, and the distance L2 of the development pad DP satisfies the following formula (1). 0 [mm] ≦ L2 ≦ 2 [mm] … (1)

[0110] By satisfying the above formula (1), the development pad DP does not exhibit phenomena such as non-specific reactions or blank spots, has good backing, and allows high line intensities to be observed in the first test line TL1, the second test line TL2, and the control line CL. Furthermore, by providing the development pad DP so as to satisfy the above formula (1), the line detection time can be shortened compared to conventional test strips that do not have a development pad DP, while the line intensities in the first test line TL1, the second test line TL2, and the control line CL can be increased compared to conventional ones.

[0111] (7) Length of the deployment pad (7-1) Verification test Next, we will explain the length of the spreading pad DP in the longitudinal direction of the test strip 1. The length of the spreading pad DP in the longitudinal direction of the test strip 1 is the sum of the distance L1 where the spreading pad DP overlaps with the membrane M, the distance L2 where the spreading pad DP overlaps with neither the conjugate pad CP nor the membrane M, and the distance L3 where the spreading pad DP overlaps with the conjugate pad CP.

[0112] Here, multiple test strips were prepared, each with a development pad DP of different lengths in the range of 4 mm to 14 mm in the longitudinal direction of the test strip, and a verification test was conducted to check the A line intensity, B line intensity, C line intensity, the occurrence of non-specific reactions, the occurrence of blank spots, and the quality of backing for each test strip. The line detection time was set to 3 minutes, shorter than the conventional 5 minutes. As a result, the results shown in Figure 9 were obtained.

[0113] The test strips of Examples 41 to 44 and Comparative Examples 41 and 42 were prepared using the sample pad SP, conjugate pad CP, spreading pad DP, membrane M, and absorbent pad AbP used in the above-mentioned "(4) Performance comparison when the configuration of the test kit is changed." The length of the sample pad SP in the longitudinal direction of the test strip was 14 [mm], the length of the conjugate pad CP in the longitudinal direction of the test strip was 14 [mm], and the length of the spreading pad DP in the longitudinal direction of the test strip was 4 [mm] or more and 14 [mm] or less.

[0114] The distance L1 where the spreading pad DP overlaps with the membrane M is set to 2 [mm], and the distance L2 where the spreading pad DP overlaps with neither the conjugate pad CP nor the membrane M is set to 0 [mm]. The distance L3 where the spreading pad DP overlaps with the conjugate pad CP is set to 2 [mm] or more and 12 [mm] or less.

[0115] Distance L0 from the downstream end of the deployment pad DP to the first test line TL1 1 is 9.0 [mm], and the distance L0 from the downstream end of the deployment pad DP to the second test line TL2 is 2 was 12.5 [mm]. The spreading pad DP was configured to dry and retain the sensitizer, and the amount of the sensitizer dried and retained in the spreading pad DP was 21 [μg]. In this verification test, as in the above-mentioned verification test, a positive test for influenza A virus antigen was performed using a self-controlled specimen positive for influenza A virus, a positive test for influenza B virus antigen was performed using a self-controlled specimen positive for influenza B virus, and a negative test for influenza A and B virus antigens was performed using a self-controlled specimen negative for influenza A and B.

[0116] When evaluating the A line intensity, B line intensity, and C line intensity, the line intensity of each sample (Example 41 shown in FIG. 9, etc.) was measured using an immunochromatography reader (C10066-10, Hamamatsu Photonics) in the same manner as in the above-mentioned verification test, and the highest line intensity value among the measurement results was set as the reference value for each of the A line intensity, B line intensity, and C line intensity. Then, the ratio of the line intensity of the remaining samples to the reference value was obtained, and evaluation was performed using "+++", "++", "+", and "±" based on Table 1. In the A line intensity column, the line intensity value of Example 42 was the highest, so the A line intensity value of Example 42 was set as the reference value. In the B line intensity column and the C line intensity column, the line intensity value of Example 41 was the highest, so the B line intensity value and the C line intensity value of Example 41 were set as the reference value, respectively. The occurrence of whiteout and non-specific reaction, and the quality of backing out were visually confirmed.

[0117] In this verification test, the samples were designated as "Examples" because they had a spreading pad DP that kept the sensitizer dry, and showed high line intensities at least for the A line and B line even in 3 minutes, which is shorter than the conventional detection time, and did not show non-specific reactions or white spots, and showed good backing. From the evaluation results of Examples 41 to 44 and Comparative Examples 41 and 42, sufficient A line and B line intensities were observed in each case, and the C line intensity in the control line was also sufficiently visible. However, in Comparative Examples 41 and 42, in which the length of the spreading pad (distance L3 + distance L2 + distance L1) was 12 mm or more, backing was poor.

[0118] Therefore, taking into consideration the verification results shown in Figure 9 as well as the verification results shown in Figures 6 and 8 described above, it was confirmed that when the distance L2 of the expansion pad DP is 0 mm or more and 2 mm or less, the optimal length of the expansion pad DP is 4 mm or more and 10 mm or less.

[0119] (7-2) Optimal range In view of the above, it is preferable that in the test strip 1, the distance L1 over which the development pad DP overlaps the membrane M in the longitudinal direction, the distance L2 over which the development pad DP overlaps neither the conjugate pad CP nor the membrane M, and the distance L3 over which the development pad DP overlaps the conjugate pad CP satisfy the following formula (2). 4[mm]≦L1+L2+L3≦10[mm]…(2)

[0120] By satisfying the above formula (2), the development pad DP does not exhibit phenomena such as non-specific reactions or blank spots, has good backing, and allows high line intensities to be observed in the first test line TL1, the second test line TL2, and the control line CL. Furthermore, by providing the development pad DP so as to satisfy the above formula (2), the line detection time can be shortened compared to conventional test strips that do not have a development pad DP, while the line intensities in the first test line TL1, the second test line TL2, and the control line CL can be increased compared to conventional ones.

[0121] (8) Distance L0 from the downstream end of the expansion pad to the inspection area (8-1) Verification test Next, a description will be given of the distance L0 from the downstream end of the development pad DP to the test area in the longitudinal direction of the test strip 1. Here, the distance L0 from the downstream end of the development pad DP to the first test line TL1 in the longitudinal direction of the test strip is 1 and the distance L0 to the second test line TL2 2 A verification test was conducted to check the A line intensity, B line intensity, the occurrence of non-specific reactions, the occurrence of blank spots, and the quality of backing for each test strip. The line detection time was set to 3 minutes, shorter than the conventional 5 minutes. As a result, the results shown in Figure 10 were obtained. In this verification test, the distance L0 from the downstream end of the development pad DP to the first test line TL1 and the second test line TL2 was set to 3 minutes. 1 , distance L0 2We adjusted the A-line intensity and the B-line intensity.

[0122] The test strips of Examples 51 to 55 and Comparative Example 51 were prepared using the sample pad SP, conjugate pad CP, spreading pad DP, membrane M, and absorbent pad AbP used in the above-mentioned "(4) Performance comparison when the configuration of the test kit is changed". The length of the sample pad SP in the longitudinal direction of the test strip was 14 [mm], the length of the conjugate pad CP in the longitudinal direction of the test strip was 14 [mm], and the length of the spreading pad DP in the longitudinal direction of the test strip was 8 [mm]. In addition, the length of the absorbent pad in the longitudinal direction of the test strip was 31 [mm].

[0123] The distance L1 where the spreading pad DP overlaps with the membrane M was set to 4 [mm], and the distance L2 where the spreading pad DP does not overlap with either the conjugate pad CP or the membrane M was set to 1 [mm]. The distance L3 where the spreading pad DP overlaps with the conjugate pad CP was set to 3 [mm]. The spreading pad DP was configured to retain a sensitizer in a dried state, and the amount of sensitizer retained in the spreading pad DP was set to 21 [μg].

[0124] Distance L0 from the downstream end of the deployment pad DP to the first test line TL1 1 The distance L0 from the downstream end of the deployment pad DP to the second test line TL2 was set to a range of -2.0 [mm] to 13.0 [mm]. 2 The distance between the first test line TL1 and the control line CL was set to a range of 1.5 mm to 16.5 mm. The first test line TL1 and the control line CL were set to a position 3.5 mm away from the second test line TL2. The absorbent pad AbP was set to a position 3 mm away from the control line CL.

[0125] In addition, a test strip having the same configuration as that of Examples 51 to 55 and Comparative Example 51, but without drying and retaining the sensitizer on the development pad DP, was prepared as a comparison test strip (a test strip with a development pad DP with 0 [μg] of sensitizer used). Then, similarly to Examples 51 to 55 and Comparative Example 51, 78 [μL] of specimen extract was dropped into the specimen drop opening 105 of each test kit containing the comparison test strip, and the case 101 was opened at the timing when the line detection time came, and the sample pad SP, the conjugate pad CP, the development pad DP without drying and retaining the sensitizer, and the absorption pad AbP were peeled off from the backing sheet 11, and the remaining membrane M was dried for 30 minutes in a thermostatic oven at 37 degrees. Then, the intensity of each line was measured with an immunochromato reader (C10066-10, Hamamatsu Photonics).

[0126] When evaluating the line intensity, the line intensity value of the corresponding comparison test strip with 0 [μg] of sensitizer was determined as the reference value for each sample. For example, in Example 51, the line intensity value of the comparison test strip corresponding to Example 51 measured with an immunochromatograph (C10066-10, Hamamatsu Photonics) was used as the reference value, and the ratio of the line intensity value of Example 51 measured with an immunochromatograph (C10066-10, Hamamatsu Photonics) was calculated. Then, based on the calculated ratio, the line intensity was evaluated according to Table 2 below. For example, when the line intensity value of the sample is higher than the reference value of the comparison test strip with 0 [μg] of sensitizer, it was determined as "improvement effect present", and when the line intensity value of the sample is lower than the reference value, it was determined as "no improvement effect present".

[0127] [Table 2]

[0128] In FIG. 10, the line strength is marked with "◯" if there is an "improvement effect" and marked with "×" if there is no "improvement effect." In this verification test, the optimal distance L0 from the downstream end of the deployment pad DP to the second test line TL2, which is the furthest from the end, is 2 In order to verify this, the samples were selected as "Examples" from samples that had a development pad DP that had a dried and retained sensitizer, had both A-line intensity and B-line intensity "improved" (marked with "◯"), and had no non-specific reaction or white spots, and also had good backing. Samples were selected as "Comparative Examples" from samples that had "no improvement effect" (marked with "x") in B-line intensity. As shown in FIG. 10, in Examples 51 to 55, the line intensity was improved in all of the A-line intensity and B-line intensity, compared to the corresponding control test strips (with 0 μg of sensitizer used).

[0129] However, the distance L0 from the downstream end of the deployment pad DP to the nearest first test line TL1 1 If the length is too short, the flow rate of the specimen extract at the position of the first test line TL1 will be too fast, which may make it difficult for the line-forming reaction to occur, and may result in a decrease in line strength.

[0130] On the other hand, the distance L0 from the downstream end of the deployment pad DP to the second test line TL2 2 In Comparative Example 51, where the distance L0 from the downstream end of the development pad DP to the nearest first test line TL1 was 16.5 [mm], no non-specific reaction or blank spots occurred, but sufficient line strength for evaluation was not obtained (i.e., the effect of improving the B line strength was not obtained). 1 and the distance L0 from the downstream end of the deployment pad DP to the farthest second test line TL2. 2 If this is too long, for example, the time it takes for the first test line TL1 or the second test line TL2 to appear (line detection time) will be longer than expected, so it is preferable that the upper limit be 13.5 mm or less.

[0131] From the evaluation results of Examples 51 to 55 and Comparative Example 51, it was confirmed that in the test strip 1, by setting all of the test areas of the first test line TL1, the second test line TL2, and the control line CL in a range of 1 mm or more and 13.5 mm or less, the intensity of each line can be increased even if the line detection time is 3 minutes, and further, no non-specific reactions or white spots occur, and back bleed is also good.

[0132] Therefore, it was found that it is preferable to provide all of the test areas of the first test line TL1, the second test line TL2, and the control line CL in a range of 1 mm to 13.5 mm in the test strip 1. In addition, when focusing on the first test line TL1 and the second test line TL2 without focusing on the control line CL, the distance L0 from the downstream end of the development pad DP to the second test line TL2, which is the furthest from the downstream end of the development pad DP, is 13.5 mm. 2 It is preferable that the distance be 13.5 mm or less.

[0133] (8-2) Optimal range In view of the above, it is preferable that in the test strip 1, the distance L0 from the downstream end of the development pad DP to the test area satisfies the following formula (3). 1[mm]≦L0≦13.5[mm]…(3)

[0134] By satisfying the above formula (3), the test strip 1 does not exhibit phenomena such as non-specific reactions or blank spots, has good backing, and allows high line intensity to be observed at the first test line TL1, the second test line TL2, etc. Furthermore, by having a configuration that satisfies the above formula (3), it is possible to shorten the line detection time compared to conventional test strips that do not have a development pad DP, while increasing the line intensity at the first test line TL1, the second test line TL2, etc.

[0135] In addition, the distance L0 from the downstream end of the development pad DP to the second test line TL2, which is the furthest from the end of the development pad DP, 2It is preferable that at least the following formula (4) is satisfied. L0 2 ≦ 13.5 [mm] … (4)

[0136] Furthermore, the distance L0 from the downstream end of the development pad DP to the control line CL that is the furthest from the end of the development pad DP 3 It is preferable to provide the sensor at a position 13.5 mm or less apart.

[0137] (9) Relationship between distance L1 and distance L2 of the deployment pad From the results shown in Figures 6, 8, 9 and 10, it was confirmed that by setting the distance L1 at which the spreading pad DP overlaps with the membrane M to 2 mm or more and 4 mm or less, and by setting the distance L2 at which the spreading pad DP does not overlap with either the conjugate pad CP or the membrane M to 0 mm or more and 2 mm or less, the intensity of each line can be increased even if the line detection time is set to 3 minutes, and further, no non-specific reactions or whiteout occurs, and good backbleed is achieved.

[0138] From the above, it was found that the optimal range of the distance L1 at which the expansion pad DP overlaps the membrane M is 2 mm or more and 4 mm or less, and the optimal range of the distance L2 of the expansion pad DP at which the expansion pad DP overlaps neither the conjugate pad CP nor the membrane M is 0 mm or more and 2 mm or less, and therefore it is preferable that the relationship between the distances L1 and L2 of the expansion pad DP satisfy the following formula (5). 1 ≦ L1 / L2 …(5)

[0139] (10) Relationship between distance L2 and distance L3 of the deployment pad From the results shown in Figures 6, 8, 9 and 10, it was confirmed that by setting the distance L2 of the spreading pad DP, where the spreading pad DP does not overlap either the conjugate pad CP or the membrane M, to 0 mm or more and 2 mm or less, and setting the distance L3 where the spreading pad DP overlaps with the conjugate pad CP to 3 mm or more and 6 mm or less, the intensity of each line can be increased even if the line detection time is set to 3 minutes, and further, no non-specific reactions or whiteout occurs, and good backbleed is achieved.

[0140] From the above, it was found that the optimal range of the distance L2 of the expansion pad DP, where the expansion pad DP does not overlap either the conjugate pad CP or the membrane M, is 0 mm or more and 2 mm or less, and the optimal range of the distance L3, where the expansion pad DP overlaps the conjugate pad CP, is 3 mm or more and 6 mm or less, so that it is preferable that the relationship between the distances L2 and L3 of the expansion pad DP satisfy the following formula (6). 1.5 ≦ L3 / L2 …(6)

[0141] (11) Sensitizers consisting of compounds having a phosphorylcholine group with a hydrophilic side chain (11-1) Verification test Next, the optimal amount of a compound having a phosphorylcholine group with a hydrophilic side chain is described as a sensitizer to be dried and retained on the spreading pad DP. For example, Lipidure (registered trademark)-BL103 (manufactured by NOF Corp.) is an example of a compound having a phosphorylcholine group with a hydrophilic side chain, and in this verification test, Lipidure (registered trademark)-BL103 (manufactured by NOF Corp.) was used as the sensitizer.

[0142] Here, we prepared several test strips with different amounts of sensitizer dried and retained on the development pad DP, and performed verification tests to check the A line intensity, B line intensity, C line intensity, development failure, and blank spots for each test strip. The line detection time was set to 3 minutes, shorter than the conventional 5 minutes. As a result, the results shown in Figure 11 were obtained.

[0143] The test strips of Examples 61 to 65 and Comparative Examples 61 and 62 were prepared using the sample pad SP, conjugate pad CP, spreading pad DP, membrane M, and absorbent pad AbP used in the above-mentioned "(4) Performance comparison when the configuration of the test kit is changed." The test strips of Examples 61 to 65 and Comparative Examples 61 and 62 had the same configuration, and only the amount of sensitizer used to be dried and retained in the spreading pad DP was changed.

[0144] The length of the sample pad SP in the longitudinal direction of the test strip was 14 [mm], and the length of the conjugate pad CP in the longitudinal direction of the test strip was 8 [mm]. The spreading pad DP had a length of 8 [mm] in the longitudinal direction of the test strip, a width of 4 [mm], a thickness of 0.43 [mm], and a volume of the spreading pad DP of 13.76 [mm]. 3 ](0.01376[cm 3 ]).

[0145] The distance L1 where the spreading pad DP overlaps with the membrane M was set to 4 [mm], and the distance L2 where the spreading pad DP does not overlap with either the conjugate pad CP or the membrane M was set to 1 [mm]. The distance L3 where the spreading pad DP overlaps with the conjugate pad CP was set to 3 [mm].

[0146] Distance L0 from the downstream end of the deployment pad DP to the first test line TL1 1 is set to 7 mm, and the distance L0 from the downstream end of the deployment pad DP to the second test line TL2 is set to 2 was set to 10.5 mm.

[0147] The spreading pad DP was designed to keep the sensitizer dry, and the amount of sensitizer kept dry in the spreading pad DP was changed within the range of 0 μg to 75 μg. In Figure 11, the amount of sensitizer kept dry in the spreading pad DP is expressed in μg, which is the theoretical value of the weight of the sensitizer after it is applied in solution and dried.

[0148] When evaluating the line intensity, the line intensity value of Comparative Example 61 (a test strip in which the sensitizer is not dried and retained on the development pad DP) in which the amount of the sensitizer used is 0 [μg] was determined as a reference value for each of the A line intensity, B line intensity, and C line intensity, and the ratio of the line intensity value of the other samples was calculated from this reference value. Then, based on the calculated ratio, the line intensity was evaluated according to the above Table 2. Specifically, for example, the result of measuring the line intensity of Comparative Example 61 with an immunochromatograph reader (C10066-10, Hamamatsu Photonics) was used as the reference value, and the line intensity values ​​of Examples 61 to 65 and Comparative Example 62 were measured with an immunochromatograph reader (C10066-10, Hamamatsu Photonics) to the reference value, and the ratios of the line intensity values ​​to the reference value were calculated. Then, based on the calculated ratio, the line intensity was evaluated according to the above Table 2. For example, when the line intensity was higher than the reference value obtained in Comparative Example 61 in which the amount of sensitizer used was 0 [μg], it was judged as "improvement effect present," and when the line intensity was lower than the reference value, it was judged as "no improvement effect." The occurrence of poor development and blank spots was confirmed by visual inspection.

[0149] In this verification test, a sample having a development pad DP that keeps the sensitizer dry, at least both the A-line strength and the B-line strength are "improved" and no development failure or whiteout occurs was determined as an "Example". A sample having an improvement effect on line strength but with unacceptable development failure or whiteout occurs was determined as a "Comparative Example". From FIG. 11, it was confirmed that when a compound having a phosphorylcholine group with a hydrophilic side chain is used as a sensitizer, the line strength of both the A-line strength and the B-line strength improves depending on the amount of sensitizer used. In Example 65, in which the amount of sensitizer used was 56 [μg], some development failure was confirmed at the end of the membrane M. However, this development failure was within an acceptable range. In Comparative Example 62, in which the amount of sensitizer used was 75 [μg], it was confirmed that unacceptable development failure occurred in the membrane M and whiteout also occurred.

[0150] (11-2) Optimal range From the above, when a compound having a phosphorylcholine group with a hydrophilic side chain is used as a sensitizer, the amount of the sensitizer used is preferably 5 [μg] or more and 56 [μg] or less. Since the amount of the sensitizer used here is a theoretical value, if the amount of the sensitizer used is calculated as a calculated value, the calculated value of 5 [μg] (0.025 [%]) is 4.6675 [μg] per test. The calculated value is calculated as follows. Since the amount of the sensitizer solution applied per test (the amount of the sensitizer solution applied to the development pad DP per test) is 18.67 [μL], if the weight (amount used) of the sensitizer is X [μg], then {(X [μg] / 1000) / 18.67 [μL]} × 100 = 0.025 [%]. Therefore, X = (0.025 / 100) × 18.67 [μL] × 1000 = 4.6675 [μg].

[0151] The volume of the deployment pad DP is 13.76 mm 3 ](0.01376[cm 3 ]), so if the concentration of sensitizer in the development pad DP when the sensitizer is 5 [μg] is expressed as a concentration of approximately 339 [μg / cm ] using the calculated value of 4.6675 [μg] above. 3 ]

[0152] On the other hand, since the amount of sensitizer used here is a theoretical value, if we calculate the amount of sensitizer used for this upper limit value as a calculated value, the calculated value of 56 [μg] (0.30 [%]) becomes 56.01 [μg] per test ((0.30 / 100) × 18.67 [μL] × 1000 = 56.01 [μg]).

[0153] The volume of the deployment pad DP is 13.76 mm 3 ](0.01376[cm 3 ]), so if the concentration of the sensitizer in the development pad DP is expressed as 56 [μg], using the calculated value of 56.01 [μg] above, it is approximately 4070 [μg / cm 3 ]

[0154] Therefore, the optimum range when a compound having a phosphorylcholine group with a hydrophilic side chain is used as a sensitizer, expressed as the concentration of the sensitizer in the spreading pad DP, preferably satisfies the following formula (7): Note that hereinafter, the sensitizer consisting of a compound having a phosphorylcholine group with a hydrophilic side chain is referred to as "sensitizer 1". 339 [μg / cm 3 ] ≦ Concentration of sensitizer 1 ≦ 4070 [μg / cm 3 ]…(7)

[0155] (12) Sensitizers consisting of compounds having a phosphorylcholine group with a hydrophobic side chain (12-1) Verification test Next, the optimal amount of a compound having a phosphorylcholine group with a hydrophobic side chain is described as a sensitizer to be dried and retained on the spreading pad DP. For example, Lipidure (registered trademark)-BL203 (manufactured by NOF Corp.) is an example of a compound having a phosphorylcholine group with a hydrophobic side chain, and in this verification test, Lipidure (registered trademark)-BL203 (manufactured by NOF Corp.) was used as the sensitizer.

[0156] Here, we prepared several test strips with different amounts of sensitizer dried and retained on the development pad DP, and performed verification tests to check the A line intensity, B line intensity, C line intensity, development failure, and whiteout occurrence for each test strip. The line detection time was set to 3 minutes, shorter than the conventional 5 minutes. As a result, the results shown in Figure 12 were obtained.

[0157] The test strips of Examples 71 to 76 and Comparative Example 71 were prepared using the sample pad SP, conjugate pad CP, spreading pad DP, membrane M, and absorbent pad AbP used in the above-mentioned "(4) Performance comparison when the configuration of the test kit was changed." The test strips of Examples 71 to 76 and Comparative Example 71 had the same configuration, and only the amount of sensitizer used to be kept dry in the spreading pad DP was changed.

[0158] The length of the sample pad SP in the longitudinal direction of the test strip was 14 [mm], and the length of the conjugate pad CP in the longitudinal direction of the test strip was 8 [mm]. The spreading pad DP had a length of 8 [mm] in the longitudinal direction of the test strip, a width of 4 [mm], a thickness of 0.43 [mm], and a volume of the spreading pad DP of 13.76 [mm]. 3 ](0.01376[cm 3 ]).

[0159] The distance L1 where the spreading pad DP overlaps with the membrane M is set to 4 [mm], and the distance L2 where the spreading pad DP does not overlap with either the conjugate pad CP or the membrane M is set to 1 [mm]. The distance L3 where the spreading pad DP overlaps with the conjugate pad CP is set to 3 [mm]. These values ​​are the same as those of the test strip sample used in the above-mentioned "(11) Regarding the sensitizer composed of a compound having a phosphorylcholine group with a hydrophilic side chain."

[0160] Distance L0 from the downstream end of the deployment pad DP to the first test line TL1 1 is set to 7 mm, and the distance L0 from the downstream end of the deployment pad DP to the second test line TL2 is set to 2 was set to 10.5 mm.

[0161] The spreading pad DP is configured to keep the sensitizer dry, and the amount of sensitizer kept dry in the spreading pad DP is changed within the range of 0 μg to 75 μg. In Figure 12, the amount of sensitizer kept dry in the spreading pad DP is expressed in μg, which is the theoretical value of the weight of the sensitizer after it is applied in solution and dried.

[0162] When evaluating the line intensity, the line intensity values ​​of the A line intensity, B line intensity, and C line intensity of each sample were measured using an immunochromatography reader (C10066-10, Hamamatsu Photonics). For each of the A line intensity, B line intensity, and C line intensity, the line intensity value in Comparative Example 71 (a test strip in which the sensitizer is not dried and retained on the development pad DP) in which the amount of sensitizer used is 0 [μg] was determined as a reference value. Next, for each of Examples 71 to 76, the ratio of the line intensity value to this reference value was calculated. Then, the line intensity was evaluated based on the obtained ratio (evaluation of "improvement effect" or "no improvement effect") based on Table 2 in the same manner as in the above-mentioned verification test. The occurrence of poor development and blank spots was confirmed by visual inspection.

[0163] In this verification test, the samples having a development pad DP that keeps the sensitizer dry, at least both the A line intensity and the B line intensity "has an improvement effect", and no development failure or whiteout occurred were designated as "Examples". From FIG. 12, it was confirmed that when a compound having a phosphorylcholine group with a hydrophobic side chain was used as a sensitizer, the line intensity of both the A line intensity and the B line intensity improved depending on the amount of the sensitizer used. In addition, in Examples 74 to 76 in which the amount of the sensitizer used was 37 [μg] or more, it was confirmed that the line intensity of the C line intensity, which is the control line CL, was also improved. It was also confirmed that in Example 76 in which the amount of the sensitizer used was 75 [μg], no development failure or whiteout occurred, and all of the line intensities of the A line intensity, the B line intensity, and the C line intensity were improved.

[0164] (12-2) Optimal range From the above, when a compound having a phosphorylcholine group with a hydrophobic side chain is used as a sensitizer, the amount of the sensitizer used is preferably 5 [μg] or more and 75 [μg] or less. As described above, the calculated amount of the sensitizer used at the lower limit is 4.6675 [μg], and the concentration of the sensitizer in the development pad DP when the sensitizer is 5 [μg] is approximately 339 [μg / cm 3 ]

[0165] On the other hand, since the amount of sensitizer used here is a theoretical value, if we calculate the amount of sensitizer used for this upper limit value as a calculated value, the calculated value of 75 [μg] (0.40 [%]) becomes 74.68 [μg] per test ((0.40 / 100) × 18.67 [μL] × 1000 = 74.68 [μg]).

[0166] The volume of the deployment pad DP is 13.76 mm 3 ](0.01376[cm 3 ]), so if the concentration of the sensitizer in the development pad DP when the sensitizer is 75 [μg] is expressed as a concentration of approximately 5427 [μg / cm ] using the calculated value of 74.68 [μg] above. 3 ]

[0167] Therefore, the optimum range when a compound having a phosphorylcholine group with a hydrophobic side chain is used as a sensitizer, expressed as the concentration of the sensitizer in the spreading pad DP, preferably satisfies the following formula (8): Hereinafter, the sensitizer made of a compound having a phosphorylcholine group with a hydrophobic side chain is referred to as "sensitizer 2". 339 [μg / cm 3 ] ≦ Concentration of sensitizer 2 ≦ 5427 [μg / cm 3 ]…(8)

[0168] (13) Sensitizers consisting of compounds having a phosphorylcholine group with an anionic side chain (13-1) Verification test Next, the optimal amount of a compound having a phosphorylcholine group with an anionic side chain is described as a sensitizer to be dried and retained on the spreading pad DP. For example, Lipidure (registered trademark)-BL405 (manufactured by NOF Corp.) is an example of a compound having a phosphorylcholine group with an anionic side chain, and in this verification test, Lipidure (registered trademark)-BL405 (manufactured by NOF Corp.) was used as the sensitizer.

[0169] Here, we prepared several test strips with different amounts of sensitizer dried and retained on the development pad DP, and performed verification tests to check the A line intensity, B line intensity, C line intensity, development failure, and whiteout occurrence for each test strip. The line detection time was set to 3 minutes, shorter than the conventional 5 minutes. As a result, the results shown in Figure 13 were obtained.

[0170] The test strips of Examples 81 to 86 and Comparative Examples 81 to 83 were prepared using the sample pad SP, conjugate pad CP, spreading pad DP, membrane M, and absorbent pad AbP used in the above-mentioned "(4) Performance comparison when the configuration of the test kit was changed." The test strips of Examples 81 to 86 and Comparative Examples 81 to 83 had the same configuration, and only the amount of sensitizer used to be dried and retained in the spreading pad DP was changed.

[0171] The length of the sample pad SP in the longitudinal direction of the test strip was 14 [mm], and the length of the conjugate pad CP in the longitudinal direction of the test strip was 8 [mm]. The spreading pad DP had a length of 8 [mm] in the longitudinal direction of the test strip, a width of 4 [mm], a thickness of 0.43 [mm], and a volume of the spreading pad DP of 13.76 [mm]. 3 ](0.01376[cm 3 ]).

[0172] The distance L1 where the spreading pad DP overlaps with the membrane M was set to 4 [mm], and the distance L2 where the spreading pad DP does not overlap with either the conjugate pad CP or the membrane M was set to 1 [mm]. The distance L3 where the spreading pad DP overlaps with the conjugate pad CP was set to 3 [mm].

[0173] Distance L0 from the downstream end of the deployment pad DP to the first test line TL1 1 is set to 7 mm, and the distance L0 from the downstream end of the deployment pad DP to the second test line TL2 is set to 2These values ​​are the same as those of the test strip sample used in the above "(11) Sensitizers composed of compounds having phosphorylcholine groups with hydrophilic side chains."

[0174] The spreading pad DP was designed to dry and retain the sensitizer, and the amount of sensitizer used to dry and retain it in the spreading pad DP was changed within the range of 0 μg to 75 μg. In Figure 13, the amount of sensitizer to dry and retain in the spreading pad DP is expressed in μg, which is the theoretical value of the weight of the sensitizer after it is applied in solution and dried.

[0175] When evaluating the line intensity, the line intensity values ​​of the A line intensity, B line intensity, and C line intensity of each sample were measured using an immunochromatography reader (C10066-10, Hamamatsu Photonics). For each of the A line intensity, B line intensity, and C line intensity, the line intensity value in Comparative Example 81 (a test strip in which the sensitizer is not dried and retained on the development pad DP) in which the amount of sensitizer used is 0 [μg] was determined as a reference value, and the ratio of the line intensity value to this reference value was calculated for each of Examples 81 to 86 and Comparative Examples 82 and 83. Then, the line intensity was evaluated based on the obtained ratio (evaluation of "improvement effect" or "no improvement effect") based on Table 2 in the same manner as in the above-mentioned verification test. The occurrence of poor development and blank spots was confirmed by visual inspection.

[0176] In this verification test, a sample having a development pad DP that keeps a sensitizer dry, in which at least the A-line strength among the A-line strength, B-line strength, and C-line strength is "improved" and no development failure or whiteout occurs was determined as an "Example". A sample having an unacceptable development failure or whiteout occurs even if there is an improvement effect of the line strength was determined as a "Comparative Example". From FIG. 13, it was confirmed that when a compound having a phosphorylcholine group with an anionic side chain is used as a sensitizer (when the amount of sensitizer used is 1 [μg] or more), the A-line strength improves depending on the amount of sensitizer used. It was confirmed that the B-line strength and C-line strength improve when the amount of sensitizer used is 5 [μg] or more.

[0177] In Example 86, in which the amount of sensitizer used was 37 [μg], some poor development was observed at the edge of the membrane M, and some white spots were also observed. However, these poor development and white spots were within the acceptable range. It was confirmed that when the amount of sensitizer used was 56 [μg] or more, unacceptable poor development occurred in the membrane M, and unacceptable white spots also occurred.

[0178] (13-2) Optimal range From the above, when a compound having a phosphorylcholine group with an anionic side chain is used as a sensitizer, the amount of sensitizer used is preferably 1 [μg] or more and 37 [μg] or less in consideration of the improvement of the line intensity of the A line intensity. Since the amount of sensitizer used here is a theoretical value, if the amount of sensitizer used for this lower limit is calculated as a calculated value, the calculated value of 1 [μg] (0.00625 [%]) is 1.1669 [μg] per test ((0.00625 / 100) × 18.67 [μL] × 1000 = 1.1669 [μg]).

[0179] The volume of the deployment pad DP is 13.76 mm 3 ](0.01376[cm 3 ]), so if the concentration of sensitizer in the development pad DP is expressed as 1 [μg], using the calculated value of 1.1669 [μg] above, it is approximately 84 [μg / cm 3 ]

[0180] On the other hand, since the amount of sensitizer used here is a theoretical value, if we calculate the amount of sensitizer used for this upper limit value as a calculated value, the calculated value of 37 [μg] (0.20 [%]) becomes 37.34 [μg] per test ((0.20 / 100) × 18.67 [μL] × 1000 = 37.34 [μg]).

[0181] The volume of the deployment pad DP is 13.76 mm 3 ](0.01376[cm 3 ]), so if the concentration of the sensitizer in the development pad DP is expressed as 37 [μg], using the calculated value of 37.34 [μg] above, it is approximately 2714 [μg / cm 3 ]

[0182] Therefore, the optimum range when a compound having a phosphorylcholine group with an anionic side chain is used as a sensitizer, expressed as the concentration of the sensitizer in the spreading pad DP, preferably satisfies the following formula (9): Hereinafter, the sensitizer made of a compound having a phosphorylcholine group with a hydrophobic side chain is referred to as "sensitizer 3". 84 [μg / cm 3 ] ≦ Concentration of sensitizer 3 ≦ 2714 [μg / cm 3 ] …(9)

[0183] In addition, when a compound having a phosphorylcholine group with an anionic side chain is used as a sensitizer, the amount of sensitizer used is preferably 5 μg or more and 37 μg or less, taking into consideration the improvement of the line intensity of the B line intensity and the C line intensity. As described above, the calculated amount of sensitizer used at the lower limit is 4.6675 μg, and the concentration of the sensitizer in the development pad DP when the sensitizer is 5 μg is approximately 339 μg / cm. 3 ]

[0184] Therefore, when a compound having a phosphorylcholine group with an anionic side chain is used as a sensitizer, the optimal range taking into consideration the line intensity improvement effect of A line intensity, B line intensity, and C line intensity, expressed as the concentration of the sensitizer in the development pad DP, preferably satisfies the following formula (10). 339 [μg / cm 3 ] ≦ Concentration of sensitizer 3 ≦ 2714 [μg / cm 3 ]…(10)

[0185] (14) Other sensitizers In the above-mentioned verification test, a compound having a phosphorylcholine group with a hydrophilic side chain, a hydrophobic side chain or an anionic side chain was used as a sensitizer, and the A-line intensity, B-line intensity, C-line intensity, the presence or absence of development defects, and the occurrence or absence of white spots were confirmed.

[0186] Here, a compound having a phosphorylcholine group with a cationic side chain or a hydrogen-bonding side chain was used as a sensitizer, and a verification test was performed to confirm the A-line intensity, B-line intensity, the presence or absence of development failure, the presence or absence of white spots, and the presence or absence of non-specific reactions. Also, a compound having a phosphorylcholine group with a hydrophobic side chain other than Lipidure (registered trademark)-BL203 (manufactured by NOF Corporation) was used as a sensitizer, and a verification test was performed to confirm the A-line intensity, B-line intensity, the presence or absence of development failure, the presence or absence of white spots, and the presence or absence of non-specific reactions.

[0187] An example of a compound having a phosphorylcholine group with a cationic side chain is Lipidure (registered trademark)-BL502 (manufactured by NOF Corporation), and in this verification test, Lipidure (registered trademark)-BL502 (manufactured by NOF Corporation) was used as a sensitizer. In addition, an example of a compound having a phosphorylcholine group with a hydrogen-bonding side chain is Lipidure (registered trademark)-BL702 (manufactured by NOF Corporation), and in this verification test, Lipidure (registered trademark)-BL702 (manufactured by NOF Corporation) was used as a sensitizer. In addition, an example of a compound having a phosphorylcholine group with a hydrophobic side chain is Lipidure (registered trademark)-BL1201 (manufactured by NOF Corporation) and Lipidure (registered trademark)-BL1301 (manufactured by NOF Corporation), and in this verification test, Lipidure (registered trademark)-BL1201 (manufactured by NOF Corporation) and Lipidure (registered trademark)-BL1301 (manufactured by NOF Corporation) were used as sensitizers.

[0188] Here, the amount of sensitizer used to be dried and retained on the development pad DP was set to 21 [μg], and test strips were prepared in which each of the above-mentioned sensitizers was dried and retained on the development pad DP, and verification tests were conducted to check the A line intensity, B line intensity, the occurrence of blank spots, the occurrence of non-specific reactions, and the occurrence of development failure for each test strip. The line detection time was set to 3 minutes, which is shorter than the conventional 5 minutes.

[0189] The test strips of Examples 121 to 124 and Comparative Example 121 were prepared using the sample pad SP, conjugate pad CP, spreading pad DP, membrane M, and absorbent pad AbP used in the above-mentioned "(4) Performance comparison when the configuration of the test kit was changed." The test strips of Examples 121 to 124 and Comparative Example 121 had the same configuration, and only the amount of sensitizer used to be dried and retained in the spreading pad DP was changed.

[0190] The length of the sample pad SP in the longitudinal direction of the test strip was 14 [mm], and the length of the conjugate pad CP in the longitudinal direction of the test strip was 8 [mm]. The spreading pad DP had a length of 8 [mm] in the longitudinal direction of the test strip, a width of 4 [mm], a thickness of 0.43 [mm], and a volume of the spreading pad DP of 13.76 [mm]. 3 ](0.01376[cm 3 ]).

[0191] The distance L1 where the spreading pad DP overlaps with the membrane M is set to 4 [mm], and the distance L2 where the spreading pad DP does not overlap with either the conjugate pad CP or the membrane M is set to 1 [mm]. The distance L3 where the spreading pad DP overlaps with the conjugate pad CP is set to 3 [mm]. These values ​​are the same as those of the test strip sample used in the above-mentioned "(11) Regarding the sensitizer composed of a compound having a phosphorylcholine group with a hydrophilic side chain."

[0192] Distance L0 from the downstream end of the deployment pad DP to the first test line TL1 1 is set to 7 mm, and the distance L0 from the downstream end of the deployment pad DP to the second test line TL2 is set to 2 was set to 10.5 mm.

[0193] In Examples 121 to 124, the sensitizer was dried and retained in the development pad DP, and the amount of sensitizer dried and retained in the development pad DP was 21 [μg] as described above. In Fig. 14, the amount of sensitizer dried and retained in the development pad DP is expressed in "μg", which is the theoretical value of the weight of the sensitizer after the sensitizer is applied in a solution state and dried.

[0194] When evaluating the line intensity, the line intensity values ​​of the A line intensity and the B line intensity of each sample were measured using an immunochromatography reader (C10066-10, Hamamatsu Photonics). For each of the A line intensity and the B line intensity, the line intensity value of Comparative Example 121 (a test strip in which the sensitizer is not dried and retained on the development pad DP) in which the amount of the sensitizer used is 0 [μg] was determined as a reference value, and the ratio of the line intensity values ​​of the other samples was calculated from this reference value. Then, based on the calculated ratio, the line intensity was evaluated according to Table 2 above. The occurrence of poor development, non-specific reaction, and white spots was visually confirmed.

[0195] In this verification test, the samples having the spreading pad DP that keeps the sensitizer dry, both the A-line strength and the B-line strength "improved", and no spreading failure, non-specific reaction, or whiteout occurred were designated as "Examples". From FIG. 14, it was confirmed that Example 121, in which a compound having a phosphorylcholine group with a cationic side chain was used as a sensitizer, Example 122, in which a compound having a phosphorylcholine group with a hydrogen-bonding side chain was used as a sensitizer, and Examples 123 and 124, in which a compound having a phosphorylcholine group with a hydrophobic side chain was used as a sensitizer, had improved line strengths for both the A-line strength and the B-line strength compared to Comparative Example 121. Moreover, in Examples 121 to 124, no spreading failure occurred, and no non-specific reaction or whiteout occurred.

[0196] From the above, it was confirmed that even if a compound having a phosphorylcholine group with a cationic side chain, a compound having a phosphorylcholine group with a hydrogen-bonding side chain, or a compound having a phosphorylcholine group with various hydrophobic side chains is used as a sensitizer to be dried and retained on the developing pad DP, the time from when the specimen extract is applied to the sample pad SP until the reaction result appears (a line appears) on the first test line TL1, etc. can be shortened compared to the conventional method, as with a compound having a phosphorylcholine group with a hydrophilic side chain. It was also confirmed that even if a compound having a phosphorylcholine group with a cationic side chain or a hydrogen-bonding side chain is used as a sensitizer, the line intensity on the first test line TL1, etc. can be improved. Therefore, it was confirmed that a compound having a phosphorylcholine group with a cationic side chain, a compound having a phosphorylcholine group with a hydrogen-bonding side chain, or a compound having a phosphorylcholine group with various hydrophobic side chains can be used as a sensitizer to be dried and retained on the developing pad DP, as with a compound having a phosphorylcholine group with a hydrophilic side chain.

[0197] (15) Actions and Effects In the above configuration, the test kit 100 has a test strip 1 including a sample pad SP, a conjugate pad CP, a spreading pad DP, and a membrane M, which is placed in a case 101. A specimen extract containing a specimen is applied to the sample pad SP. The conjugate pad CP holds a labeling substance that reacts specifically with the specimen developed from the sample pad SP by capillary action to form a complex in a wet and releasable state. The membrane M is provided with a first test line TL1, a second test line TL2, and a control line CL as detection regions, on which a capture substance that reacts specifically with the complex developed from the upstream spreading pad DP by capillary action is immobilized.

[0198] In addition, the developing pad DP holds a sensitizer that can be contained in the sample extract developed from the upstream conjugate pad CP by capillary action. The developing pad DP holds a compound having a phosphorylcholine group in a dry state as the sensitizer.

[0199] In this way, by providing the development pad DP in which a compound having a phosphorylcholine group is held in a dry state as a sensitizer, the test strip 1 can shorten the time from when the specimen extract is applied to the sample pad SP to when the reaction result appears (a line appears) on the first test line TL1, etc., compared to the conventional method.

[0200] In addition, the amount of the sensitizer, which is a compound having a phosphorylcholine group and is dried and retained on the development pad DP, is preferably 1 μg or more and 75 μg or less, so that the test strip 1 does not suffer from poor development or blank spots, and can improve the line intensity of at least the first test line TL1 even if the line detection time is shortened from the conventional line detection time of 5 minutes to 3 minutes.

[0201] Because the amount of sensitizer used here is a theoretical value, the lower and upper limits of the amount of sensitizer used can be calculated to be between 1.1669 μg ((0.00625 / 100) × 18.67 μL × 1000 = 1.1669 μg) and 74.68 μg ((0.40 / 100) × 18.67 μL × 1000 = 74.68 μg) per test.

[0202] Therefore, the optimum range of the sensitizer is expressed as the concentration of the sensitizer in the development pad DP based on the lower and upper limits calculated as the amount of the sensitizer used, as given by the following formula (11). 84 [μg / cm 3 ] ≦ Sensitizer concentration ≦ 5427[μg / cm 3 ]…(11)

[0203] As described above, in the test strip 1, the concentration of the sensitizer in the development pad DP is adjusted to satisfy the above formula (11), and a configuration can be realized that prevents development failures and blank spots that may cause erroneous determination of line appearance in the first test line TL1, the second test line TL2, and the control line CL. As a result, in the test strip 1, while preventing development failures and blank spots, it is possible to improve the line intensity in the first test line TL1, the second test line TL2, and the control line CL even if the line detection time is shortened compared to the conventional case.

[0204] <Second embodiment> (1) Regarding test strips having a laminated portion in which the spreading pad overlaps both the conjugate pad and the membrane In the above-described first embodiment, the test strip 1 in which the development pad DP does not overlap both the conjugate pad CP and the membrane M has been described, but the present invention is not limited thereto. For example, as shown in 1011 in Fig. 15, another test strip 1B may be configured to include a laminated portion 3f in which the development pad DP overlaps both the conjugate pad CP and the membrane M.

[0205] In this case, in the test strip 1B, the development pad DP does not overlap either the conjugate pad CP or the membrane M, and the distance L2 of the development pad DP is 0 [mm]. When the distance at which the development pad DP overlaps both the conjugate pad CP and the membrane M in the longitudinal direction of the test strip 1B is L4, it is preferable that the distance L4 satisfies the following formula (12). 0[mm]< L4 ≦ 2[mm] …(12)

[0206] By satisfying the above formula (12), the development pad DP does not exhibit phenomena such as non-specific reactions or white spots, has good backing, and allows high line intensity to be observed at least in the first test line TL1 and the second test line TL2.

[0207] Furthermore, even if an expansion pad DP is provided so as to satisfy the above formula (12), as in the first embodiment described above, the line detection time can be shortened compared to conventional test strips that do not have an expansion pad DP, while the line intensity of at least the first test line TL1 can be increased more than in the conventional case.

[0208] Furthermore, as in the first embodiment described above, the test strip 1B also has a development pad DP in which a compound having a phosphorylcholine group is held in a dry state as a sensitizer, so that the time from when a specimen extract is applied to the sample pad SP until the reaction results appear on the first test line TL1 and the second test line TL2 can be shortened more than in the conventional method.

[0209] (2) Verification Test Next, a verification test in which the distance L4 of the spreading pad DP in the longitudinal direction of the test strip 1B was compared will be described. Here, a test strip without a spreading pad DP, a test strip 1 (FIG. 4) in which the distance L4 of the spreading pad DP in the longitudinal direction of the test strip is 0 [mm] (i.e., a configuration in which the spreading pad DP does not have a laminated portion 3f in which the spreading pad DP overlaps both the conjugate pad CP and the membrane M) and the distance L2 of the spreading pad DP is 1 [mm], and a test strip 1B in which the distance L4 of the spreading pad DP in the longitudinal direction of the test strip is 2 [mm] and the distance L2 of the spreading pad DP is 0 [mm] were prepared, and a verification test was performed to check the A line intensity, B line intensity, C line intensity, the occurrence of non-specific reactions, the occurrence of blank spots, and the quality of backing for each test strip 1 and 1B. As a result, the results shown in FIG. 16 were obtained.

[0210] The test strips of Examples 91, 92 and Comparative Example 91 were prepared using the sample pad SP, conjugate pad CP, spreading pad DP, membrane M and absorbent pad AbP used in the above-mentioned "(4) Performance comparison when the configuration of the test kit is changed."

[0211] The length of the sample pad SP in the longitudinal direction of the test strip was 14 [mm], and the length of the conjugate pad CP in the longitudinal direction of the test strip was 8 [mm]. The spreading pad DP had a length of 8 [mm] in the longitudinal direction of the test strip, a width of 4 [mm], a thickness of 0.43 [mm], and a volume of the spreading pad DP of 13.76 [mm]. 3 ](0.01376[cm 3 ]).

[0212] The overlapping distance between the sample pad SP and the conjugate pad CP was 2 [mm]. In Example 91, the overlapping distance L1 between the spreading pad DP and the membrane M was 4 [mm], and in Example 92, the overlapping distance L1 was 2 [mm]. In Example 92, the overlapping distance L4 between the spreading pad DP and both the conjugate pad CP and the membrane M was 2 [mm], and the overlapping distance L3 between the spreading pad DP and only the conjugate pad CP was 4 [mm]. That is, in Example 92, the overlapping length between the spreading pad DP and the conjugate pad CP (distance L3 + distance L4) was 6 [mm].

[0213] Distance L0 from the downstream end of the deployment pad DP to the first test line TL1 1 is set to 7 mm, and the distance L0 from the downstream end of the deployment pad DP to the second test line TL2 is set to 2 The distance L0 from the downstream end of the deployment pad DP to the control line CL was set to 10.5 mm. 3 was set to 14 mm.

[0214] The spreading pad DP was configured to dry and retain Lipidure (registered trademark)-BL103 (manufactured by NOF Corporation) as a sensitizer, and the amount of sensitizer used to dry and retain in the spreading pad DP was 21 [μg]. In FIG. 16, the amount of sensitizer to dry and retain in the spreading pad DP is expressed in "μg", which is the theoretical value of the weight of the sensitizer after the sensitizer is applied in a solution state and dried. The line detection time of Comparative Example 91 was 5 minutes, the same as the conventional one, and the line detection time of Examples 91 and 92 was 3 minutes, which is shorter than the conventional 5 minutes.

[0215] When evaluating line intensity, the line intensity values ​​of the A line intensity, B line intensity, and C line intensity of each sample were measured using an immunochromatography reader (C10066-10, Hamamatsu Photonics). As a result, the A line intensity, B line intensity, and C line intensity of Comparative Example 91, which had a line detection time of 5 minutes and used a conventional test strip, were higher than those of Examples 91 and 92. For this reason, the line intensity values ​​of the A line intensity, B line intensity, and C line intensity of Comparative Example 91 were determined as the reference values ​​of the A line intensity, B line intensity, and C line intensity. Then, the ratios of the line intensity values ​​of Examples 91 and 92 to the respective reference values ​​were calculated for each of the A line intensity, B line intensity, and C line intensity. From the obtained ratios, the line intensity was evaluated based on Table 1 in the same manner as in the above-mentioned verification test. The occurrence or non-occurrence of non-specific reactions, the occurrence or non-occurrence of white spots, and the quality of back spots were visually confirmed.

[0216] In this verification test, the sample having the developing pad DP that holds the sensitizer in a dried state, in which at least both the A line intensity and the B line intensity are observed with high line intensity, and in which non-specific reactions and white spots do not occur and the backing is also good was designated as the "Example". From FIG. 16, it was confirmed that in Example 92 in which the developing pad DP overlaps both the conjugate pad CP and the membrane M, high line intensities can be observed for both the A line intensity and the B line intensity, as in Example 91. In addition, in Example 92, as in Example 91, it was confirmed that high line intensity can be observed even when the line detection time is set to 3 minutes, which is shorter than the conventional 5 minutes, and furthermore, non-specific reactions and white spots do not occur and the backing is also good.

[0217] (3) Regarding test strips in which the spreading pad overlaps both the sample pad and the conjugate pad In the above-mentioned second embodiment, the test strip 1B in which the development pad DP does not overlap both the sample pad SP and the conjugate pad CP has been described, but the present invention is not limited thereto. As another embodiment, for example, as shown in 1012 of Fig. 15, the development pad DP may have a laminated portion 3f in which the development pad DP overlaps both the conjugate pad CP and the membrane M, and the development pad DP may have a laminated portion 3g in which the development pad DP overlaps both the sample pad SP and the conjugate pad CP.

[0218] In this type of test strip 1C, as in the first embodiment described above, a development pad DP is provided in which a compound having a phosphorylcholine group is held in a dry state as a sensitizer, so that the time from when a specimen extract is applied to the sample pad SP to when a reaction result is expressed at least at the first test line TL1 can be shortened more than in the conventional method.

[0219] <Third embodiment> (1) Dipstick-type test strips In the above-mentioned first and second embodiments, the test kit 100 in which the test strips 1, 1B, and 1C are placed in the case 101 has been described, but the present invention is not limited to this. For example, the test strip may be a dipstick-type test strip in which the tip of the sample pad SP is dipped in a specimen extract in a container without being placed in the case 101, and the specimen extract is sucked up from the sample pad SP.

[0220] In this test strip, similar to the first and second embodiments described above, a configuration can be realized in which a sample pad SP, a conjugate pad CP, a spreading pad DP, and a membrane M are provided, and a compound having a phosphorylcholine group is held in a dry state in the spreading pad DP as a sensitizer. As a result, in the dipstick type test strip, similar to the first and second embodiments, the time from when the specimen extract is applied to the sample pad SP until the reaction result appears at least at the first test line TL1 can be shortened compared to the conventional method.

[0221] (2) Verification Test Next, a verification test will be described in which a dipstick-type test strip is provided with a development pad DP to compare performance. Here, a dipstick-type test strip without a development pad DP and a dipstick-type test strip with a development pad DP that holds a dried sensitizer were prepared, and a verification test was carried out to check the A line intensity, B line intensity, C line intensity, the occurrence of non-specific reactions, the occurrence of white spots, and the quality of backing spots for each test strip. As a result, the results shown in Figure 17 were obtained. Comparative Examples 101 and 102 have the same configuration and are the same as those of the existing product "StatMark TM "FLU Stick II" (Kainos Corporation) was used as a sample. In Comparative Example 101, the line detection time was set to the conventional 5 minutes, and in Comparative Example 102, the line detection time was set to 3 minutes, which is shorter than the conventional 5 minutes. In Example 101, the line detection time was set to 3 minutes, which is shorter than the conventional 5 minutes.

[0222] The test strips of Example 101 and Comparative Examples 101 and 102 were prepared using the sample pad SP, conjugate pad CP, spreading pad DP, membrane M, and absorbent pad AbP used in the above-mentioned "(4) Performance comparison when the configuration of the test kit is changed." The only difference between Example 101 and Comparative Examples 101 and 102 is whether or not they have a spreading pad DP that holds the sensitizer in a dried state, and the other configurations were the same.

[0223] In Example 101, the sample pad SP, conjugate pad CP, spreading pad DP, membrane M and absorbent pad AbP were arranged as shown in Figures 3 and 4. In Example 101, the length of the sample pad SP in the longitudinal direction of the test strip was 16 [mm]. The length of the conjugate pad CP in the longitudinal direction of the test strip was 8 [mm]. The spreading pad DP had a length in the longitudinal direction of the test strip of 8 [mm], a width of 4 [mm] and a thickness of 0.43 [mm], and a volume of the spreading pad DP of 13.76 [mm]. 3 ](0.01376[cm 3 ]).

[0224] The overlapping distance between the sample pad SP and the conjugate pad CP was 2 [mm]. In Example 101, the overlapping distance L1 between the spreading pad DP and the membrane M was 2 [mm]. In Example 101, the overlapping distance L2 between the spreading pad DP, which does not overlap either the conjugate pad CP or the membrane M, was 2 [mm], and the overlapping distance L3 between the spreading pad DP and the conjugate pad CP was 4 [mm].

[0225] Distance L0 from the downstream end of the deployment pad DP to the first test line TL1 1 is 9 [mm], and the distance L0 from the downstream end of the deployment pad DP to the second test line TL2 is 2 The distance L0 from the downstream end of the deployment pad DP to the control line CL was set to 12.5 mm. 3 was set to 16 mm.

[0226] The spreading pad DP contained 0.11 [wt%] Lipidure (registered trademark)-BL103 (manufactured by NOF Corporation) as a sensitizer and was dried and maintained. The specimen extract was 400 [μl] per test, and the tip of the sample pad SP of each test strip was immersed in the specimen extract in the container with the test strip standing upright, and the A-line intensity, etc. were confirmed after the line detection time had elapsed.

[0227] When evaluating line intensity, the line intensity values ​​of the A line intensity, B line intensity, and C line intensity of each sample were measured using an immunochromatography reader (C10066-10, Hamamatsu Photonics). As a result, the A line intensity, B line intensity, and C line intensity of Comparative Example 101, an existing product with a line detection time of 5 minutes, were higher than those of Comparative Example 102 and Example 101. For this reason, the line intensity values ​​of the A line intensity, B line intensity, and C line intensity of Comparative Example 101 were determined as the reference values ​​of the A line intensity, B line intensity, and C line intensity. Then, the ratios of the line intensity values ​​of Comparative Example 102 and Example 101 to the respective reference values ​​were calculated for each of the A line intensity, B line intensity, and C line intensity. From the obtained ratios, the line intensity was evaluated based on Table 1 in the same manner as in the above-mentioned verification test. The occurrence or non-occurrence of non-specific reactions, the occurrence or non-occurrence of white spots, and the quality of back spots were visually confirmed.

[0228] From Figure 17, it was confirmed that in Example 101, as in Comparative Example 101 of an existing product, even if the line detection time was shortened from the conventional 5 minutes to 3 minutes, high line intensity could be observed for all of the A line intensity, B line intensity and C line intensity, and further, no non-specific reaction or whiteout occurred, and good backing was also achieved.

[0229] <Other embodiments> In the above-described embodiment, a first test line TL1, a second test line TL2, and a control line CL are provided as detection areas in which a capture substance that specifically reacts with the complex developed from the development pad is immobilized. However, the present invention is not limited to this, and for example, only the first test line TL1 may be provided, or the first test line TL1, the second test line TL2, the third test line TL3, and a control line CL may be provided, or one or more detection areas may be provided.

[0230] In the above-described embodiment, an immunochromatographic test strip utilizing an antigen-antibody reaction is described, in which an antigen is used as the specimen, a labeled antibody bound to colored particles is used as the labeling substance, and an antigen capture antibody and a labeled capture antibody are used as the capture substance. However, the present invention is not limited to this, and the present invention may be a chromatographic test strip utilizing, for example, a complementary nucleic acid reaction or a ligand-receptor reaction by using various other specimens, labeling substances, and capture substances. [Explanation of symbols]

[0231] 1, 1B, 1C Test Strips 101 cases AbP Absorption Pad SP Sample Pad CP Conjugate Pad DP Expanding Pad M Membrane TL1 First test line (detection area) TL2 Second test line (detection area) CL Control line (detection area)

Claims

1. A test kit having a test strip disposed within a case, The test strip comprises: a sample pad to which a specimen extract containing a specimen is applied; a conjugate pad on which a labeling substance that reacts specifically with the specimen developed from the sample pad by capillary action to form a complex is retained in a wet, releasable manner; a developing pad holding a sensitizer that can be contained in the specimen extract developed from the conjugate pad by capillary action; a membrane having a detection region in which a capture substance that specifically reacts with the complex developed from the developing pad by capillary action is immobilized; Equipped with A test kit, wherein the spreading pad holds a compound having a phosphorylcholine group in a dry state as the sensitizer.

2. In the longitudinal direction of the test strip, the spreading pad does not overlap either the conjugate pad or the membrane. In this case, the distance of the spreading pad is L2. 0 [mm] ≦ L2 ≦ 2 [mm] The test kit of claim 1 .

3. The distance by which the spreading pad overlaps the membrane in the longitudinal direction of the test strip is L1, In the longitudinal direction of the test strip, the spreading pad does not overlap either the conjugate pad or the membrane. In this case, the distance of the spreading pad is L2. 1≦L1 / L2 The test kit of claim 1 .

4. A distance of the spreading pad in the longitudinal direction of the test strip where the spreading pad does not overlap either the conjugate pad or the membrane is defined as L2; When the distance by which the spreading pad overlaps the conjugate pad in the longitudinal direction of the test strip is L3, 1.5≦L3 / L2 The test kit of claim 1 .

5. In the longitudinal direction of the test strip, the spreading pad overlaps both the conjugate pad and the membrane. The test kit of claim 1.

6. When the distance by which the spreading pad overlaps both the conjugate pad and the membrane in the longitudinal direction of the test strip is L4, 0 [mm] < L4 ≦ 2 [mm] The test kit according to claim 5 .

7. The distance by which the spreading pad overlaps the membrane in the longitudinal direction of the test strip is L1, A distance of the spreading pad in the longitudinal direction of the test strip where the spreading pad does not overlap either the conjugate pad or the membrane is defined as L2; When the distance by which the spreading pad overlaps the conjugate pad in the longitudinal direction of the test strip is L3, 4 [mm]≦L1+L2+L3≦10[mm] The test kit of claim 1 .

8. When the distance from the downstream end of the development pad to the detection area in the longitudinal direction of the test strip is L0, 1 [mm] ≦ L0 ≦ 13.5 [mm] The test kit of claim 1 .

9. the membrane has a plurality of detection regions; The plurality of detection regions have different types of capture substances immobilized thereon, The distance from the downstream end of the development pad to the detection area farthest downstream from the end of the development pad in the longitudinal direction of the test strip is defined as L0. ｎ When L0 ｎ ≦ 13.5[mm] The test kit of claim 1 .

10. The compound having a phosphorylcholine group has at least one side chain selected from the group consisting of a hydrophilic side chain, a hydrophobic side chain, an anionic side chain, a cationic side chain, and a hydrogen-bonding side chain. The test kit of claim 1.

11. The spreading pad has a concentration of the sensitizer of 84 [μg / cm ３ ] or more, 5427 [μg / cm ３ ] or less.

12. a sample pad to which a specimen extract containing a specimen is applied; a conjugate pad on which a labeling substance that reacts specifically with the specimen developed from the sample pad by capillary action to form a complex is retained in a wet, releasable manner; a developing pad holding a sensitizer that can be contained in the specimen extract developed from the conjugate pad by capillary action; a membrane having a detection region in which a capture substance that specifically reacts with the complex developed from the developing pad by capillary action is immobilized; Equipped with A test strip, wherein the spreading pad holds a compound having a phosphorylcholine group in a dry state as the sensitizer.

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