Immunochromatography device, its manufacturing method, and method for detecting target bacteria using the same

Abstract

An immunochromatography device 10 that holds a labeling antibody-carrying member, which elutably carries an antibody for labeling, disposed thereon in such a manner that at least a part of the labeling antibody-carrying member is immersed in a specimen solution. Owing to this arrangement, the labeling antibody elutes and diffuses into the specimen solution when the immunochromatography device 10 is immersed in the specimen solution. By diffusing the labeling antibody into the specimen solution, an antigen-antibody reaction occurs in the specimen solution so that the reaction time can be prolonged compared with the case of using conventional techniques, thereby improving the detection sensitivity.

Description

[Technical field]

[0001] The present invention relates to an immunochromatographic device for detecting target bacteria in a sample liquid by antigen-antibody reaction, a method for producing such an immunochromatographic device, and a method for detecting target bacteria using the immunochromatographic device. [Background technology]

[0002] Conventionally, methods for detecting the presence or absence of various bacteria in various specimens include the culture method, in which the specimen is cultured to confirm bacterial growth, and the ATP method, in which intracellular ATP (adenosine triphosphate) of bacteria in the specimen is detected. However, the former culture method has the problem that it requires a long time for culture and is laborious. Furthermore, the latter ATP method has the problem that it is difficult to distinguish bacteria from eukaryotic cells derived from living organisms, foods, beverages, and the environment, because ATP is present not only in bacterial cells but also in eukaryotic cells (animal cells, plant cells, etc.).

[0003] Therefore, in order to test for bacterial contamination in samples quickly and with high sensitivity, simplified measurement devices using chromatography (chromatographic devices) that can quickly and easily detect specific target substances in samples by utilizing a specific reaction caused by a specific binding substance specific to the target substance have become widespread. Various substances are used as the specific binding substance, but among them, immunochromatographic devices that use antibodies as the specific binding substance can detect various target substances and are used in various fields such as research, medicine, and food.

[0004] A lateral flow type is widely used as a representative type of immunochromatography device. Lateral flow type immunochromatography devices are mainly divided into dipstick type and cassette type. In food or livestock production sites, where simple operation and low cost are required, dipstick type immunochromatography devices are used, which do not require a housing and can be tested by simply inserting a strip into a sample liquid (Patent Document 1: Japanese Patent No. 6325895). In addition, in order to increase the sensitivity of bacterial detection, a technology is known in which bacteria are lysed using a lytic agent or enzyme and the target substance is extracted (Patent Document 2: Japanese Patent No. 6280567, Patent Document 3: Japanese Patent No. 6314156, Patent Document 4: Japanese Patent No. 6370807). In addition, a technology is known in which a lytic enzyme is incorporated into an immunochromatography strip to detect the target substance with high sensitivity (Patent Document 5: Japanese Patent No. 6081457). Such an immunochromatographic device is usually produced by immobilizing a capture reagent containing a specific binding substance specific to the substance to be detected in the detection region of a chromatographic development membrane carrier.

[0005] In addition, milk samples from livestock, etc., can be used as a measurement target for such immunochromatography devices. However, livestock milk is not sterile and may contain some microorganisms due to disease or the environment. In particular, it is known that diseases caused by microbial infection of the udder often result in the excretion of many microorganisms into the milk. A typical livestock disease caused by microbial infection is mastitis. Mastitis is an inflammation of the milk duct system and mammary gland tissue, and is mainly caused by microorganisms invading, settling, and multiplying in the udder. Many animals are affected by mastitis, but bovine mastitis in dairy cows is said to affect 15 to 40% of all dairy cows, and is one of the most important diseases for dairy farmers. When dairy cows are affected by mastitis, the milk synthesis function is inhibited, resulting in a decrease in milk production and in some cases the cessation of milk production, as well as causing dairy farmers to incur significant economic losses such as medical expenses and penalties on milk prices due to reduced milk quality. Furthermore, the labor burden on dairy farmers increases, such as milking the mastitis-affected quarters individually to prevent infection.

[0006] Mastitis is caused by infection with various microorganisms. Among the causative bacteria, coliform bacteria, such as E. coli and Klebsiella, cause systemic symptoms such as fever, and local symptoms such as heat, swelling, and induration in the breast. Culture methods are widely used to detect coliform bacteria from milk. Culture methods require several days to obtain results and are not suitable for quickly identifying the causative bacteria. On the other hand, identification methods based on antigen-antibody reactions using antibodies against components specific to the causative bacteria, especially immunochromatography, can give results in a few tens of minutes and are therefore widely used at home and in examination rooms as a quick and simple test method.

[0007] Methods for detecting milk samples using immunochromatography include the various methods described above. However, a problem specific to milk samples is that the large amounts of fat globules contained in milk inhibit the development of the chromatographic membrane, making it necessary to remove the fat globules (Patent Document 6: Patent No. 5693938). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 6325895 [Patent Document 2] Patent No. 6280567 [Patent Document 3] Patent No. 6314156 [Patent Document 4] Patent No. 6370807 [Patent Document 5] Patent No. 6081457 [Patent Document 6] Patent No. 5693938 Summary of the Invention [Problem to be solved by the invention]

[0009] In conventional immunochromatography devices, there has been a demand for improved detection sensitivity and / or detection efficiency for a target substance. That is, one object of the present invention is to improve the detection sensitivity and / or detection efficiency for a target substance in an immunochromatography device.

[0010] Furthermore, according to the study of the present invention, it was found that, although conventional immunochromatographic devices are manufactured by fixing a capture reagent containing a specific binding substance specific to a substance to be detected in a detection region of a chromatographic development membrane carrier, the sensitivity of the chromatographic device manufactured in this manner increases over time during storage after manufacture. That is, one of the objects of the present invention is to reduce the change in sensitivity over time during storage from the viewpoint of quality control during the manufacture of the immunochromatographic device.

[0011] Furthermore, when a conventional immunochromatography device is used for a milk sample, there has been a demand for an improvement in poor development caused by the fat globule removal member (hereinafter also referred to as the "filter member" as appropriate). That is, one of the objects of the present invention is to improve poor development caused by the fat globule removal member when an immunochromatography device is used for a milk sample. [Means for solving the problem]

[0012] As a result of intensive research, the present applicants have found that, when using an immunochromatographic device, by diffusing a labeling antibody in a sample liquid, an antigen-antibody reaction occurs in the sample liquid, and thus a longer reaction time can be secured than in the conventional technology, resulting in improved detection sensitivity and / or detection efficiency. In addition, when fixing a specific binding substance to the detection region of a chromatographic development membrane carrier, by performing a drying process at a higher temperature than the drying temperature normally used for a longer time, the change in sensitivity over time during the storage period of the obtained immunochromatographic device is reduced. Furthermore, in an immunochromatographic device in which a fat globule removal member for milk samples is arranged, the amount of liquid to be subjected to chromatographic development (hereinafter also referred to as "total water absorption amount Y") and the overlap of multiple fat globule removal members have a significant effect on the development characteristics of the chromatographic membrane during use, and by optimizing these parameters, poor development caused by the fat globule removal member can be improved. As a result of further intensive research based on these findings, the present applicants have completed the present invention.

[0013] That is, the present invention relates to: [Item 1] A method for detecting a target bacterium in a sample liquid by an antigen-antibody reaction using an immunochromatography device, 1) a step of immersing at least a part of a labeling antibody-attached member to which a labeling antibody has been attached in the sample liquid, and at least a part of an antigen-antibody reaction between an antigen derived from the target bacterium and the labeling antibody in the sample liquid is carried out to form a first complex; 2) a step of forming a second complex by causing an antigen-antibody reaction between the capture antibody immobilized on the immunochromatography device and the first complex, and detecting bacteria based on the label of the labeling antibody: A method for detecting bacteria, comprising: [Item 2] The method according to item 1, wherein at least a portion of the labeling antibody-attached member and at least a portion of the lytic agent-attached member to which the lytic agent has been attached are immersed in the specimen liquid. [Item 3] The method according to Item 2, wherein at least a portion of the labeling antibody-applied member and at least a portion of the lytic agent-applied member to which the lytic agent has been applied are simultaneously immersed in the specimen liquid. [Item 4] The method according to any one of Items 1 to 3, further comprising stirring the sample liquid after the immersion and before the detection. [Item 5] The method according to any one of Items 1 to 4, wherein the antigen of the target bacterium is an intracellular antigen. [Item 6] The method according to Item 5, wherein the intracellular antigen is a ribosomal protein. [Item 7] The method according to Item 6, wherein the ribosomal protein is an L7 / L12 protein. [Item 8] The method according to any one of Items 1 to 7, wherein after the immersion, at least a part of the labeling antibody attached to the labeling antibody-attached member and the lytic agent attached to the lytic agent-attached member are eluted into the sample liquid. [Item 9] An immunochromatographic device for detecting a target bacterium in a sample liquid by an antigen-antibody reaction, comprising: (a) a labeling antibody-attached member to which a labeling antibody that forms a first complex by an antigen-antibody reaction with an antigen derived from a target bacterium is attached; and (b) a chromatographic development member including an immersion area to be immersed in a sample liquid, and a detection area in which a capture antibody that forms a second complex by an antigen-antibody reaction with the first complex is immobilized. Including, wherein, when the immunochromatography device is in use, at least a part of the labeling antibody-attached member and the immersion area of ​​the chromatographic development member are both immersed in a sample liquid; Here, the detection of target bacteria in a sample liquid by the immunochromatographic device is performed by detecting the second complex captured in the detection area of ​​the chromatographic development member based on the label of the labeling antibody. [Item 10] An immunochromatography device according to Item 9, wherein at least a portion of the labeling antibody-attached member is positioned so as to be included in the immersion area. [Item 11] The immunochromatography device comprises: (c) A bacteriolytic agent-impregnated member to which a bacteriolytic agent has been impregnated Further comprising: Here, the immunochromatography device according to item 9 or 10 is configured so that at least a portion of the lytic agent-applied member is immersed in a sample liquid when the immunochromatography device is in use. [Item 12] An immunochromatographic device according to Item 11, wherein the lytic agent-attached member and the labeling antibody-attached member are each formed as separate members. [Item 13] An immunochromatographic device according to Item 11, wherein the lytic agent-attached member and the labeling antibody-attached member are formed as a single member. [Item 14] An immunochromatography device according to any one of Items 11 to 13, wherein the lytic agent-applied member is disposed so that at least a portion of the lytic agent-applied member is included in the immersion area. [Item 15] An immunochromatography device described in any one of Items 11 to 14, wherein one or both of the lytic agent-applied member and the labeling antibody-applied member are provided as separate members from the chromatographic development member. [Item 16] The immunochromatographic device comprises: (d) Sample contact member Further comprising: Item 16. The immunochromatographic device according to any one of Items 11 to 15, wherein at least a part of the sample contact member is immersed in a sample liquid when the immunochromatographic device is in use. Effect of the Invention

[0014] According to one aspect of the present invention, there is provided an excellent immunochromatography device having improved detection sensitivity and / or detection efficiency for a target substance.

[0015] Furthermore, according to one aspect of the present invention, there is provided an excellent immunochromatography device in which the change in sensitivity over time during storage is reduced.

[0016] Furthermore, according to one aspect of the present invention, there is provided an excellent immunochromatography device for use with milk samples, in which poor development caused by a fat globule removal member is improved. [Brief description of the drawings]

[0017] [Figure 1] 1 is a cross-sectional view showing the configuration of an immunochromatography device according to one embodiment of the present invention (Examples I-1, I-7, and I-13). A labeling antibody-adhered member 1 is placed on a base material 8 on the back side of an immunochromatography device 10, and a lytic agent-adhered member 2 is placed on the base material 8 on the front side of the immunochromatography device 10. The labeling antibody-adhered member 1 and the lytic agent-adhered member 2 are exposed, and are characterized in that the labeling antibody-adhered member 1 and the lytic agent-adhered member 2 are placed in an immersion area 9. [Diagram 2] 2 is a cross-sectional view showing the configuration of an immunochromatography device according to one embodiment of the present invention (Examples I-2, I-8, and I-14). The labeling antibody-attached member 1 and the lytic agent-attached member 2 are provided separately from the immunochromatography device 10, but are immersed in the sample liquid. [Diagram 3] 3 is a cross-sectional view showing the configuration of an immunochromatography device according to one embodiment of the present invention (Examples I-3, I-9, and I-15). A labeling antibody-adhered member 1 and a lytic agent-adhered member 2 are placed on a base material 8 on the front side of an immunochromatography device 10. The labeling antibody-adhered member 1 and the lytic agent-adhered member 2 are exposed, and are characterized in that they are placed in an immersion area 9. [Figure 4] 4 is a cross-sectional view showing the configuration of an immunochromatography device according to one embodiment of the present invention (Examples I-4, I-10, and I-16). A labeling antibody-adhered member 1 and a lytic agent-adhered member 2 are placed in contact with each other on a substrate 8 on the front side of an immunochromatography device 10. A sample contact member 5 is placed so as to cover the labeling antibody-adhered member 1 and the lytic agent-adhered member 2. The labeling antibody-adhered member 1 and the lytic agent-adhered member 2 are characterized by being placed in an immersion area 9. [Diagram 5]5 is a cross-sectional view showing the configuration of an immunochromatography device according to one embodiment of the present invention (Examples I-5, I-11, and I-17). A labeling antibody-attached member 1 and a lytic agent-attached member 2 are arranged apart from each other on a base material 8 on the front side of an immunochromatography device 10. A sample contact member 5 is arranged so as to cover the labeling antibody-attached member 1 and the lytic agent-attached member 2. At least a part of the labeling antibody-attached member 1 and the lytic agent-attached member 2 are arranged in an immersion area 9. [Figure 6] 6 is a cross-sectional view showing the configuration of an immunochromatography device according to one embodiment of the present invention (Examples I-6, I-12, and I-18). A labeling antibody-adhered member 1 and a lytic agent-adhered member 2 are arranged in contact with each other on a base material 8 on the front side of an immunochromatography device 10. The lytic agent-adhered member 2 is arranged across the base material 8 and the labeling antibody-adhered member 1, and is arranged so as to completely cover the labeling antibody-adhered member 1. Furthermore, a sample contact member 5 is arranged so as to cover the lytic agent-adhered member 2. The labeling antibody-adhered member 1 and the lytic agent-adhered member 2 are characterized by being arranged in an immersion area 9. [Figure 7] FIG. 7 shows cross-sectional views of the configurations of the immunochromatography devices according to Comparative Examples I-1, I-5, and I-9. The labeling antibody-attached member 1 and the lytic agent-attached member 2 are arranged apart from each other on the substrate 8 on the front side of the immunochromatography device 10. The sample contact member 5 is divided into two, and arranged so that they partially overlap. The labeling antibody-attached member 1 is arranged so as to straddle a part of the downstream sample contact member 5 and the substrate 8, and the upstream sample contact member is arranged so as to cover the arranged labeling antibody-attached member 1. The lytic agent-attached member 2 is arranged exposed. The labeling antibody-attached member 1 is arranged outside the immersion area 9, while the lytic agent-attached member 2 is arranged inside the immersion area 9. [Figure 8]Fig. 8 is a cross-sectional view showing the configuration of the immunochromatography devices according to Comparative Examples I-2, I-6, and I-10. The labeling antibody-attached member 1 and the lytic agent-attached member 2 are arranged apart from each other on the substrate 8 on the front side of the immunochromatography device 10. The labeling antibody-attached member 1 is arranged so as to straddle a part of the chromatographic development membrane carrier 3 and the substrate 8, and the sample contact member 5 is arranged so as to cover the labeling antibody-attached member 1. The lytic agent-attached member 2 is exposed. The labeling antibody-attached member 1 is arranged outside the immersion area 9, while the lytic agent-attached member 2 is arranged inside the immersion area 9. [Figure 9] FIG. 9 shows a cross-sectional view of the configuration of the immunochromatography devices according to Comparative Examples I-3, I-7, and I-11. The labeling antibody-attached member 1 and the lytic agent-attached member 2 are arranged apart from each other on the substrate 8 on the front side of the immunochromatography device 10. The sample contact member 5 is divided into two and arranged so that they overlap. The labeling antibody-attached member 1 is arranged so as to straddle a part of the chromatographic development membrane carrier 3 and the substrate 8, and the downstream sample contact member 5 is arranged so as to cover the labeling antibody-attached member 1 arranged. The lytic agent-attached member 2 is arranged so as to straddle a part of the downstream sample contact member 5 and the substrate 8, and the upstream sample contact member 5 is arranged so as to cover the lytic agent-attached member 2 arranged. The labeling antibody-attached member 1 and the lytic agent-attached member 2 are both arranged outside the immersion area 9. [Figure 10] Fig. 10 is a cross-sectional view showing the configuration of the immunochromatographic devices according to Comparative Examples I-4 and I-8. A chromatographic development membrane carrier 3 is arranged on the front side of a substrate 8 of an immunochromatographic device 10, a labeling antibody-attached member 1 is arranged so as to straddle a part of the upstream side of the chromatographic development membrane carrier 3 and the substrate 8, and a sample contact member 5 is arranged so as to straddle a part of the upstream side of the labeling antibody-attached member 1 and the substrate 8. The immunochromatographic device is characterized in that the labeling antibody-attached member 1 is arranged outside the immersion area 9 and that it does not have a lytic agent-attached member 2. [Figure 11]FIG. 11 is a cross-sectional view showing the configuration of an immunochromatography device according to one embodiment of the present invention (Examples I-19, I-21, and I-24). A labeling antibody-attached member 1 and a lytic agent-attached member 2 are placed in contact with each other on a substrate 8 on the front side of an immunochromatography device 10. A filter member 6 is placed so as to straddle a part of the chromatographic development membrane carrier 3 and the substrate 8, and a sample contact member 5 is placed so as to straddle a part of the filter member 6 and the substrate 8. The sample contact member 5 is placed so as to cover the labeling antibody-attached member 1 and the lytic agent-attached member 2. The labeling antibody-attached member 1 and the lytic agent-attached member 2 are characterized in that they are placed in an immersion area 9. [Figure 12] FIG. 12 is a cross-sectional view showing the configuration of an immunochromatography device according to one embodiment of the present invention (Examples I-20 and I-25). The labeling antibody-attached member 1 and the lytic agent-attached member 2 are arranged in contact with each other on the substrate 8 on the front side of the immunochromatography device 10. The lytic agent-attached member 2 is arranged across the substrate 8 and the labeling antibody-attached member 1, and is arranged so as to completely cover the labeling antibody-attached member 1. Furthermore, the sample contact member 5 is arranged so as to cover the lytic agent-attached member 2. The filter member 6 is arranged so as to straddle a part of the downstream side of the sample contact member 5 and the substrate 8. Furthermore, the filter member 6 is arranged so as to straddle a part of the upstream side of the chromatographic development membrane carrier 3 and the substrate 8. The labeling antibody-attached member 1 and the lytic agent-attached member 2 are characterized by being arranged in the immersion area 9. [Figure 13] 13 is a cross-sectional view showing the configuration of the immunochromatographic devices according to Comparative Examples I-12 and I-13. A chromatographic development membrane carrier 3 is arranged on the front side of a substrate 8 of an immunochromatographic device 10, a filter member 6 is arranged so as to straddle a part of the upstream side of the chromatographic development membrane carrier 3 and the substrate 8, a labeling antibody-attached member 1 is arranged so as to straddle a part of the upstream side of the filter member 6 and the substrate 8, and a sample contact member 5 is arranged so as to straddle a part of the upstream side of the labeling antibody-attached member 1 and the substrate 8. The immunochromatographic device is characterized in that the labeling antibody-attached member 1 is arranged outside the immersion region 9 and does not have a lytic agent-attached member 2. [Figure 14]FIG. 14 is a cross-sectional view showing the configuration of the immunochromatography device according to Example I-22. A chromatographic development membrane carrier 3 is arranged on the front side of a substrate 8 of an immunochromatography device 10, and a labeling antibody-attached member 1 and a lytic agent-attached member 2 are arranged in contact with each other. The labeling antibody-attached member 1 is arranged across the substrate 8 and the lytic agent-attached member 2, and is arranged so as to completely cover the lytic agent-attached member 2. Furthermore, a sample contact member 5 is arranged so as to cover the labeling antibody-attached member 1. A filter member 6 is arranged so as to span a part of the upstream side of the chromatographic development membrane carrier 3 from the substrate 8, and a part of the downstream side of the sample contact member 5 is arranged so as to span a part of the upstream side of the filter member 6. The labeling antibody-attached member 1 and the lytic agent-attached member 2 are characterized in that they are arranged in an immersion area 9. [Figure 15] Fig. 15 is a cross-sectional view showing the configuration of the immunochromatography device according to Examples I-23 and I-29. A chromatographic development membrane carrier 3 is arranged on the front side of a substrate 8 of an immunochromatography device 10, and a labeling antibody-attached member 1 and a lytic agent-attached member 2 are arranged apart from each other. A sample contact member 5 is arranged so as to completely cover the labeling antibody-attached member 1 and the lytic agent-attached member 2. A filter member 6 is arranged so as to straddle a part of the upstream side of the chromatographic development membrane carrier 3 from the substrate 8, and a part of the downstream side of the sample contact member 5 is arranged so as to straddle a part of the upstream side of the filter member 6. The labeling antibody-attached member 1 and the lytic agent-attached member 2 are characterized by being arranged in an immersion area 9. [Figure 16]FIG. 16 is a cross-sectional view showing the configuration of the immunochromatography device according to Example I-26. A chromatographic development membrane carrier 3 is arranged on the front side of a substrate 8 of an immunochromatography device 10, and a labeling antibody-attached member 1 and two lytic agent-attached members 2-1 and 2-2 are also arranged. The two lytic agent-attached members 2-1 and 2-2 are arranged so that the lytic agent-attached member 2-2 covers a part of the lytic agent-attached member 2-1. The labeling antibody-attached member 1 is arranged away from the two lytic agent-attached members 2-1 and 2-2. The sample contact member 5 is arranged so as to completely cover the labeling antibody-attached member 1 and the lytic agent-attached members 2-1 and 2-2. The filter member 6 is arranged so as to straddle a part of the upstream side of the substrate 8 and the chromatographic development membrane carrier 3, and a part of the downstream side of the sample contact member 5 is arranged so as to straddle a part of the upstream side of the filter member 6. The labeling antibody-attached member 1 and the lytic agent-attached members 2-1 and 2-2 are characterized by being placed in an immersion area 9. [Figure 17] FIG. 17 is a cross-sectional view showing the configuration of an immunochromatography device according to Example I-27. A chromatographic development membrane carrier 3 is arranged on the front side of a substrate 8 of an immunochromatography device 10, and a labeling antibody-attached member 1 and two lytic agent-attached members 2-1 and 2-2 are arranged. The two lytic agent-attached members 2-1 and 2-2 are arranged so that the lytic agent-attached member 2-2 covers a part of the lytic agent-attached member 2-1. The labeling antibody-attached member 1 is arranged so as to completely cover the lytic agent-attached member 2-1 and to contact the lytic agent-attached member 2-2. The sample contact member 5 is arranged so as to completely cover the labeling antibody-attached member 1 and the lytic agent-attached member 2-2. The filter member 6 is arranged so as to straddle a part of the upstream side of the substrate 8 and the chromatographic development membrane carrier 3, and a part of the downstream side of the sample contact member 5 is arranged so as to straddle a part of the upstream side of the filter member 6. The labeling antibody-attached member 1 and the lytic agent-attached members 2-1 and 2-2 are characterized by being placed in an immersion area 9. [Figure 18]FIG. 18 is a cross-sectional view showing the configuration of an immunochromatography device according to Example I-28. A chromatographic development membrane carrier 3 is arranged on the front side of a substrate 8 of an immunochromatography device 10, and a labeling antibody-attached member 1 and two lytic agent-attached members 2-1 and 2-2 are arranged. The two lytic agent-attached members 2-1 and 2-2 are arranged so that the lytic agent-attached member 2-2 covers a part of the lytic agent-attached member 2-1. The labeling antibody-attached member 1 is arranged so as to completely cover the lytic agent-attached member 2-1 and to cover a part of the lytic agent-attached member 2-2. The sample contact member 5 is arranged so as to completely cover the labeling antibody-attached member 1 and the lytic agent-attached member 2-2. The filter member 6 is arranged so as to straddle a part of the upstream side of the substrate 8 and the chromatographic development membrane carrier 3, and a part of the downstream side of the sample contact member 5 is arranged so as to straddle a part of the upstream side of the filter member 6. The labeling antibody-attached member 1 and the lytic agent-attached members 2-1 and 2-2 are characterized by being placed in an immersion area 9. [Figure 19] FIG. 19 is a cross-sectional view showing the configuration of an immunochromatography device according to Example I-30. A chromatographic development membrane carrier 3 is arranged on the front side of a substrate 8 of an immunochromatography device 10, and a labeling antibody-attached member 1 and a lytic agent-attached member 2 are arranged in contact with each other. The labeling antibody-attached member 1 is arranged across the substrate 8 and the lytic agent-attached member 2, and is arranged so as to completely cover the lytic agent-attached member 2. A filter member 6 is arranged so as to straddle a part of the upstream side of the chromatographic development membrane carrier 3 from the substrate 8, and a sample contact member 5 is arranged so as to cover the labeling antibody-attached member 1 and straddle a part of the upstream side of the filter member 6 and the substrate 8. Furthermore, the labeling antibody-attached member 1 and the lytic agent-attached member 2 are characterized in that they are arranged in an immersion area 9. [Figure 20] FIG. 20 is a schematic diagram showing a scene in which the immunochromatographic device of FIG. 1 is used by immersing it in a sample liquid. [Figure 21] FIG. 21 is a schematic diagram showing a scene in which the immunochromatography device of FIG. 2, the labeling antibody-affixed member 1, and the lytic agent-affixed member 2 are immersed in a sample liquid for use. [Figure 22]FIG. 22 is a cross-sectional view showing the configuration of an example of a chromatography device produced by a production method according to one embodiment of the present invention. [Figure 23] FIG. 23 is a schematic diagram showing a scene in which the immunochromatographic device of FIG. 22 is used by immersing it in a sample liquid. [Figure 24] FIG. 24 is a cross-sectional view showing the configuration of an immunochromatography device 10 according to one embodiment of the present invention. [Diagram 25] 25 is a cross-sectional view showing the configuration 10 of an immunochromatography device according to one embodiment of the present invention, excluding the sample contact member 5. Note that (A) and (B) each show a diagram in which the distance of overlap (indicated by reference symbol 100) between the first filter member 6-1 and the second filter member 6-2 (hereinafter also referred to as overlap distance X) is changed. [Figure 26] 26 is a schematic diagram showing the dimensions of the immunochromatographic devices used in the examples of the present invention, in which (A) shows the immunochromatographic device of Example III-1, (B) shows the immunochromatographic device of Example III-5, and (C) shows the immunochromatographic device of Example III-6. [Figure 27] FIG. 27 is a schematic diagram showing an immunochromatographic device 10 according to one embodiment of the present invention being used while immersed in a milk sample. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present invention will be described in detail below with reference to specific embodiments. However, the present invention is not limited to the following embodiments, and can be embodied in any form without departing from the spirit of the present invention.

[0019] [First aspect] A first aspect of the present invention relates to an immunochromatographic device for detecting target bacteria in a sample liquid through an antigen-antibody reaction, and also to a method for detecting target bacteria in a sample liquid through an antigen-antibody reaction using the immunochromatographic device.

[0020] [Immunochromatography device] The immunochromatographic device refers to a kit including an immunochromatographic device for detecting a detection target (target bacteria) in a sample liquid by an antigen-antibody reaction. When using the immunochromatographic device, the immersion area 9 of the immunochromatographic device 10 is immersed in a sample liquid contained in a sample liquid holding container 20, and the sample liquid is developed from the sample contact member 5 to the absorption member (absorption pad) 7 via the chromatographic development membrane carrier 3 by utilizing the capillary principle. A complex formed by a labeling antibody and an antigen contained in the developed sample liquid is captured by a capture antibody fixed to the detection area on the chromatographic development membrane carrier 3, and the label of the labeling antibody is detected, and the presence or absence of the target bacteria is detected. In order to prevent the development of the sample liquid from places other than the immersion area 9, all or part of the area other than the immersion area 9 may be covered with a water-impermeable cover member. In addition, if the structure is such that the labeling antibody and / or the bacteriolytic agent can be dissolved in the sample liquid, a part of the immersion area 9 may be covered with a water-impermeable cover member. The immunochromatographic device may be composed of only the immunochromatographic device 10, or may include a sample liquid holding container 20 for holding a sample liquid, and may further include an instruction manual.

[0021] The immunochromatographic device according to this embodiment includes (a) a labeling antibody-attached member 1 to which a labeling antibody is attached, and (b) an immunochromatographic device 10 having an immersion area 9 and a detection area to which a capture antibody is immobilized. When the immunochromatographic device is used, at least a part of the labeling antibody-attached member 1 and the immersion area 9 are both immersed in a sample liquid. The labeling antibody-attached member 1 may be disposed in the immersion area of ​​the immunochromatographic device 10, or may be provided separately from the immunochromatographic device 10. When the labeling antibody-attached member 1 is immersed in the sample liquid, the labeling antibody attached in an elutable manner is eluted and diffused into the sample liquid. Since an antigen-labeling antibody reaction occurs in the sample liquid, a sufficiently long reaction time can be ensured. Since a sufficiently long reaction time can be ensured, the amount of a complex formed from the antigen-labeling antibody increases. The formed complex is captured by the capture antibody immobilized in the detection area, and high sensitivity can be achieved. By including a bacteriolytic agent in the sample solution, the bacteriolytic reaction occurs prior to the antigen-labeled antibody reaction. This allows the bacteriolytic reaction and the antigen-labeled antibody reaction to occur in the sample solution, ensuring a longer reaction time for each reaction than in conventional technology, and as a result, the detection sensitivity is further improved.

[0022] The lytic agent in the specimen liquid may be added separately to the specimen liquid, or may be eluted from the lytic agent-applied member 2 included in the immunochromatography device. When the lytic agent is added separately, the immunochromatography device may contain a lytic agent separate from the immunochromatography device 10. The immunochromatography device according to this embodiment may further include (c) a lytic agent-applied member 2 to which a lytic agent is applied. The lytic agent-applied member 2 may be placed in advance in the specimen liquid holding container 20, or may be placed in the immunochromatography device 10. At least a part of the lytic agent-applied member 2 is immersed in the specimen liquid when the immunochromatography device is used. The lytic agent-applied member 2 may be placed in the immersion area of ​​the immunochromatography device 10, or may be provided separately from the immunochromatography device 10. When the lytic agent-applied member 2 is immersed in the specimen liquid, the lytic agent is eluted and diffused in the specimen liquid. This causes a lytic reaction in the specimen liquid. By disposing the lytic agent-applied member 2 in the immunochromatographic device 10, both the lytic agent and the detection antibody are eluted into the sample liquid by immersing the immunochromatographic device 10 in the sample liquid. This causes a lytic reaction in the sample liquid, and further causes an antigen-labeling antibody reaction in the sample liquid. By ensuring a sufficiently long reaction time for both reactions, the amount of complex formed from the antigen-labeling antibody increases, making it possible to achieve high sensitivity. Furthermore, by disposing the lytic agent-applied member 2 in the immunochromatographic device 10, bacteria can be detected by the immunochromatographic device 10 alone, further simplifying the configuration of the immunochromatographic device.

[0023] (a) The labeling antibody-attached member 1 may be made of any material as long as the labeling antibody is attached to it. The labeling antibody is preferably attached to the labeling antibody-attached member 1 in a manner that allows it to be dissolved. The labeling antibody-attached member may be manufactured by impregnating a water-absorbent member with a solution containing the labeling antibody and, if necessary, drying it. Examples of water-absorbent members include paper, woven fabric, nonwoven fabric, fiber aggregates such as glass fiber, and porous bodies such as sponges. In another embodiment, the labeling antibody-attached member 1 may be obtained by directly applying or adhering a solution containing the labeling antibody to a member constituting the immunochromatographic device 10. When adhering, the labeling antibody-attached member 1 may be made into a water-soluble adhesive substance by adding a thickener or the like. When the labeling antibody-attached member 1 is arranged in the immunochromatographic device 10, it may be arranged on any member of the immunochromatographic device 10 as long as it is arranged so that at least a part of the labeling antibody-attached member 1 is immersed in the sample liquid when the immunochromatographic device is in use.

[0024] The amount of the labeling antibody to be applied can be appropriately selected depending on the volume of the sample liquid, the ability to form a complex with the target substance, etc., and is, for example, 0.01 to 1000 μg. At least a portion of the applied labeling antibody, for example, 10% or more, preferably 50% or more, and more preferably 90% or more, can diffuse into the sample liquid.

[0025] (b) The immunochromatographic device 10 may include any of the configurations of a normal immunochromatographic device 10 used in immunochromatographic devices. The immunochromatographic device 10 has an immersion region 9 arranged upstream and a detection region arranged downstream. The detection region of the immunochromatographic device 10 has a capture site 4 to which a capture antibody is immobilized. Furthermore, a control site can be formed downstream of the capture site 4 by immobilizing a substance that binds to the labeling antibody. The capture site 4 and the control site can be formed, for example, linearly on the chromatographic development membrane carrier 3. The immunochromatographic device 10 includes a chromatographic development membrane carrier 3, an absorption member (absorption pad) 7, and a sample contact member 5 on a base material 8, and may further include one or more filter members 6. A labeling antibody-attached member 1 and a bacteriolytic agent-attached member 2 can be arranged in the immersion region 9 of the above-mentioned immunochromatographic device 10. The labeling antibody-affixed member 1 and / or the lytic agent-affixed member 2 may be disposed on any member as long as a part of them is disposed in an immersion area 9 that is immersed in a sample liquid during use. An immunochromatography device 10 is usually formed by arranging each component on a substrate 8, but does not have to include a substrate 8. Detection is performed by immersing the immersion area of ​​the immunochromatography device 10 in a sample liquid holding container 20 containing a sample liquid.

[0026] (c) The lytic agent-imparted member 2 may be made of any material as long as the lytic agent is eluted. The lytic agent-imparted member 2 may be manufactured by impregnating a water-absorbent member with a solution containing the lytic agent and optionally drying it. Examples of water-absorbent members include paper, woven fabric, nonwoven fabric, fiber aggregates such as glass fiber, and porous bodies such as sponges. In another embodiment, the lytic agent-imparted member 2 may be obtained by directly applying or adhering a solution containing the lytic agent to a member constituting the immunochromatographic device 10. When adhering, the lytic agent-imparted member 2 may be made into a water-soluble adhesive substance by adding a thickener or the like. When the lytic agent-imparted member 2 is arranged in the immunochromatographic device 10, it may be arranged at any position in the immunochromatographic device 10 as long as it is arranged so that at least a part of the lytic agent-imparted member 2 is immersed in the sample liquid when the immunochromatographic device is in use. The labeling antibody-attached member 1 and the lytic agent-attached member 2 may be formed as a single member by impregnating a water-absorbent member with a solution containing the labeling antibody and the lytic agent. The type of lytic agent (described below) is selected depending on the type of bacteria to be detected, and examples thereof include, but are not limited to, lytic enzymes and surfactants.

[0027] The amount of the lytic agent to be applied can be appropriately selected depending on the volume of the sample liquid, the lytic ability, the type of bacteria, etc. For example, in the case of a lytic enzyme, 0.01 to 100,000 μg is used, and in the case of a surfactant, 0.1 to 100 mg is used. At least a part of the applied lytic agent, for example 10% or more, preferably 50% or more, and more preferably 90% or more, diffuses into the sample liquid.

[0028] [Arrangement of components of immunochromatography device 10] The arrangement of each component of the immunochromatography device 10 will be described with reference to Figs. 1 to 21. Each component of the immunochromatography device 10 is arranged on a substrate 8 and / or other members in a superimposed manner. The side on which each component, particularly the chromatographic development membrane carrier 3, is arranged is the front side of the substrate 8. Each component of the immunochromatography device 10 can be adhered to each other using an adhesive substance. An adhesive substance may be arranged on the substrate 8. After the components are arranged on the substrate 8, the substrate 8 may be removed. As an example, the chromatographic development membrane carrier 3 is arranged on the substrate 8, and an absorbing member (absorbent pad) 7 is arranged so as to span at least a part of the downstream end of the chromatographic development membrane carrier 3 and the substrate 8 (Figs. 1 to 19). A sample contact member 5 is arranged so as to span at least a part of the upstream end of the chromatographic development membrane carrier 3 and the substrate 8 (Figs. 1 to 10). With the above basic configuration, when the immersion area 9 of the immunochromatography device 10 is immersed in the sample liquid, the sample liquid is developed on the chromatographic development membrane carrier 3 through the sample contact member 5, and the sample liquid is finally absorbed by the absorption member (absorption pad) 7. The sample contact member 5 may be divided into two or more members (FIGS. 7 and 9), and a labeling antibody-attached member 1 and / or a bacteriolytic agent-attached member 2 may be disposed between the two sample contact members. In addition, other members may be present between the sample contact member 5 and the chromatographic development membrane carrier 3. In that case, the sample contact member 5 and the chromatographic development membrane carrier 3 do not need to be in contact with each other, and the sample liquid may be developed on the chromatographic development membrane carrier 3 through the other members. For example, the filter member 6 may be disposed across at least a part of the chromatographic development membrane carrier 3 and the substrate 8, and the sample contact member 5 may be disposed across at least a part of the filter member 6 and the substrate 8 (FIGS. 11 to 19). As a result, the specimen liquid is developed on the chromatographic development membrane carrier 3 from the sample contact member 5 in the immersion region through the filter member 6 .

[0029] The labeling antibody-attached member 1 may be placed in any position as long as at least a part of it is immersed in the sample liquid during use. The labeling antibody-attached member 1 may be placed on the immunochromatography device 10 (Figs. 1 and 3-19), or may be placed away from the immunochromatography device 10 (Fig. 2). When the labeling antibody-attached member 1 is placed on the immunochromatography device 10, it can be placed on any member as long as a part of it is placed in the immersion area. The labeling antibody-attached member 1 may be placed, for example, on the back side (Fig. 1) or front side (Figs. 3-19) of the base material 8, or may be placed on the sample contact member 5, filter member 6, lytic agent-attached member 2, or chromatographic development membrane carrier 3 as long as a part of it is placed in the immersion area. The labeling antibody-attached member 1 may be placed across these base materials or members. A part or the whole of the labeling antibody-attached member 1 may be covered by another member, for example, the sample contact member 5 (Figs. 4 to 19), or the labeling antibody-attached member 1 may be exposed (Figs. 1 to 3). From the viewpoint of allowing the lytic reaction and the antigen-antibody reaction to proceed in close proximity, it is preferable to arrange the labeling antibody-attached member 1 and the lytic agent-attached member 2 so that they are both covered by the sample contact member 5 (Figs. 4 to 6, 11, 12, and 14 to 19). In Figs. 7 to 10 and 13, the labeling antibody-attached member 1 is arranged on the immunochromatography device 10 but is not arranged in the immersion area 9. Therefore, the immunochromatography devices 10 in Figs. 7 to 10 and 13 do not constitute this embodiment in terms of the arrangement of the labeling antibody-attached member 1.

[0030] The lytic agent-attached member 2 may be disposed in any position as long as at least a part of it is immersed in the sample liquid during use. The lytic agent-attached member 2 may be disposed on the immunochromatography device 10 (FIGS. 1, 3-12, and 14-19), or may be disposed apart from the immunochromatography device 10 (FIG. 2). The lytic agent-attached member 2 may be disposed, for example, on the back side or front side of the substrate 8 (FIGS. 1, 3-9, 11-12, and 14-19), or may be disposed on the sample contact member 5, the filter member 6, the labeling antibody-attached member 1, or the chromatographic development membrane carrier 3, as long as a part of it is disposed in the immersion region 9. The lytic agent-attached member 2 may be disposed across these substrates or members. The lytic agent-imparted member 2 and the labeling antibody-imparted member 1 are both placed in the immersion area 9, but they may be in contact with each other (Figs. 3, 4, 6, 11, 12, 14, 17, 18, and 19), or may be placed apart (Figs. 2, 5, 7-10, 15, and 16). When the labeling antibody-imparted member 1 and the lytic agent-imparted member 2 are in contact with each other, their walls may be in contact (Figs. 3, 4, and 11), or they may be partially or entirely overlapped (Figs. 6, 12, 14, 17, 18, and 19). As an example, the lytic agent-imparted member 2 may be placed across the substrate 8 and the labeling antibody-imparted member 1 (Figs. 6 and 12). In this case, the lytic agent-imparted member 2 may cover only a part of the labeling antibody-imparted member 1, or may completely cover the labeling antibody-imparted member 1 as shown in Figs. 6 and 12. A part or the whole of the lytic agent-imparted member 2 may be covered by another member, for example, the sample contact member 5 (FIGS. 4, 5, 6, 9, 11, 12, and 15-19), or may be exposed (FIGS. 1-3, 7, and 8). As another example, the labeling antibody-imparted member 1 may be disposed across the substrate 8 and the lytic agent-imparted member 2. The labeling antibody-imparted member 1 may cover only a part of the lytic agent-imparted member 2 (FIGS. 16-18), or may completely cover the lytic agent-imparted member 2. In FIG. 9, the lytic agent-imparted member 2 is disposed on the immunochromatography device 10, but is not disposed in the immersion area 9. Therefore, the immunochromatography device 10 in FIG. 9 does not constitute the present embodiment in terms of the arrangement of the lytic agent-imparted member 2.

[0031] The chromatographic development membrane carrier 3 refers to a member that performs chromatographic membrane development using the principle of capillary tubes. The chromatographic development membrane carrier 3 is not particularly limited as long as it can perform immunochromatography, but is preferably nitrocellulose, mixed nitrocellulose ester, polyvinylidene fluoride, nylon, etc. As an example, a nitrocellulose membrane is used. A capture antibody can be immobilized on the chromatographic development membrane carrier 3 to form a capture site 4. Furthermore, a substance that binds to the labeling antibody can be immobilized downstream of the capture site 4 to form a control site. The capture region and the control region can be formed, for example, linearly on the chromatographic development membrane carrier 3, and together they constitute the detection region.

[0032] The sample contact member 5 is a member that comes into contact with the sample, and may be any member that allows the solution to pass through. As the sample contact member 5, a fiber assembly, such as filter paper, cloth, or nonwoven fabric, is used. Examples of fibers used in the sample contact member 5 include chemical fibers such as cellulose fiber and glass fiber, and natural fibers such as cotton. The sample contact member 5 may also function as the filter member 6 by adjusting it to have an exclusion size due to the fiber assembly.

[0033] The filter member 6 is a member that removes some of the substances in the sample liquid. The sample liquid contains substances that clog the chromatographic development membrane carrier 3, and in order to remove such substances, the filter member 6 can be arranged upstream of the chromatographic development membrane carrier. The filter member 6 is an aggregate of fibers having a predetermined retention particle size. The fibers used for the filter member 6 can be chemical fibers, particularly glass fibers. As an example, when milk is used as the sample, a fat globule removing member that removes fat globules in milk can be used as the filter member. Fat globules in milk have a diameter of about 1 to 10-odd μm, and as an example, a filter having a retention particle size of, for example, 1 μm to 10 μm, preferably 1 to 5 μm, that can remove milk fat globules can be used as the filter member. The filter member 6 does not have to be arranged depending on the type of sample liquid, but one or more filter members 6 can be arranged.

[0034] The absorbing member 7 is a water-absorbent member disposed at the downstream end, and is a member for continuing chromatographic development by absorbing the solution chromatographically developed downstream. Any water-absorbent material may be used, such as cotton, cloth, or paper. The absorbing member 7 is disposed so as to straddle a part of the downstream end of the chromatographic development membrane carrier 3 and the substrate 8.

[0035] The substrate 8 is a water-impermeable thin layer member used for superposing each component thereon in manufacturing the immunochromatography device 10. As an example, it may be made of polystyrene, and an adhesive substance may be attached to fix each component.

[0036] [Detection method of target bacteria] The method of detecting target bacteria in a sample liquid by antigen-antibody reaction using an immunochromatography device is as follows: The immersion area 9 of the immunochromatography device 10 is immersed in a sample liquid, The second complex captured in the detection region of the immunochromatography device 10 is detected based on the label of the labeling antibody. The method includes immersing the immersion area 9 of the immunochromatography device 10 in the sample liquid, so that at least a part of the labeling antibody-attached member 1 is immersed in the sample liquid, and the labeling antibody is eluted in the sample liquid. This causes an antigen-antibody reaction in the sample liquid, and the reaction time can be extended, resulting in improved detection sensitivity. In addition, when the immunochromatography device includes the lytic agent-attached member 2, at least a part of the lytic agent-attached member 2 is immersed in the sample liquid, similarly to the labeling antibody-attached member 1, and the labeling antibody and the lytic agent are eluted in the sample liquid. In this case, the timing at which the labeling antibody-attached member 1 and the lytic agent-attached member 2 are immersed in the sample liquid is not limited, and they may be immersed simultaneously, or may be immersed sequentially with a short time interval (for example, within 5 minutes, within 1 minute, or within 30 seconds) between them (i.e., one of the labeling antibody-attached member 1 and the lytic agent-attached member 2 is immersed, and then the other is immersed). The presence of the labeling antibody and the bacteriolytic agent in the sample liquid causes a bacteriolytic reaction and an antigen-antibody reaction to occur in the sample liquid, and the time for both reactions can be long, resulting in improved detection sensitivity. The immersion time is a time sufficient for the second complex to be captured by the capture antibody immobilized on the immunochromatography device 10. The immersion time may be until the labeling antibody binds to the control region and the label is detected. In the detection region where the capture antibody is immobilized, the second complex is formed, making it possible to detect it based on the label of the labeling antibody.

[0037] From the above viewpoint, in order to obtain a higher detection sensitivity, it is preferable that at least a part of the labeling antibody-attached member 1 and at least a part of the lytic agent-attached member 2 are close to each other. More specifically, when the immersion area side or the sample contact member side of the chromatography device is called the upstream side and the detection area side is called the downstream side, and the direction from the upstream side to the downstream side is called the chromatographic development direction, it is preferable that the labeling antibody-attached member 1 and the lytic agent-attached member 2 are arranged so that their positions contact or overlap when projected in a direction perpendicular to the chromatographic development direction (i.e., the labeling antibody-attached member 1 and the lytic agent-attached member 2 include the same position in at least a part of the chromatographic development direction). The "overlapping" arrangement referred to here is not limited to the case where the labeling antibody-attached member 1 and the lytic agent-attached member 2 contact or overlap each other on the same surface of the chromatography device, but also includes a state in which, for example, one is placed on the back side of the chromatography device and they are separated from each other. With this arrangement, when the immersion area 9 of the immunochromatography device 10 is immersed in the sample liquid, at least a part of the labeling antibody-attached member 1 and at least a part of the lytic agent-attached member 2 are immersed in close proximity to the sample liquid, the labeling antibody and the lytic agent are dissolved in close proximity to the sample liquid, and the presence of the labeling antibody and the lytic agent in the sample liquid causes a lytic reaction and an antigen-antibody reaction to occur in the sample liquid, ensuring a long reaction time for both reactions and, as a result, improving the detection sensitivity.

[0038] The detection method of this embodiment may further include a step of stirring the detection solution (specimen liquid) using the immunochromatography device 10. Stirring promotes the elution of the labeling antibody and the lytic agent into the specimen liquid, and promotes the lytic reaction and / or the antibody-antigen reaction. After stirring, the immunochromatography device 10 is left standing, and the first complex formed by the antibody-antigen is developed on the chromatographic development membrane carrier 3 together with the specimen liquid via the sample contact member 5 (filter member 6 depending on the structure) of the immunochromatography device 10.

[0039] When the immunochromatographic device does not include the lytic agent-applied member 2, the detection method for target bacteria of this embodiment may include a step of separately adding a lytic agent to the specimen liquid. The lytic agent may be placed in advance in the specimen liquid holding container 20 of the immunochromatographic device, and in that case, the lytic agent can be dispersed in the specimen liquid by the step of putting the specimen liquid into the holding container.

[0040] The step of detecting the second complex captured in the detection region of the immunochromatography device 10 based on the label of the labeling antibody can be performed by an appropriate means depending on the label. When gold colloid is used as the label, the coloring of the discrimination part can be confirmed with the naked eye. The coloring of the control part where the substance to which the labeling antibody binds is fixed indicates that the labeling antibody has been properly developed on the chromatographic development membrane carrier 3.

[0041] [Labeling antibody] The labeling antibody is an antibody to which a detection label is attached, and is an antibody that forms a complex by an antigen-antibody reaction with an antigen derived from the target bacterium released from the lysed target bacterium. The complex thus formed is called the first complex. The type of detection label used for the labeling antibody is not particularly limited, and may be appropriately selected according to the detection method. Specific examples of the detection label include metal colloids such as gold colloid, platinum colloid, and palladium colloid; non-metal colloids such as selenium colloid, alumina colloid, and silica colloid; insoluble granular substances such as colored resin particles, dye colloid, and colored liposome; color reaction catalytic enzymes such as alkaline phosphatase, peroxidase, and luciferase; fluorescent dyes, radioisotopes; chemiluminescent labels, bioluminescent labels, and electrochemiluminescent labels. The method of adding a label to an antibody is not particularly limited, and specific examples of the detection label include physical adsorption using the hydrophobicity of the antibody and chemical binding using the functional groups of the antibody.

[0042] The first complex formed by an antigen-antibody reaction between the target bacterium-derived antigen and the labeling antibody is developed from the immersion region of the immunochromatography device 10 to the detection region of the immunochromatography device.

[0043] [Capture antibody] The capture antibody is an antibody immobilized in the detection region of the immunochromatography device 10, and forms a complex with the first complex through an antigen-antibody reaction. The complex thus formed is referred to as the second complex. More specifically, the capture antibody is immobilized on a chromatographic development membrane carrier 3, and the immobilized site is defined as a capture site 4 in the detection region. There are no particular limitations on the method for linking the antibody to the solid phase member, but specific examples include immobilization by physical adsorption utilizing the hydrophobicity of the antibody, and immobilization by chemical bonding utilizing the functional groups of the antibody.

[0044] The second complex is retained at the fixed position of the capture antibody by an antigen-antibody reaction with the immobilized capture antibody, where the label of the labeling antibody is detected. When the label is detected at the capture site 4 where the capture antibody is immobilized, the target bacterium is detected in the sample liquid.

[0045] [antibody] In the present invention, an "antibody" refers to a protein that recognizes and binds to a specific antigen or substance, and is sometimes called immunoglobulin (Ig). A typical antibody usually has two light chains and two heavy chains that are mutually bonded by disulfide bonds. There are two types of light chains, called λ chains and κ chains, and there are five types of heavy chains, called γ chains, μ chains, α chains, δ chains, and ε chains. Depending on the type of heavy chain, there are five isotypes of antibodies, called IgG, IgM, IgA, IgD, and IgE.

[0046] Each heavy chain contains a heavy chain constant (CH) region and a heavy chain variable (VH) region. Each light chain contains a light chain constant (CL) region and a light chain variable (VL) region. The light chain constant (CL) region is composed of a single domain. The heavy chain constant (CH) region is composed of three domains, namely CH1, CH2 and CH3. Each light chain variable (VL) region and heavy chain variable (VH) region are composed of four highly conserved regions (FR-1, FR-2, FR-3, FR-4) called framework regions (FR) and three hypervariable regions (CDR-1, CDR-2, CDR-3) called complementarity determining regions (CDR). The heavy chain constant (CH) region has three CDRs (CDR-H1, CDR-H2, CDR-H3) and four FRs (FR-H1, FR-H2, FR-H3, FR-H4), which are arranged from the amino terminus to the carboxy terminus in the following order: FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4. The light chain constant (CL) region has three CDRs (CDR-L1, CDR-L2, CDR-L3) and four FRs (FR-L1, FR-L2, FR-L3, FR-L4), which are arranged from the amino terminus to the carboxy terminus in the following order: FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, FR-L4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

[0047] The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody. A polyclonal antibody is usually prepared from the serum of an animal immunized with an antigen, and is a mixture of various antibody molecular species with different structures. On the other hand, a monoclonal antibody refers to an antibody consisting of a single type of molecule containing a combination of a light chain variable (VL) region and a heavy chain variable (VH) region having a specific amino acid sequence. A monoclonal antibody can be produced from a clone derived from an antibody-producing cell, but it can also be produced by genetic engineering using a nucleic acid molecule obtained having a gene sequence that codes for the amino acids of the antibody protein. In addition, modification to improve the binding and specificity of an antibody using genetic information such as heavy and light chains, or their variable regions and CDRs, is a technique well known to those skilled in the art in this field.

[0048] The antibody of the present invention may be an antibody fragment and / or derivative. Examples of antibody fragments include F(ab')2, Fab, Fv, etc. Examples of antibody derivatives include antibodies in which amino acid mutations have been artificially introduced into the constant region of the light chain and / or heavy chain, antibodies in which the domain configuration of the constant region of the light chain and / or heavy chain has been modified, antibodies having two or more Fc regions per molecule, sugar chain-modified antibodies, bispecific antibodies, antibody conjugates in which an antibody or an antibody fragment is bound to a protein other than an antibody, antibody enzymes, tandem scFvs, bispecific tandem scFvs, diabodies, etc. Furthermore, when the antibody or a fragment or derivative thereof is derived from a non-human animal, the antibody of the present invention also includes chimeric antibodies or humanized antibodies in which a part or all of the sequence other than the CDR is replaced with the corresponding sequence of a human antibody. Unless otherwise specified, the term "antibody" in the present invention also includes antibody fragments and / or derivatives.

[0049] The antibody of the present invention undergoes an antigen-antibody reaction with a certain bacterium means that the antibody specifically binds to a certain component of the bacterium as an antigen. The bacterial component that serves as an antigen for the antibody of the present invention is not limited. It may be a component contained in the cell wall or cell membrane exposed outside the bacterial cell, or a component contained in the cytoplasm, organelles, nucleus, etc. that is not exposed outside the bacterial cell, i.e., an intracellular antigen.

[0050] According to one embodiment, the antibody of the present invention preferably undergoes an antigen-antibody reaction with an intracellular antigen of a bacterium, more preferably undergoes an antigen-antibody reaction with a bacterial ribosomal protein, and particularly preferably undergoes an antigen-antibody reaction with ribosomal protein L7 / L12. In the present invention, "ribosomal protein L7 / L12" or simply "L7 / L12" is a type of ribosomal protein essential for protein synthesis in microorganisms, and is a protein commonly possessed by various bacteria. For the antibody that undergoes an antigen-antibody reaction with bacterial ribosomal protein L7 / L12 and a method for producing the same, reference can be made to, for example, the description in International Publication No. WO 2000 / 006603, which is a patent publication relating to a previous patent application filed by the present inventors.

[0051] There are no particular limitations on the level of the antigen-antibody reaction between the antibody of the present invention and the bacteria to be detected, as long as the antigen-antibody reaction occurs to an extent that can be detected by any known detection method.

[0052] [Bacteria to be detected] The bacteria to be detected may be any bacteria, but examples thereof include the genus Escherichia, Staphylococcus, Klebsiella, Proteus, Enterococcus, Citrobacter, Pseudomonas, Bacillus, Serratia, Rahnella, Listeria, Enterobacter, Salmonella, Moraxella, Aeromonas, Lactobacillus, Micrococcus, and the like. Examples of bacteria that may be used include bacteria of the genera Crococcus, Acinetobacter, Campylobacter, Vibrio, Streptococcus, Haemophilus, Mycoplasma, Legionella, Bordetella, Chlamydia, and the genera Neisseria, Yersinia, Erwinia, Hafnia, Morganella, Providencia, Shigella, and Pectobacterium.

[0053] Examples of bacteria in the genus Escherichia include Escherichia coli (E. coli, EC) and Escherichia albertii.

[0054] Examples of bacteria in the genus Staphylococcus include Staphylococcus aureus (S. aureus, SA), S. epidermidis, and S. argenteus.

[0055] Examples of bacteria in the genus Streptococcus include Streptococcus uberis (S. uberis, SU), S. agalactiae, S. dysgalactiae, and S. pneumoniae.

[0056] Examples of bacteria in the genus Klebsiella include Klebsiella pneumoniae (K. pneumoniae, KP), Klebsiella oxytoca (K. oxytoca), and Klebsiella aerogenes (K. aerogenes).

[0057] Examples of bacteria in the genus Proteus include Proteus milabilis (P. milabilis, PM), Proteus morganii (P. morganii), Proteus vulgaris (P. vulgaris), and Proteus rettgeri (P. rettgeri).

[0058] Examples of bacteria belonging to the genus Enterococcus include Enterococcus faecalis and Enterococcus faecium.

[0059] Examples of bacteria in the genus Citrobacter include Citrobacter freundii (C. freundii, CF), Citrobacter amalonaticus (C. amalonaticus), Citrobacter diversus (C. diversus), etc.

[0060] Examples of bacteria in the genus Pseudomonas include Pseudomonas aeruginosa (P. aeruginosa, PA), P. fluorescens, P. phosphorescence, and P. putida.

[0061] Examples of bacteria in the genus Bacillus include Bacillus subtilis (B. subtilis, BS), Bacillus cereus (B. cereus), Bacillus licheniformis (B. licheniformis), and Bacillus megaterium (B. megaterium).

[0062] Bacteria in the genus Serratia include Serratia liquefaciens (S. liquefaciens, SL), Serratia marcescens (S. marcescens), and Serratia fonticola (S. fonticola).

[0063] Examples of bacteria in the genus Rahnella include Rahnella aquatilis (R. aquatilis, RA), Rahnella victoriana (R. victoriana), Rahnella bruchi (R. bruchi), and the like.

[0064] Examples of bacteria belonging to the genus Listeria include Listeria monocytogenes (L. monocytogenes, LM), Listeria innocua (L. innocua), and Listeria ivanovii (L. ivanovii).

[0065] Examples of bacteria in the genus Enterobacter include Enterobacter cloacae (E. cloacae, ECL), Enterobacter aerogenes (E. aerogenes), and Enterobacter sakazakii (E. sakazakii).

[0066] Examples of bacteria belonging to the genus Salmonella include Salmonella enteritidis (S. enteritidis, SE), Salmonella infantis (S. infantis), Salmonella typhimurium (S. typhimurium), and the like.

[0067] [Lysing agent] The lytic agent may be any substance used in the art as long as it can lyse the target bacterium. Examples of the lytic agent include lytic enzymes and surfactants. The lytic enzymes and surfactants may be used alone, or multiple surfactants and / or multiple lytic enzymes may be used in combination.

[0068] The lytic enzyme can be appropriately selected depending on the type of target bacteria, and examples thereof include lysozyme, lysostaphin, labiase, pepsin, glucosidase, galactosidase, achromopeptidase, β-N-acetylglucosaminidase, lysostaphin, acetylglucoaminidase, and achromopeptidase. These enzymes may be natural enzymes or genetically modified enzymes. The amount of the lytic enzyme to be added is appropriately selected depending on the desired final concentration and the volume of the sample liquid. As an example, the lytic enzyme is used at a final concentration of 0.01 μg / mL or more to 100 mg / mL, and therefore the amount to be added is 0.01 to 100,000 μg, preferably 0.1 to 10,000 μg, and more preferably 1 to 1,000 μg.

[0069] The surfactant can be appropriately selected depending on the target bacteria. As the surfactant, a nonionic surfactant, a cationic surfactant, an anionic surfactant, and an amphoteric surfactant may be used alone or in combination. The amount of the surfactant to be attached is appropriately selected depending on the desired final concentration and the volume of the sample liquid. As an example, the surfactant is used at a final concentration of 0.01% to 10%, so that the amount to be attached is 0.1 to 100 mg, preferably 0.1 to 10 mg, and more preferably 1 to 10 mg.

[0070] As the nonionic surfactant, any of ester ether type, ester type, and ether type can be suitably used, and more specifically, polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, fatty acid sorbitan ester, alkyl polyglucoside, fatty acid diethanolamide, alkyl monoglyceryl ether, polysorbates (fatty acid sorbitan ester condensed with several tens of molecules of ethylene oxide), etc. are listed, but are not limited thereto. Particularly preferred examples are polysorbates, more specifically polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (60) sorbitan monostearate, polyoxyethylene (65) sorbitan tristearate, and polyoxyethylene (80) sorbitan monooleate, and polyoxyethylene alkyl phenyl ether, more specifically polyoxyethylene (10) octyl phenyl ether, etc.

[0071] Anionic surfactants include carboxylates, sulfonates, sulfates, and others. Any of the above can be suitably used. More specifically, alkyl ether carbo Examples of the phosphate include phosphates, linear alkylbenzene sulfonates (LAS), α-olefin sulfonates (AOS), dialkyl sulfosuccinates, formaldehyde condensates of naphthalene sulfonates, alkyl sulfates (AS), polyoxyethylene alkyl sulfates (AES) obtained by adding ethylene oxide to higher alcohols to sulfate, and phosphates of higher alcohols and their ethylene oxide adducts. Further specific examples include sodium alkyl sulfates such as sodium dodecyl sulfate and sodium myristyl sulfate, sodium N-acyl sarcosinates such as sodium N-lauroyl sarcosinate and sodium N-myristoyl sarcosinate, sodium dodecylbenzene sulfonate, sodium hydrogenated coconut fatty acid monoglyceride monosulfate, sodium lauryl sulfoacetate, N-acyl glutamates such as sodium N-palmitoyl glutamate, sodium N-methyl-N-acylalanine, and sodium α-olefin sulfonate.

[0072] As cationic surfactants, either amine salt type or quaternary ammonium salt type is suitable. More specific examples of the compounds that can be used include distearyl dimethyl ammonium chloride, salts Benzalkonium chloride, hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide Methyl ammonium bromide, myristyl trimethyl ammonium bromide, etc. This can be done.

[0073] Amphoteric surfactants include amino acid-based (alkylamino fatty acid salts), betaine-based (a Examples include alkyl betaines, amine oxides (alkyl amine oxides), etc. More specific examples include dimethylammoniopropanesulfonate. , dodecyl dimethyl ammonium phosphate, betaine laurate, and amidopropyl Betaine can be given.

[0074] [Second aspect] A second aspect of the present invention relates to a method for producing a chromatography device comprising a chromatographic development membrane carrier in which a capture reagent containing a specific binding substance (capture antibody) that specifically binds to a substance to be detected is immobilized in a detection region.

[0075] [Manufacturing method] The present embodiment is a method for producing a chromatography device including a membrane carrier for chromatographic development in which a capture reagent containing a specific binding substance (capture antibody) that specifically binds to a substance to be detected is immobilized in a detection region, the method comprising the steps of: applying a capture reagent solution containing a specific binding substance (capture antibody) to a detection area of ​​a chromatographic development membrane carrier; drying the chromatographic membrane carrier including the detection area to which the capture reagent solution has been applied; The drying step is carried out at 55° C. or higher for 3 hours or more and 30 hours or less. By carrying out the drying step at 55° C. or higher for 3 to 30 hours in this manner, it is possible to suppress the change in sensitivity over time during the storage period of the manufactured chromatography device.

[0076] The application step refers to applying the capture reagent solution to the detection area of ​​the chromatographic development membrane carrier. In the application step, the capture reagent solution may be applied using a brush, stamp, roller, or the like, or may be applied by dripping using a dispensing device such as an inkjet. In the detection area, the capture reagent solution is usually applied in a line shape, but may be applied in any shape.

[0077] The capture reagent solution used in the application step contains 0.1 mg / mL or more and 20 mg / mL or less of a specific binding substance (capture antibody). From the viewpoint of achieving sufficient sensitivity for detection, 0.3 mg / mL or more is preferable, and 1 mg / mL or more is more preferable. From the viewpoint of suppressing false positives, 10 mg / mL or less is preferable, and 5 mg / mL is more preferable. The capture reagent solution may further contain a buffer, sugar, and / or protein. By applying a capture reagent solution containing at least one of sugar, buffer, and protein to a membrane carrier for chromatographic development and passing through a drying step, the manufacturing method of this embodiment can suppress non-specific binding without including additional blocking and washing steps. This can reduce the number of steps in manufacturing a chromatographic device.

[0078] As the buffer, inorganic salts such as phosphate, acetate, borate, etc., Good's buffers such as CAPSO, MOPSO, TAPS, ACES, Tris hydrochloride, etc. can be used according to the target pH, and the concentration can be 1 mM or more and 500 mM or less. By including a buffer in the capture reagent solution, the stability of the specific binding substance can be increased and the detection performance of the specific binding substance can be improved. From the viewpoint of exerting buffering ability, 3 mM or more is preferable, and 5 mM or more is more preferable. From the viewpoint that the specific binding substance becomes unstable when the ionic strength is high, 300 mM or less is preferable, and 100 mM or less is more preferable.

[0079] As the saccharide, a monosaccharide, disaccharide, or polysaccharide is used, for example, sucrose, maltose, trehalose, etc., and the concentration may be 0.1% by mass or more and 10% by mass or less. By including a saccharide in the capture reagent solution, deterioration of the specific binding substance during storage can be prevented. From the viewpoint of suppressing deterioration, 0.3% by mass or more is preferable, and 1% by mass or more is more preferable. From the viewpoint of suppressing hygroscopicity after production, 7.5% by mass or less is preferable, and 5% by mass or less is more preferable.

[0080] The protein may be casein, albumin, or skim milk, and may be present at a concentration of 0.01% by mass or more and 1.00% by mass or less. By including a protein in the capture reagent solution, it is possible to exert an effect of suppressing non-specific reactions (blocking effect). From the viewpoint of exerting a blocking effect, the concentration is preferably 0.05% by mass or more, and more preferably 0.1% by mass or more. From the viewpoint of not interfering with the binding of a specific binding substance to a chromatographic development membrane carrier, the concentration is preferably 0.5% by mass or less, and more preferably 0.25% by mass or less.

[0081] The application step of the manufacturing method of this embodiment may further include applying another reagent solution to the detection region of the chromatographic development membrane carrier. Such another reagent solution may be a capture reagent solution containing a different specific binding substance, or a control reagent solution. By applying another reagent solution containing a different specific binding substance, a chromatographic device capable of detecting two or more target substances in a sample liquid can be provided. When a control reagent solution is applied, the control reagent solution contains a substance that binds to a labeling antibody. By applying the control reagent solution to the chromatographic development membrane carrier and going through a drying process, the substance that binds to the labeling antibody is immobilized in the detection region of the chromatographic development membrane carrier to form a control site. The control site can bind to the labeling antibody, and when labeling at the control site is observed, it indicates that chromatographic development has been performed appropriately. The control site is usually located downstream of the capture site on the chromatographic development membrane carrier.

[0082] The drying step refers to a step of drying the detection area to which the capture reagent solution has been applied. The temperature of the drying step is 55° C. or higher, preferably 57° C. or higher, and more preferably 59° C. or higher. From the viewpoint of avoiding denaturation of the specific binding substance contained in the capture reagent, the temperature of the drying step is 80° C. or lower, preferably 70° C. or lower, and more preferably 65° C. or lower. The drying step is performed for 3 hours or more and 30 hours or less. From the viewpoint of suppressing sensitivity changes, the drying time is preferably 6 hours or more, and more preferably 10 hours or more. From the viewpoint of suppressing deterioration of detection performance, it is preferably 24 hours or less, and more preferably 18 hours or less. As an example, the drying step can be performed by using a constant temperature dryer. In the conventional technology, the drying step was avoided at a high temperature exceeding 50° C. in order to avoid denaturation of the specific binding substance contained in the capture reagent.

[0083] The drying process utilizes the hydrophobicity of the specific binding substance to immobilize it on the chromatographic development membrane carrier by physical adsorption. Immobilization is achieved by applying a capture reagent solution containing the specific binding substance to the detection area of ​​the chromatographic development membrane carrier and drying it. The immobilized specific binding substance remains on the chromatographic development membrane carrier even when the sample liquid is chromatographically developed on the chromatographic development membrane carrier during use of the chromatography device, and is able to capture a complex between the target substance and the labeling antibody in the sample liquid. This makes it possible to determine the presence or absence of the target substance by detecting the label of the complex.

[0084] The method for manufacturing a chromatography device includes a step of assembling a chromatographic development membrane carrier that has been subjected to an immobilization step and a drying step. As an example, a chromatographic development membrane carrier to which a specific binding substance is bound is laminated on a substrate, and necessary members for the chromatographic device, such as a sample contact member, an absorption member, a filter member, and a cover member, are laminated to assemble the chromatographic device. An adhesive substance may be disposed on the substrate to fix each member, or an adhesive substance may be disposed between the members. The above-mentioned members are disposed in succession in the order described above so that the sample liquid can be developed on the chromatographic membrane from the sample contact member through the filter member and the chromatographic development membrane carrier to the absorption member. Here, "disposed in succession in succession" may mean that each member is partially overlapped and in contact with each other, or may be disposed side by side so that the wall surfaces are in contact with each other without overlapping. Each member is in the form of a substantially rectangular sheet, and the thickness may vary depending on each member, but is, for example, 50 to 2000 μm. The sample contact member side of the chromatographic device is called the upstream side, and the detection region side is called the downstream side. The direction from upstream to downstream is also called the chromatographic development direction.

[0085] [Chromatography equipment] The chromatography device manufactured in this embodiment is a device (also called a strip) for developing a sample liquid on a chromatographic development membrane carrier by utilizing the capillary principle and detecting a target substance in the sample liquid. An example of the chromatography device 10 of this embodiment will be described with reference to FIG. 22. The chromatography device 10 includes, from upstream to downstream, a sample contact member 5, a chromatographic development membrane carrier 3, and an absorption member 7 (also called an absorption pad). The chromatography device 10 may further include any member that can be included in the chromatography device 10, such as a base material 8, a filter member 6, and a cover member 11. When the sample liquid contacts the sample contact member 5, the sample liquid can be developed from the sample contact member 5 through the chromatographic development membrane member 3 to the absorption member 7 by utilizing the capillary principle. The members may be in contact with each other by contacting their walls, or may be in contact by being partially stacked. Depending on the type of specimen liquid, one or more filter members 6 may be disposed between the sample contact member 5 and the chromatographic development membrane member 3. The target substance contained in the developed specimen liquid is captured by a specific binding substance fixed to the capture site 4 of the detection region of the chromatographic development membrane member 3. The captured target substance forms a complex with a labeling antibody, and the presence or absence of the target substance is detected by detecting the label. The labeling antibody that binds to the target substance may be added to the specimen liquid in advance, or a labeling antibody-attached member 1 to which a labeling antibody is attached may be provided in the chromatographic device 10 to cause an antigen-antibody reaction between the target substance and the labeling antibody during use. When the target substance is bacteria, a bacteriolytic agent-attached member 2 may be provided in the chromatographic device 10 to cause a bacteriolytic reaction during use. In order to prevent the development of the specimen liquid from places other than the sample contact member 5, the chromatographic device may be entirely or partially covered with a water-impermeable cover member 11. The filter member 6 can remove impurities from the sample liquid and prevent clogging of the chromatographic development membrane carrier. The chromatography device 10 is manufactured by laminating each member on the substrate 8.

[0086] The sensitivity of a chromatography device changes over the storage period after manufacture. In the case of a chromatography device manufactured by a conventional technology (i.e., a manufacturing method including a drying step at 50°C or less), the sensitivity immediately after manufacture (hereinafter referred to as initial sensitivity) is low, and the sensitivity increases after a storage period of several months. In order to test the change in sensitivity during storage, a heating accelerated test is performed. The temperature for the heating accelerated test can be a predetermined temperature selected from 45 to 55°C, and the test can be performed for a predetermined period of 1 day to 4 weeks. One week at 50°C can be used as an example of a condition for the heating accelerated test. A heating accelerated test of 50°C for 1 week is usually considered to be equivalent to a storage period of 3 to 6 months at room temperature. In order to evaluate the change in sensitivity from the initial sensitivity, the change in sensitivity is expressed as the rate of change in sensitivity after the heating accelerated test (hereinafter referred to as sensitivity after accelerated test) from the initial sensitivity:

number

[0087] The chromatographic development membrane carrier 3 is not particularly limited as long as it can perform chromatographic development, but is preferably nitrocellulose, mixed nitrocellulose ester, polyvinylidene fluoride, nylon, etc. One example is a nitrocellulose membrane. A capture reagent solution containing a specific binding substance is applied to the detection region of the chromatographic development membrane carrier and dried, whereby the specific binding substance is immobilized to form a capture site. Furthermore, a substance that binds to the labeling antibody can be immobilized downstream of the capture site to form a control site. The capture site 4 and the control site can be formed, for example, linearly, in the detection region of the chromatographic development membrane carrier.

[0088] The sample contact member 5 is a member that comes into contact with the sample (analyte liquid) and may be any member that allows the sample liquid to pass through. As the sample contact member 5, a fiber assembly, such as filter paper, cloth, or nonwoven fabric, is used. Examples of fibers used in the sample contact member 5 include chemical fibers such as cellulose fiber and glass fiber, and natural fibers such as cotton. The sample contact member 5 may also function as a filter member by adjusting it to have an exclusion size due to the fiber assembly. A portion of the sample contact member is made into a sample contact area (also called an immersion area 9), and this area comes into contact with the sample liquid.

[0089] The filter member 6 is a member that removes some substances in the sample liquid. The sample liquid contains impurities that clog the chromatographic development membrane carrier 3, and in order to remove such substances, the filter member 6 can be arranged upstream of the chromatographic development membrane carrier 3. The filter member 6 is an aggregate of fibers having a predetermined retention particle size. As the fibers used for the filter member, chemical fibers, particularly glass fibers, can be used. As an example, when milk is used as the sample, the filter member 6 can be a fat globule removing member that removes fat globules in milk. The fat globules in milk have a diameter of about 1 to 10-odd μm, and as an example, a filter having a retention particle size of, for example, 1 μm to 10 μm, preferably 1 to 5 μm, that can remove milk fat globules can be used. The filter member 6 does not have to be arranged depending on the type of sample liquid, but one or more filter members 6 can be arranged.

[0090] The absorbing member 7 is a water-absorbent member arranged at the downstream end, and is a member for continuing chromatographic development by absorbing the solution chromatographically developed downstream. Any water-absorbent member may be used, and cotton, cloth, paper, etc. may be used. The absorbing member 7 is arranged so as to straddle a part of the downstream end of the chromatographic development membrane carrier and the substrate 8.

[0091] The substrate 8 is a water-impermeable thin layer member used for laminating each component thereon in manufacturing the chromatography device 10. For example, it may be made of polyester or polystyrene, and may have an adhesive attached thereto in order to fix each component.

[0092] The labeling antibody-attached member 1 refers to a member to which a labeling antibody is attached. The labeling antibody-attached member 1 may be arranged in any position as long as at least a portion of the member is immersed in the sample liquid during use. When the chromatography device 10 is in use, at least a portion of the labeling antibody-attached member 1 arranged in the immersion area 9 of the chromatography device 10 is immersed in the sample liquid, and a portion of the labeling antibody attached in a dissolvable manner is eluted and diffused into the sample liquid. This causes an antigen-antibody reaction in the sample liquid, ensuring a sufficiently long reaction time. By ensuring a sufficiently long reaction time, the amount of complex formed between the analyte and the labeling antibody increases.

[0093] The labeling antibody-attached member 1 may be made of any material as long as the labeling antibody is attached to it. The labeling antibody is preferably attached to the labeling antibody-attached member 1 in a manner that allows it to be dissolved. The labeling antibody-attached member 1 may be manufactured by impregnating a water-absorbent member with a solution containing the labeling antibody, and optionally drying it. Examples of water-absorbent members include fiber aggregates such as paper, woven fabric, and nonwoven fabric, and porous bodies such as sponges. In another embodiment, the labeling antibody-attached member 1 may be one in which a solution containing the labeling antibody is directly attached to a member constituting the chromatography device 10. When attaching, the labeling antibody-attached member 1 may be made into a water-soluble adhesive substance by adding a thickener or the like. From the viewpoint of improving storage stability, the labeling antibody-attached member may contain sugars such as sucrose and trehalose.

[0094] The amount of the labeling antibody to be added can be appropriately selected depending on the amount of the test solution, the ability to form a complex with the complex, etc. As an example, an antibody solution with an antibody concentration adjusted so that the absorbance at a wavelength of 540 nm when the antibody solution is diluted 21 times is 0.1 to 2.0 can be used.

[0095] The lytic agent-attached member 2 refers to a member to which a lytic agent is attached. The lytic agent-attached member 2 may be arranged in any position as long as at least a part of it is immersed in the specimen liquid during use. The lytic agent-attached member 2 may be arranged between the substrate 8 and another member, or may be arranged on the sample contact member 5, the filter member 6, the labeling antibody-attached member 1, the cover member 11, or on the back side of the substrate 8 as long as a part of it is arranged in the immersion area 9. The labeling antibody-attached member 1 and the lytic agent-attached member 2 are both arranged in the immersion area 9, but may be in contact with each other or may be arranged separately. When the labeling antibody-attached member 1 and the lytic agent-attached member 2 are in contact with each other, their walls may be in contact with each other, or they may be partially or entirely overlapped. As an example, the lytic agent-attached member 2 may be arranged straddling the substrate 8 and the labeling antibody-attached member 1. In this case, the lytic agent-attached member 2 may cover only a part of the labeling antibody-attached member 1, or may completely cover the labeling antibody-attached member 1. A sample contact member 5 or a filter member 6 may be further disposed on the lytic agent-attached member 2. To detect bacteria in a sample liquid with high sensitivity, it is preferable to lyse the bacteria with a lytic agent and extract the substance to be detected from the bacteria.

[0096] The lytic agent-imparted member 2 may be made of any material as long as the lytic agent is eluted. The lytic agent-imparted member 2 may be manufactured by impregnating a water-absorbent member with a solution containing the lytic agent and, if necessary, drying the same. Examples of water-absorbent members include fiber aggregates such as paper, woven fabric, and nonwoven fabric, and porous bodies such as sponges. In another embodiment, the lytic agent-imparted member 2 may be a member that is directly fixed with a solution containing the lytic agent to a member that constitutes the immunochromatographic device 10. When fixing the lytic agent-imparted member 2, a thickener or the like may be added to make the lytic agent-imparted member 2 a water-soluble adhesive substance. From the viewpoint of improving storage stability, the lytic agent-imparted member may contain sugars such as sucrose and trehalose. When the lytic agent-imparted member 2 is arranged in the immunochromatographic device 10, the lytic agent-imparted member 2 may be arranged at any position in the immunochromatographic device 10 as long as it is arranged so that at least a part of the lytic agent-imparted member 2 is immersed in the sample liquid when the immunochromatographic device is in use. The labeling antibody-attached member 1 and the lytic agent-attached member 2 may be formed as a single member by impregnating a water-absorbent member with a solution containing the labeling antibody and the lytic agent.

[0097] [Lysing agent] The lytic agent may be any substance used in the art as long as it can lyse the target bacterium. Examples of the lytic agent include lytic enzymes and surfactants. The lytic enzymes and surfactants may be used alone, or multiple surfactants and / or multiple lytic enzymes may be used in combination.

[0098] [Lytic enzyme] As the lytic enzyme, any lytic enzyme used in the present technical field can be used as long as it is a substance capable of lysing the target bacterium. The lytic enzyme can be appropriately selected according to the type of the target bacterium, and examples thereof include lysozyme, lysostaphin, pepsin, glucosidase, galactosidase, achromopeptidase, β-N-acetylglucosaminidase, acetylglucoaminidase, and achromopeptidase. These enzymes may be natural enzymes or genetically modified enzymes. The amount of the lytic enzyme to be applied is appropriately selected according to the desired final concentration and the volume of the sample liquid. As an example, the lytic enzyme is used at a final concentration of 0.01 μg / mL or more and 100 mg / mL or less, and therefore the amount to be applied is 0.01 μg or more and 100 mg or less, preferably 0.1 μg or more and 10 mg or less, and more preferably 1 μg or more and 3 mg or less.

[0099] [Surfactants] The surfactant can be appropriately selected depending on the target bacteria. As the surfactant, a nonionic surfactant, a cationic surfactant, an anionic surfactant, and an amphoteric surfactant may be used alone or in combination. The amount of the surfactant to be attached is appropriately selected depending on the desired final concentration and the volume of the sample liquid. As an example, the surfactant is used at a final concentration of 0.01% by mass or more and 10% by mass or less, so that the amount to be attached is 0.1 mg to 100 mg or less, preferably 0.1 mg to 10 mg or less, and more preferably 1 mg to 10 mg or less.

[0100] As the nonionic surfactant, any of ester ether type, ester type, and ether type can be suitably used, and more specifically, polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, fatty acid sorbitan ester, alkyl polyglucoside, fatty acid diethanolamide, alkyl monoglyceryl ether, polysorbates (fatty acid sorbitan ester condensed with several tens of molecules of ethylene oxide), etc. are listed, but are not limited thereto. Particularly preferred examples are polysorbates, more specifically, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (60) sorbitan monostearate, polyoxyethylene (65) sorbitan tristearate, and polyoxyethylene (80) sorbitan monooleate, and polyoxyethylene alkyl phenyl ether, more specifically, polyoxyethylene (10) octyl phenyl ether, etc.

[0101] Anionic surfactants include carboxylates, sulfonates, sulfates, and others. Any of the above can be suitably used. More specifically, alkyl ether carbo Examples of the phosphate include phosphates, linear alkylbenzene sulfonates (LAS), α-olefin sulfonates (AOS), dialkyl sulfosuccinates, formaldehyde condensates of naphthalene sulfonates, alkyl sulfates (AS), polyoxyethylene alkyl sulfates (AES) obtained by adding ethylene oxide to higher alcohols to sulfate, and phosphates of higher alcohols and their ethylene oxide adducts. Further specific examples include sodium alkyl sulfates such as sodium dodecyl sulfate and sodium myristyl sulfate, sodium N-acyl sarcosinates such as sodium N-lauroyl sarcosinate and sodium N-myristoyl sarcosinate, sodium dodecylbenzene sulfonate, sodium hydrogenated coconut fatty acid monoglyceride monosulfate, sodium lauryl sulfoacetate, N-acyl glutamates such as sodium N-palmitoyl glutamate, sodium N-methyl-N-acylalanine, and sodium α-olefin sulfonate.

[0102] As cationic surfactants, either amine salt type or quaternary ammonium salt type is suitable. More specific examples of the compounds that can be used include distearyl dimethyl ammonium chloride, salts Benzalkonium chloride, hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide Methyl ammonium bromide, myristyl trimethyl ammonium bromide, etc. This can be done.

[0103] Amphoteric surfactants include amino acid-based (alkylamino fatty acid salts), betaine-based (a Examples include alkyl betaines, amine oxides (alkyl amine oxides), etc. More specific examples include dimethylammoniopropanesulfonate. , dodecyl dimethyl ammonium phosphate, betaine laurate, and amidopropyl Betaine can be given.

[0104] A specific binding substance refers to a substance that can specifically bind to a substance to be detected. Examples of specific binding substances include physiologically active substances or analogs thereof that have interactions such as a ligand and a receptor, substrates or analogs thereof in an enzyme reaction, and antigens or analogs thereof in an antigen-antibody reaction. There are no particular limitations on the specific binding substance as long as it falls within these categories, and for example, proteins, peptides, amino acids, sugar chains, lipids, or complexes thereof (glycoproteins, glycolipids, fatty acid-modified proteins, and nucleic acids such as DNA and RNA) can be used as the specific binding substance. In addition, from the viewpoint of binding to any substance to be detected, antibodies are preferred as the specific binding substance. When an antibody is used as the specific binding substance, the chromatography device can be called an immunochromatography device.

[0105] Any substance can be used as the substance to be detected, so long as there is a specific binding substance that specifically binds to the substance to be detected. Examples of the substance to be detected include physiologically active substances or their analogs that have interactions such as a ligand and a receptor, substrates or their analogs in an enzyme reaction, and antigens or their analogs in an antigen-antibody reaction. There are no particular limitations on the substance as long as it falls within these categories, and the substance to be detected may be, for example, a protein, a peptide, an amino acid, a sugar chain, a lipid, or a complex thereof (glycoprotein, glycolipid, fatty acid-modified protein, DNA, RNA, and other nucleic acid). In addition, the substance may be an artificial substance such as a low molecular weight compound intended as a therapeutic drug or a part of an antibody (Fab' fragment, etc.).

[0106] The sample liquid may be any liquid containing the substance to be detected. As an example, it is preferable that the sample liquid is a food or environmental sample liquid. The food sample liquid refers to any sample liquid obtained from any food or drink. As an example, the food sample may be the food or drink itself, or the sample liquid obtained by diluting, crushing, wiping the surface of the food or drink with a swab and / or suspending the food or drink. The environmental sample refers to any sample liquid obtained from the environment. As an example, there is a sample liquid obtained by wiping the surface of a substance with a swab and dispersing it in a solution. When bacteria are detected as the substance to be detected, milk can be used as the sample liquid in particular.

[0107] When an antibody is used as a specific binding substance, not only proteins, peptides, sugars, lipids, nucleic acids, or complexes thereof, but also any substance that can be recognized by an antibody, such as metals or compounds, can be used as the detection substance. In particular, from the viewpoint of use in the food and livestock fields, bacteria are used as the detection substance, and antibodies that bind to proteins derived from bacteria are used as the specific binding substance. When such an antibody specifically binds to some component of a certain bacterium as an antigen, it can be said to be an antibody capable of detecting bacteria. The bacterial component that serves as the antigen of the antibody of the present invention is not limited. It may be a component contained in the cell wall or cell membrane exposed outside the bacterial cell, or a component contained in the cytoplasm, organelles, nucleus, etc. that is not exposed outside the bacterial cell, that is, an intracellular antigen. According to one embodiment, the antibody of the present invention preferably undergoes an antigen-antibody reaction with an intracellular antigen of the bacteria, more preferably undergoes an antigen-antibody reaction with a ribosomal protein of the bacteria, and particularly preferably undergoes an antigen-antibody reaction with ribosomal protein L7 / L12.

[0108] The substance to be detected forms a complex with the labeling antibody in advance, and when the specific binding substance captures the substance to be detected at the capture site, the label can be detected at the capture site. The labeling antibody that does not form a complex with the substance to be detected is captured at the control site, and the label is detected at the control site, thereby indicating that the chromatographic development has been performed appropriately. The label may be appropriately selected depending on the detection method, and specific examples of the label include metal colloids such as gold colloid, platinum colloid, and palladium colloid; non-metal colloids such as selenium colloid, alumina colloid, and silica colloid; insoluble granular substances such as colored resin particles, dye colloids, and colored liposomes; color reaction catalytic enzymes such as alkaline phosphatase, peroxidase, and luciferase; fluorescent dyes, radioisotopes; chemiluminescent labels, bioluminescent labels, and electrochemiluminescent labels. The method of labeling an antibody to obtain a labeled antibody is not particularly limited, and specific examples of the labeling method include physical adsorption using the hydrophobicity of the antibody and chemical binding using the functional group of the antibody.

[0109] [Third aspect] A third aspect of the present invention relates to an immunochromatographic device for detecting a target substance in a milk sample through an antigen-antibody reaction, and a method for detecting a target substance in a sample liquid through an antigen-antibody reaction using the immunochromatographic device.

[0110] [Immunochromatography device] The immunochromatographic device is an immunochromatographic device 10 (also called a strip) for detecting a substance to be detected by an antigen-antibody reaction. In this embodiment, the immunochromatographic device particularly relates to a dipstick-type immunochromatographic device. The dipstick-type immunochromatographic device relates to an immunochromatographic device that induces chromatographic membrane development by immersing the immersion area 9 of the immunochromatographic device 10 in a solution. When using the immunochromatographic device of this embodiment, the immersion area 9 of the immunochromatographic device 10 is immersed in a milk sample contained in a milk sample holding container 20, and the milk sample solution is developed using the principle of a chromatogram. A complex formed between a labeling antibody and an antigen contained in the developed milk sample solution is captured by a capture antibody fixed on the chromatographic development membrane member 3 in the detection area 4, and the label of the labeling antibody is detected, and the presence or absence of the substance to be detected is detected. The dipstick-type immunochromatographic device may be composed of only the immunochromatographic device 10, or may include a sample liquid holding container 20 for holding a milk sample, and may further include the usual components included in an immunochromatographic device, such as an instruction manual.

[0111] The immunochromatography device 10 according to the present embodiment includes an immersion region 9 and a detection region 4, and is configured as follows: a labeling antibody-attached member 1 to which a labeling antibody is attached, the labeling antibody forming a first complex by an antigen-antibody reaction with a substance to be detected in a milk sample; A first filter member 6-1 having a retention particle size capable of removing milk fat globules from milk; a second filter member 6-2 having a particle size smaller than that of the first filter member 6-1; and A chromatographic development membrane member 3 having a detection region in which a capture antibody for a substance to be detected is fixed The immunochromatography device 10 may further include members used in the immunochromatography device 10, such as a lytic agent-applied member 2 to which a lytic agent is applied, a sample contact member 5, an absorbing member 7, a substrate 8, and a cover member 11.

[0112] When the immunochromatographic device 10 of this embodiment is used, the above-mentioned components are arranged in the order described above so that the milk sample is developed from the upstream to the downstream in the order of the first filter member, the second filter member, and the chromatographic development membrane member. Here, "arranged in order" means that the components may be partially overlapped and in contact with each other, or may be arranged side by side so that the wall surfaces are in contact without overlapping. Each component is in the form of a substantially rectangular sheet, and the thickness may vary depending on the component, but is, for example, 50 to 2000 μm. In this embodiment, in order to promote chromatographic development, the first filter member 6-1 and the second filter member 6-2 are partially overlapped and in contact with each other, and the overlap is represented by the symbol 100, and the distance is the overlap distance X. When the absorbing member 7 is arranged, the solution is finally absorbed by the absorbing member 7. The immersion area 9 side of the immunochromatographic device 10 is called the upstream side, and the detection area 4 side is called the downstream side. The direction from the upstream to the downstream is also called the chromatographic development direction.

[0113] The arrangement of each component of the immunochromatography device 10 will be described with reference to the drawings. Each component of the immunochromatography device 10 is arranged on a substrate 8 and / or other components in a superimposed manner. The side on which each component, particularly the chromatographic development membrane member 3, is arranged is the front side of the substrate 8. Each component of the immunochromatography device 10 may be adhered to each other using an adhesive substance. An adhesive substance may be arranged on the substrate 8. In addition, the substrate 8 may be removed after being arranged on the substrate 8. As an example, the chromatographic development membrane member 3 is arranged on the substrate 8, and the absorbing member 7 (absorption pad) is arranged so as to straddle at least a part of the downstream end of the chromatographic development membrane member 3 and the substrate 8 (FIG. 24). The second filter member 6-2 is arranged so as to straddle at least a part of the upstream end of the chromatographic development membrane member 3 and the substrate 8, and the first filter member 6-1 is arranged on at least a part of the upstream end of the second filter member 6-2 and the substrate 8. A sample contact member 5 may be disposed on at least a part of the first filter member 6-1 and the substrate 8 (FIG. 24). The sample contact member 5 may not be disposed (FIG. 25). When the sample contact member 5 is not disposed, the first filter member functions as the sample contact member 5 and contacts the specimen sample. A cover member 11 is disposed on the outermost surface of the sample contact member 5 or the first filter member 6-1 formed on the substrate 8 in this manner, except for a part of the sample contact member 5 or the first filter member 6-1. The cover member 11 is a water-impermeable member, and prevents the members below the cover member 11 from contacting the solution. In FIGS. 24 to 27, the cover member 11 is shown not to be in contact with each member for understanding the configuration, but in reality, it contacts and adheres to each member to provide the immunochromatography device 10. The area where the cover member 11 is not present and the members are exposed can be the water-immersed area 9. With the above basic configuration, when the immersion area 9 of the immunochromatography device 10 is immersed in a sample liquid, the sample liquid is spread on the chromatographic development membrane member 3 through the first filter member 6-1 and then the second filter member 6-2 via the sample contact member 5. A labeling antibody-attached member 1 and / or a lytic agent-attached member 2 may be disposed in the immersion area.As a result, when the immunochromatographic device 10 is immersed in a milk sample, the labeling antibody and / or lytic agent dissolves from the immersion area and diffuses into the milk sample, causing a lytic reaction and / or an antigen-antibody reaction in the milk sample, and the formed complex is developed on the chromatographic development membrane member 3 via the first filter member 6-1 and the second filter member 6-2.

[0114] The immunochromatographic device 10 is in the form of a long, approximately rectangular sheet, particularly a rectangle, with the long side in the direction of chromatographic development. As an example, the length of the long side is 40 mm to 100 mm, and the length of the short side is 3 mm to 20 mm. From the viewpoint of increasing the distance of chromatographic development, the long side of the immunochromatographic device is preferably 50 mm or more, more preferably 60 mm or more. From the viewpoint of facilitating operation of the device, the long side of the immunochromatographic device is preferably 90 mm or less, more preferably 80 mm or less. The thickness of the thickest part of the immunochromatographic device may vary depending on the type of overlapping member, but is, as an example, 0.5 mm to 3 mm.

[0115] [Filter material] The filter member is a member for removing fat globules in milk, and can also be called a fat globule removing member. In this embodiment, a plurality of filter members are used, including a first filter member 6-1 and a second filter member 6-2 in particular. The first filter member 6-1 is disposed upstream of the second filter member. These filter members may be any member capable of removing fat globules in milk. As an example, a filter made of glass fiber may be used. Fat globules in milk have a diameter of about 1 to 10 μm. Therefore, it is preferable that the filter member has a retention particle size capable of removing milk fat globules of these sizes. Conventionally, one filter was used, but there was a limit to the fat globules that could be removed with one filter, and clogging occurred in some cases. Therefore, in this embodiment, two or more filters are used, and the retention particle size of the second filter member 6-2 may be smaller than that of the first filter member 6-1. The overall length of the filter member used can be appropriately selected, but from the viewpoint of miniaturization of the device, it is preferable that it does not exceed half the length of the immunochromatography device 10. As an example, the first filter member 6-1 and the second filter member 6-2 are used so that the total length of the overlapped first filter member 6-1 and second filter member 6-2 (also referred to as the total length of the filter member) is 70 mm or less, preferably 55 mm or less, 40 mm or less. From the viewpoint of removing fat globules, a filter member having a total length of usually 5 mm or more, preferably 10 mm or more, more preferably 15 mm or more, and even more preferably 25 mm or more is used. The total length of the overlapped first filter member 6-1 and the second filter member 6-2 is 10% to 70% of the total length of the immunochromatography device. From the viewpoint of removing fat globules well, the total length is preferably 20% or more of the total length of the immunochromatography device, more preferably 25% or more, and even more preferably 30% or more. From the viewpoint of reducing dead volume and improving sensitivity, the total length is preferably 60% or less of the total length of the immunochromatography device, and even more preferably 50% or less. The particle size retention of the filter member can be measured by a method for measuring the particle size that can be retained by 98% or more in a liquid.

[0116] The particle size retention of the first filter can be appropriately selected as long as it is capable of removing milk fat globules, and as an example, a member having a particle size retention of 3 to 10 μm, preferably 3 to 8 μm, and more preferably 3 to 4 μm can be used as the first filter member 6-1. The particle size retention of the second filter member 6-2 may be any size smaller than that of the first filter member 6-1, and a member achieving a particle size retention of, for example, 0.5 to 3 μm, and preferably 1 to 2.5 μm can be used. Fat globules of relatively large size (3 μm or more) can be removed by the first filter, and then fat globules of smaller size (less than 3 μm) can be removed by the second filter.

[0117] The length of the first filter member 6-1 in the upstream-downstream direction is preferably 5 mm or more, more preferably 10 mm or more, even more preferably 15 mm or more, and even more preferably 21 mm or more, from the viewpoint of exerting a fat globule removal effect. Also, from the viewpoint of suppressing loss of the amount of sample liquid to be subjected to the test, it is preferably 40 mm or less, more preferably 35 mm or less, even more preferably 32 mm or less, and even more preferably 29 mm or less.

[0118] The length of the second filter member 6-2 in the upstream-downstream direction is preferably 5 mm or more, more preferably 10 mm or more, and even more preferably 14 mm or more, from the viewpoint of exerting the fat globule removal effect, and is preferably 40 mm or less, more preferably 30 mm or less, and even more preferably 20 mm or less, from the viewpoint of suppressing loss of the sample liquid volume used in the test.

[0119] In this embodiment, it was found that poor chromatographic development occurs when the distance (overlapping distance X) of the overlap (represented by the reference symbol 100) between the first filter member 6-1 and the second filter member 6-2 is small (FIG. 25(A)). In order to obtain good chromatographic development, it is preferable that the direct overlap distance X between the first filter member and the second filter member is sufficiently secured with respect to the amount of liquid to be developed chromatographically. The amount of liquid to be developed chromatographically refers to the amount of liquid that can be absorbed by the chromatographic development membrane member 3 and the absorption member 7. The amount of liquid to be developed chromatographically can be determined by subtracting the weights of the chromatographic development membrane member 3 and the absorption member 7 before chromatographic development from the weights of the chromatographic development membrane member 3 and the absorption member 7 after chromatographic development. A member that does not absorb liquid, such as the base material 8, may also be included in the measurement. Therefore, more specifically, the ratio (X / Y mm / g) of the overlapping distance X to the total amount of absorbed liquid Y of the chromatographic development member and the absorbing member is preferably 30 or more, more preferably 70 or more, and even more preferably 100 or more. Moreover, if the overlapping distance X is too long, the loss of the amount of sample liquid used in the test increases, resulting in a decrease in sensitivity, so that X / Y is preferably 500 or less, more preferably 350 or less, and even more preferably 150 or less.

[0120] [Membrane material for chromatographic development] The chromatographic development membrane member 3 refers to a membrane member that performs chromatographic development using the principle of chromatogram. The chromatographic development membrane member 3 is not particularly limited as long as it can perform immunochromatography, but is preferably made of nitrocellulose, mixed nitrocellulose ester, polyvinylidene fluoride, nylon, etc. As an example, a nitrocellulose membrane is used. A capture antibody can be immobilized on the chromatographic development membrane member 3 to form a capture region. Furthermore, an antigen can be immobilized downstream of the capture region to form a control region. The capture region and the control region can be formed linearly on the chromatographic development membrane member 3, and together they constitute the detection region 4.

[0121] [Material for labeling antibody attachment] The labeling antibody-attached member 1 refers to a member to which a labeling antibody is attached. The labeling antibody-attached member 1 may be arranged in any position as long as at least a part of it is immersed in a milk sample during use. The labeling antibody-attached member 1 may be arranged between the base material 8 and another member (e.g., the sample contact member 5 or the first filter member 6-1) (FIGS. 24 and 25), or may be arranged on the sample contact member 5, the first filter member 6-1, the lytic agent-attached member 2, the cover member 11, or on the back side of the base material 8, as long as a part of it is arranged in the immersion area.

[0122] When the immunochromatography device is in use, at least a portion of the labeling antibody-attached member 1 arranged in the immersion area 9 of the immunochromatography device is immersed in the milk sample, and the labeling antibody attached in a dissolvable manner is eluted and diffused into the milk sample. This causes an antigen-labeling antibody reaction in the milk sample, ensuring a sufficiently long reaction time. By ensuring a sufficiently long reaction time, the amount of complex formed from the antigen-labeling antibody increases. In a dipstick-type immunochromatography device in which the labeling antibody forms a complex in the test solution and the complex is subjected to chromatographic development, the membrane member 3 for chromatographic development may become clogged with fat globules contained in the milk sample, making it important to adjust the filter member 6.

[0123] The labeling antibody-attached member 1 may be made of any material as long as the labeling antibody is attached to it. The labeling antibody is preferably attached to the labeling antibody-attached member 1 in a manner that allows it to be dissolved. The labeling antibody-attached member 1 may be manufactured by impregnating a water-absorbent member with a solution containing the labeling antibody, and optionally drying it. Examples of water-absorbent members include fiber aggregates such as paper, woven fabric, and nonwoven fabric, and porous bodies such as sponges. In another embodiment, the labeling antibody-attached member 1 may be one in which a solution containing the labeling antibody is directly attached to a member constituting the immunochromatography device 10. When attaching the labeling antibody-attached member 1, a thickener or the like may be added to make it a water-soluble adhesive substance.

[0124] The amount of the labeling antibody to be added can be appropriately selected depending on the amount of the test solution, the ability to form a complex with the complex, etc. As an example, an antibody solution with an antibody concentration adjusted so that the absorbance at a wavelength of 540 nm when the antibody solution is diluted 21 times is 0.1 to 2.0 can be used.

[0125] [Materials impregnated with lytic agent] The lytic agent-imparted member 2 refers to a member to which a lytic agent is impregnated. The lytic agent-imparted member 2 may be arranged in any position as long as at least a part of it is immersed in a milk sample during use. The lytic agent-imparted member 2 may be arranged between the base material 8 and another member (e.g., the sample contact member 5 or the first filter member 6-1) (FIGS. 24, 25(A), 25(B), 26(A), and 26(B)), or may be arranged on the sample contact member 5, the first filter member 6-1, the labeling antibody-imparted member 1, the cover member 11, or on the back side of the base material 8, as long as a part of it is arranged in the immersion area 9. The labeling antibody-imparted member 1 and the lytic agent-imparted member 2 are both arranged in the immersion area 9, but may be in contact with each other or may be arranged apart from each other. When the labeling antibody-imparted member 1 and the lytic agent-imparted member 2 are in contact with each other, their walls may be in contact with each other, or they may be partially or entirely overlapped. As an example, the bacteriolytic agent-attached member 2 may be arranged across the substrate 8 and the labeling antibody-attached member 1 (FIG. 26(C)). In this case, the bacteriolytic agent-attached member 2 may cover only a part of the labeling antibody-attached member 1, or may completely cover the labeling antibody-attached member 1 as shown in FIG. 26(C). A sample contact member 5 may be further arranged on the bacteriolytic agent-attached member 2 (FIG. 24), or a first filter member 6-1 may be arranged (FIGS. 25(A), 25(B), 26(A), 26(B), and 26(C)). To detect bacteria in milk with high sensitivity, it is preferable to lyse the bacteria with a bacteriolytic agent and extract the substance to be detected from the bacteria. When a lytic enzyme is used as the bacteriolytic agent, the lysis reaction by the enzyme takes several tens of seconds to several minutes. Therefore, when the bacteriolysis reaction and the detection operation using the immunochromatography device are performed simultaneously, it takes several tens of seconds to several minutes from the start of the detection operation until the detection substance is extracted, and the detection signal appears after several minutes. Therefore, in order to detect bacteria with high sensitivity, it is important that the development of the sample liquid in the immunochromatography device proceeds smoothly even after several minutes without causing blockage.

[0126] The lytic agent-imparted member 2 may be made of any material as long as the lytic agent is eluted. The lytic agent-imparted member 2 may be manufactured by impregnating a water-absorbent member with a solution containing the lytic agent and, if necessary, drying the member. Examples of water-absorbent members include fiber aggregates such as paper, woven fabric, and nonwoven fabric, and porous bodies such as sponges. In another embodiment, the lytic agent-imparted member 2 may be a member in which a solution containing the lytic agent is directly fixed to a member constituting the immunochromatography device 10. When fixing the lytic agent-imparted member 2, a thickener or the like may be added to make the lytic agent-imparted member 2 a water-soluble adhesive substance. When the lytic agent-imparted member 2 is arranged in the immunochromatography device 10, the lytic agent-imparted member 2 may be arranged at any position in the immunochromatography device 10 as long as at least a part of the lytic agent-imparted member 2 is immersed in a milk sample when the immunochromatography device is in use. The labeling antibody-attached member 1 and the lytic agent-attached member 2 may be formed as a single member by impregnating a water-absorbent member with a solution containing the labeling antibody and the lytic agent.

[0127] [Sample contact parts] The sample contact member 5 is a member that comes into contact with the milk sample, and may be any member that allows the solution to pass through. As the sample contact member 5, a fiber assembly, such as filter paper, cloth, or nonwoven fabric, is used. Examples of fibers used for the sample contact member 5 include fibers such as cellulose derivatives, chemical fibers such as glass fibers and polyester fibers, and natural fibers such as cotton. The sample contact member 5 does not need to be disposed, in which case the first filter member 6-1 is exposed in the immersion region and can function as the sample contact member 5 (FIGS. 25(A), 25(B), 26(A), 26(B), and 26(C)).

[0128] [Absorption material] The absorbing member 7 is a water-absorbent member disposed at the downstream end, and is a member for continuing chromatographic development by absorbing the solution chromatographically developed downstream. Any water-absorbent material may be used, such as cotton, cloth, or paper. The absorbing member 7 is disposed so as to straddle a part of the downstream end of the chromatographic development membrane member 3 and the substrate 8.

[0129] [Base material] The substrate 8 is a water-impermeable thin layer member used for superposing each component thereon in manufacturing the immunochromatography device 10. As an example, it may be made of polystyrene, and an adhesive substance may be attached to fix each component.

[0130] [Labeling antibody] The labeling antibody is an antibody to which a detection label is attached, and which forms a complex with any antigen contained in a milk sample by an antigen-antibody reaction. Such an antigen may be a protein contained in milk, or an antigen derived from a target bacterium released by lysis of bacteria contained in milk. The complex formed by the labeling antibody is called a first complex. The type of detection label used for the labeling antibody is not particularly limited, and may be appropriately selected depending on the detection method. Specific examples of the detection label include metal colloids such as gold colloid, platinum colloid, and palladium colloid; non-metal colloids such as selenium colloid, alumina colloid, and silica colloid; insoluble granular substances such as colored resin particles, dye colloid, and colored liposome; color reaction catalytic enzymes such as alkaline phosphatase, peroxidase, and luciferase; fluorescent dyes, radioisotopes; chemiluminescent labels, bioluminescent labels, and electrochemiluminescent labels. The method of adding a label to an antibody is not particularly limited, and specific examples of the detection label include physical adsorption using the hydrophobicity of the antibody and chemical binding using the functional groups of the antibody.

[0131] The first complex formed by an antigen-antibody reaction between the antigen of the detection target substance and the labeling antibody is developed from the immersion area 9 of the immunochromatography device 10 into the detection area 4 .

[0132] [Capture antibody] The capture antibody is an antibody immobilized in the detection region 4 of the immunochromatography device 10, and forms a complex with the first complex through an antigen-antibody reaction. The complex thus formed is referred to as the second complex. More specifically, the capture antibody is immobilized on the chromatographic development membrane member 3 located in the detection region 4 of the immunochromatography device 10. The method for linking the antibody to the solid phase member is not particularly limited, and specific examples include immobilization by physical adsorption utilizing the hydrophobicity of the antibody, and immobilization by chemical binding utilizing the functional group of the antibody. From the viewpoint of suppressing non-specific reactions derived from the sample, when immobilizing the capture antibody on the chromatographic development membrane member 3, a protein such as casein or BSA may be immobilized at the same time as a blocking agent.

[0133] The second complex is retained at the position where the capture antibody is fixed by an antigen-antibody reaction with the immobilized capture antibody, and the label of the labeling antibody is detected there. When the label is detected at the position where the capture antibody is immobilized, the target bacterium is detected in the sample liquid.

[0134] [Bacteria to be detected] The bacteria to be detected in this embodiment may be any bacteria, but examples thereof include bacteria of the genus Escherichia, Staphylococcus, Klebsiella, Proteus, Enterococcus, Citrobacter, Pseudomonas, Bacillus, Serratia, Rahnella, Listeria, Enterobacter, Salmonella, Moraxella, Aeromonas, Lactobacillus, Micrococcus, and the like. Examples of the genera Micrococcus, Acinetobacter, Campylobacter, Vibrio, Streptococcus, Haemophilus, Mycoplasma, Legionella, Bordetella, Chlamydia, and Neisseria, Yersinia, Erwinia, Hafnia, Morganella, Providencia, Shigella, and Pectobacterium.

[0135] Examples of bacteria of the genus Escherichia include Escherichia coli (E. coli, EC), Escherichia albertii, etc. Among them, Escherichia coli is preferred.

[0136] Examples of bacteria of the genus Staphylococcus include Staphylococcus aureus (S. aureus, SA), S. epidermidis, S. argenteus, etc. Among these, Staphylococcus aureus is preferred.

[0137] Examples of bacteria belonging to the genus Klebsiella include Klebsiella pneumoniae (K. pneumoniae, KP), Klebsiella oxytoca (K. oxytoca), Klebsiella aerogenes (K. aerogenes), etc. Among them, it is preferable to detect an antigen-antibody reaction with Klebsiella pneumoniae.

[0138] Examples of bacteria of the genus Proteus include Proteus milabilis (P. milabilis, PM), Proteus morganii (P. morganii), Proteus vulgaris (P. vulgaris), Proteus rettgeri (P. rettgeri), etc. Among them, Proteus milabilis is preferred.

[0139] Examples of bacteria of the genus Enterococcus include Enterococcus faecalis, Enterococcus faecium, etc. Among them, Enterococcus faecalis is preferable.

[0140] Examples of bacteria of the genus Citrobacter include Citrobacter freundii (C. freundii, CF), Citrobacter amalonaticus (C. amalonaticus), Citrobacter diversus (C. diversus), etc. Among them, Citrobacter freundii is preferred.

[0141] Examples of bacteria of the genus Pseudomonas include Pseudomonas aeruginosa (P. aeruginosa, PA), P. fluorescens, P. phosphorescence, P. putida, etc. Among these, Pseudomonas aeruginosa is preferred.

[0142] Examples of bacteria in the genus Bacillus include Bacillus subtilis (B. subtilis, BS), Bacillus cereus (B. cereus), Bacillus licheniformis (B. licheniformis), and Bacillus megaterium (B. megaterium).

[0143] Examples of bacteria of the genus Serratia include Serratia liquefaciens (S. liquefaciens, SL), Serratia marcescens (S. marcescens), Serratia fonticola (S. fonticola), etc. Among them, Serratia liquefaciens is preferred.

[0144] Examples of bacteria in the genus Rahnella include Rahnella aquatilis (R. aquatilis, RA), Rahnella victoriana (R. victoriana), Rahnella bruchi (R. bruchi), and the like.

[0145] Examples of bacteria belonging to the genus Listeria include Listeria monocytogenes (L. monocytogenes, LM), Listeria innocua (L. innocua), and Listeria ivanovii (L. ivanovii).

[0146] Examples of bacteria in the genus Enterobacter include Enterobacter cloacae (E. cloacae, ECL), Enterobacter aerogenes (E. aerogenes), and Enterobacter sakazakii (E. sakazakii).

[0147] Examples of bacteria belonging to the genus Salmonella include Salmonella enteritidis (S. enteritidis, SE), Salmonella infantis (S. infantis), Salmonella typhimurium (S. typhimurium), and the like.

[0148] [Lysing agent] The lytic agent may be any substance used in the art as long as it can lyse the target bacterium. Examples of the lytic agent include lytic enzymes and surfactants. The lytic enzymes and surfactants may be used alone, or multiple surfactants and / or multiple lytic enzymes may be used in combination.

[0149] [Lytic enzyme] As the lytic enzyme, any lytic enzyme used in the present technical field can be used as long as it is a substance capable of lysing the target bacterium. The lytic enzyme can be appropriately selected according to the type of the target bacterium, and examples thereof include lysozyme, lysostaphin, pepsin, glucosidase, galactosidase, achromopeptidase, β-N-acetylglucosaminidase, acetylglucoaminidase, and achromopeptidase, and particularly preferred. These enzymes may be natural enzymes or genetically modified enzymes. The amount of the lytic enzyme to be applied is appropriately selected according to the desired final concentration and the volume of the sample liquid. As an example, the lytic enzyme is used at a final concentration of 0.01 μg / mL or more to 100 mg / mL, and therefore the amount to be applied is 0.01 μg to 100 mg, preferably 0.1 μg to 10 mg, and more preferably 1 μg to 3 mg.

[0150] [Surfactants] The surfactant can be appropriately selected depending on the target bacteria. As the surfactant, a nonionic surfactant, a cationic surfactant, an anionic surfactant, and an amphoteric surfactant may be used alone or in combination. The amount of the surfactant to be attached is appropriately selected depending on the desired final concentration and the volume of the sample liquid. As an example, the surfactant is used at a final concentration of 0.01% to 10%, so that the amount to be attached is 0.1 to 100 mg, preferably 0.1 to 10 mg, and more preferably 1 to 10 mg.

[0151] As the nonionic surfactant, any of ester ether type, ester type, and ether type can be suitably used, and more specifically, polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, fatty acid sorbitan ester, alkyl polyglucoside, fatty acid diethanolamide, alkyl monoglyceryl ether, polysorbates (fatty acid sorbitan ester condensed with several tens of molecules of ethylene oxide), etc. are listed, but are not limited thereto. Particularly preferred examples are polysorbates, more specifically, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (60) sorbitan monostearate, polyoxyethylene (65) sorbitan tristearate, and polyoxyethylene (80) sorbitan monooleate, and polyoxyethylene alkyl phenyl ether, more specifically, polyoxyethylene (10) octyl phenyl ether, etc.

[0152] As the anionic surfactant, any of carboxylates, sulfonates, sulfates, etc. can be suitably used. More specific examples include alkyl ether carboxylates, linear alkylbenzene sulfonates (LAS), α-olefin sulfonates (AOS), dialkyl sulfosuccinates, formaldehyde condensates of naphthalene sulfonates, alkyl sulfates (AS), polyoxyethylene alkyl sulfates (AES) obtained by adding ethylene oxide to higher alcohols to give sulfates, and phosphates of higher alcohols and their ethylene oxide adducts. Further specific examples include sodium alkyl sulfates such as sodium dodecyl sulfate and sodium myristyl sulfate, sodium N-acyl sarcosinates such as sodium N-lauroyl sarcosinate and sodium N-myristoyl sarcosinate, sodium dodecylbenzenesulfonate, hydrogenated coconut fatty acid monoglyceride sodium monosulfate, sodium lauryl sulfoacetate, N-acyl glutamates such as sodium N-palmitoyl glutamate, sodium N-methyl-N-acylalanine, and sodium α-olefin sulfonate.

[0153] As the cationic surfactant, either an amine salt type or a quaternary ammonium salt type can be suitably used, and more specific examples include distearyldimethylammonium chloride, benzalkonium chloride, hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, myristyltrimethylammonium bromide, and the like.

[0154] Examples of amphoteric surfactants include, but are not limited to, amino acid surfactants (alkylamino fatty acid salts), betaine surfactants (alkyl betaines), amine oxide surfactants (alkyl amine oxides), etc. More specific examples include dimethylammoniopropanesulfonate, dodecyldimethylammoniobutyrate, betaine laurate, and amidopropyl betaine.

[0155] [Detection of target substances] The method for detecting a target substance in a milk sample by an antigen-antibody reaction using the immunochromatography device 10 of this embodiment is as follows: The immersion area 9 of the immunochromatography device 10 is immersed in a milk sample; The second complex captured in the detection region 4 of the immunochromatography device 10 is detected based on the label of the labeling antibody. This includes:

[0156] By immersing the immersion area 9 of the immunochromatography device in the milk sample, at least a part of the labeling antibody-attached member 1 is immersed, and the labeling antibody is eluted in the milk sample. This causes an antigen-antibody reaction in the milk sample, and the reaction time can be extended, resulting in improved detection sensitivity. In addition, when the dipstick-type immunochromatography device is provided with the bacteriolytic agent-attached member 2, at least a part of the bacteriolytic agent-attached member 2 is immersed in the milk sample, similarly to the labeling antibody-attached member 1, and the labeling antibody and the bacteriolytic agent are eluted in the milk sample. The presence of the labeling antibody and the bacteriolytic agent in the milk sample causes the bacteriolytic reaction and the antigen-antibody reaction to occur in the milk sample, and the reaction time for both reactions can be extended, resulting in improved detection sensitivity. Therefore, in order to detect bacteria with high sensitivity, it is important that the development of the sample liquid in the immunochromatography device proceeds smoothly even after several minutes without causing blockage. The immersion time is a time sufficient for the second complex to be captured by the capture antibody immobilized on the immunochromatography device 10. The immersion time may be until the labeling antibody binds to the control region and the label is detected. In the capture region where the capture antibody is immobilized, the second complex is formed, and detection becomes possible based on the label of the labeling antibody.

[0157] The detection method of this embodiment may further include a step of stirring the detection solution using the immunochromatography device 10. Stirring can promote the elution of the labeling antibody and the lytic agent into the sample liquid, and promote the lytic reaction and / or the antibody-antigen reaction. After stirring, the immunochromatography device 10 is left standing, whereby the first complex formed by the antibody-antigen is developed on the chromatographic development membrane member 3 together with the sample liquid via the sample contact member 5, the first filter member 6-1, and the second filter member 6-2 of the immunochromatography device 10.

[0158] When the immunochromatography device does not include the lytic agent-applied member 2, the detection method of the target bacteria of this embodiment may include a step of separately adding a lytic agent to the specimen liquid. The lytic agent may be placed in advance in the specimen liquid holding container 20 of the dipstick-type immunochromatography device, and in that case, the lytic agent can be dispersed in the specimen liquid by the step of putting the specimen liquid into the holding container.

[0159] The step of detecting the second complex captured in the detection region 4 of the immunochromatography device 10 based on the label of the labeling antibody can be performed by an appropriate means depending on the label. When gold colloid is used as the label, the coloring of the discrimination section can be confirmed with the naked eye. The coloring of the control section, to which the antigen to which the labeling antibody binds is fixed, indicates that the labeling antibody has been properly developed on the chromatographic development membrane member 3. On the other hand, if the filter member is not properly adjusted, clogging may occur in the chromatographic development membrane member 3. In this case, it may be judged by the fact that the control section is not colored, or it may be visually confirmed by the presence or absence of liquid wetting that the chromatographic membrane development of the milk sample has stopped halfway. Normally, when chromatographic membrane development occurs, the color of the chromatographic development membrane member 3 and the absorption member 7 changes due to liquid wetting, but when the chromatographic membrane development stops, liquid wetting does not occur. If the chromatographic membrane development of the milk sample stops halfway, a problem of reduced detection performance occurs, so it is important to adjust the filter member so that the chromatographic membrane development can be performed well.

[0160] All documents mentioned herein are incorporated by reference in their entirety. EXAMPLES

[0161] The following examples of the present invention are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. The present invention can be modified, for example, by adding, deleting, and replacing the constituent elements of the present invention, without departing from the spirit of the present invention.

[0162] [Example Group I: Immunochromatography device according to the first embodiment]

[0163] Test 1: Detection of Staphylococcus aureus using an immunochromatographic device (1) Preparation of an immunochromatographic device for detecting Staphylococcus aureus The immunochromatography devices shown in the schematic cross-sectional views of FIGS. 1 to 10 were fabricated according to the following description.

[0164] (a) Generation of ribosomal protein L7 / L12 antibodies Staphylococcus aureus ( Staphylococcus aureus For the detection of Staphylococcus aureus, a monoclonal antibody against the ribosomal protein L7 / L12 was used. The L7 / L12 ribosomal protein of Staphylococcus aureus was obtained according to the method described in Example 5 of WO 2000 / 006603, and a monoclonal antibody was produced using the protein. The monoclonal antibodies were a combination of two types (SA-1 and SA-2) that can simultaneously bind to different sites of the L7 / L12 ribosomal protein (capable of forming a sandwich-type complex with the L7 / L12 antigen).

[0165] (b) Labeling antibody attachment member Monoclonal antibody SA-2 was used as the antibody for labeling, which forms the first complex by antigen-antibody reaction with the antigen derived from the target bacteria. 0.9 mL of colloidal gold solution (particle size 60 nm) manufactured by BBI International was mixed with 0.1 M potassium phosphate (pH 7.5), 0.1 mL of monoclonal antibody SA-2 with a concentration of 100 μg / mL for labeling colloidal gold was added, and the mixture was left at room temperature for 5 minutes to bind the antibody to the surface of colloidal gold particles. Next, a 10% aqueous solution of bovine serum albumin (BSA) was added so that the final concentration in the colloidal gold solution was 1%, and the remaining surface of the colloidal gold particles was blocked with BSA to prepare a solution of monoclonal antibody SA-2 labeled with colloidal gold (hereinafter referred to as "labeling antibody" as appropriate). This solution was centrifuged (15,000×rpm, 5 minutes) to precipitate the labeling antibody, and the supernatant was removed to obtain the labeling antibody. This labeling antibody was suspended in 20 mM Tris-HCl buffer (pH 8.2) containing 0.25% BSA and 2.5% sucrose, and adjusted so that the absorbance at a wavelength of 540 nm was 0.5 when diluted 21 times to obtain a labeling antibody solution. A 5 mm x 300 mm strip of glass fiber pad was impregnated with 1 mL of the labeling antibody solution and dried under reduced pressure at room temperature to obtain a labeling antibody-attached member 1.

[0166] (c) Bacteriolytic agent-impregnated material Recombinant lysostaphin manufactured by Fujifilm Wako Pure Chemical Industries was dissolved in 20 mM sodium acetate buffer (pH 4.5) containing 5% sucrose to a concentration of 100 μg / mL, and a 5 mm x 300 mm strip of glass fiber pad was impregnated with 1 mL of the enzyme solution and air-dried at room temperature to prepare a lytic agent-applied member 2.

[0167] (d) Detection area with immobilized capture antibody A nitrocellulose membrane 25 mm wide and 300 mm long was prepared as the chromatographic development membrane carrier 3. Monoclonal antibody SA-1 was used as a capture antibody that forms a second complex by antigen-antibody reaction with the first complex. A solution containing 1.5 mg / mL of monoclonal antibody SA-1 was applied in a line shape at 1 μL / cm to a position 10 mm from the end of the chromatographic development starting point side of the chromatographic development membrane carrier 3, and this was dried at 50° C. for 30 minutes and then dried at room temperature overnight. This was used as the capture site 4 for the complex of Staphylococcus aureus ribosomal protein L7 / L12 antigen and gold colloid labeling antibody.

[0168] (e) Preparation of immunochromatography device In addition to the labeling antibody-attached member 1, the bacteriolytic agent-attached member 2, and the chromatographic development membrane carrier 3, cotton cloth was prepared as the sample contact member 5, and filter paper was prepared as the absorption member 7. These members were then attached to a base material 8 (254 μm thick, made of polystyrene with an adhesive for attaching the members), and the upper part of the figure (the surface opposite to the base material 8) except for the immersion region 9 was covered with a cover film (water-impermeable member). This was cut to a width of 5 mm to prepare the immunochromatographic devices shown in the cross-sectional views of Figures 1 to 10. The upstream 10 mm of each immunochromatographic device was designated as the immersion region 9.

[0169] (f) Preparation of reaction mixture In the immunochromatography devices of FIGS. 1 to 9, 0.1% Tween 20 was added as a surfactant for liquid development to 0.1 M MOPSO buffer solution (pH 7.4) to prepare a reaction solution for immunochromatography. Since no lytic agent was applied to the immunochromatography device in Figure 10, 16 μg / mL (75 units / mL) lysostaphin was added as a lytic agent to 0.1 M MOPSO buffer (pH 7.4) and 0.1% Tween 20 was added as a surfactant for liquid development to prepare the reaction solution for immunochromatography.

[0170] (g) Sample fluid holding container As the sample liquid holding container 20, an Eppendorf microtube with a capacity of 2 mL was used.

[0171] (2) Detection of Staphylococcus aureus by immunochromatography In a microtube, add the sample solution, i.e., 1x10 final concentration 4 cfu / mL, 1×10 5 cfu / mL, 1×10 6 300 μL of physiological saline containing cfu / mL Staphylococcus aureus (ATCC25923 strain) or physiological saline without bacteria was taken, to which 300 μL of the reaction solution was added and mixed at room temperature. For the immunochromatography device in FIG. 10, the immunochromatography device was left to stand for 30 minutes before immersion.

[0172] The immunochromatographic device was immersed in the mixed solution (sample solution) from the sample contact member 5 so that the entire immersion area was immersed in the sample solution. The appearance when the immunochromatographic device having the structure of Fig. 1 and Fig. 2 was used is shown in Figs. 20 and 21. After that, the sample solution was stirred in the immunochromatographic device, and then it was left to stand at room temperature for 60 minutes to develop, and the presence or absence of the capture of the complex between the ribosomal protein L7 / L12 antigen and the gold colloid labeling antibody captured at the capture site 4 was visually determined. In addition, for the immunochromatographic devices of Figs. 4 and 10, the time from mixing the sample solution and the reaction solution to visually confirming the capture of the complex at the capture site 4 was measured, and the time was shown in parentheses in the table.

[0173] [Table 1-1] [Table 1-2]

[0174] 1 to 6 in which the lytic agent-applied member 2 to which the lytic enzyme used as the lytic agent is applied and at least a part of the labeling antibody-applied member 1 are immersed in the sample liquid were able to detect bacterial concentrations up to 1e4cfu / mL, whereas the immunochromatographic device having the structure of Figs. 7 to 10 in which the labeling antibody-applied member and / or the lytic agent-applied member 2 are not immersed in the sample liquid were only able to detect bacterial concentrations up to 1e5cfu / mL. It is also clear that the immunochromatographic device of Example I-4 according to the present invention was able to detect the target antigen in a shorter time than the immunochromatographic device of Comparative Example I-4, which corresponds to the prior art.

[0175] Test 2: Detection of Escherichia coli by immunochromatography (1) Preparation of an immunochromatographic device for detecting E. coli The immunochromatography device shown in the schematic cross-sectional views of FIGS. 1 to 10 was fabricated according to the following description.

[0176] (a) Generation of ribosomal protein L7 / L12 antibodies To detect Escherichia coli, a monoclonal antibody against the ribosomal protein L7 / L12 of Escherichia coli was used. According to the method described in Example 5 of WO 2000 / 006603, the L7 / L12 ribosomal protein of Escherichia coli was obtained, and a monoclonal antibody was produced using the protein. The monoclonal antibody was a combination of two types (EC-1 and EC-2) that can simultaneously bind to different sites of the L7 / L12 ribosomal protein.

[0177] (b) Labeling antibody attachment member Monoclonal antibody EC-2 was used as the antibody for labeling, which forms the first complex by antigen-antibody reaction with the antigen derived from the target bacteria. 0.9 mL of gold colloid solution (particle size 60 nm) manufactured by BBI International was mixed with 0.1 M potassium phosphate (pH 7.5), 0.1 mL of monoclonal antibody EC-2 with a concentration of 100 μg / mL for labeling gold colloid was added, and the mixture was left at room temperature for 5 minutes to bind the antibody to the surface of the gold colloid particles. Next, a 10% aqueous solution of bovine serum albumin (BSA) was added so that the final concentration in the gold colloid solution was 1%, and the remaining surface of the gold colloid particles was blocked with BSA to prepare a solution of monoclonal antibody EC-2 labeled with gold colloid (hereinafter referred to as "labeling antibody" as appropriate). This solution was centrifuged (15,000×rpm, 5 minutes) to precipitate the labeling antibody, and the supernatant was removed to obtain the labeling antibody. This labeling antibody was suspended in 20 mM Tris-HCl buffer (pH 8.2) containing 0.25% BSA and 2.5% sucrose, and adjusted so that the absorbance at a wavelength of 540 nm was 0.5 when diluted 21 times to obtain a labeling antibody solution. A 5 mm x 300 mm strip of glass fiber pad was impregnated with 1 mL of the labeling antibody solution and dried under reduced pressure at room temperature to obtain a labeling antibody-attached member 1.

[0178] (c) Bacteriolytic agent-impregnated material Egg white-derived lysozyme manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. was dissolved in 20 mM Tris-HCl buffer (pH 8.0) containing 5% sucrose to a concentration of 100 mg / mL, and a 5 mm x 300 mm strip of glass fiber pad was impregnated with 1 mL of the enzyme solution and air-dried at room temperature to obtain a lytic agent-imparted member 2.

[0179] (d) Detection area with immobilized capture antibody A nitrocellulose membrane 25 mm wide and 300 mm long was prepared as the chromatographic development membrane carrier 3. Monoclonal antibody EC-1 was used as a capture antibody that forms a second complex by antigen-antibody reaction with the first complex. A solution containing 1.5 mg / mL of monoclonal antibody EC-1 was applied in a line shape at 1 μL / cm to a position 10 mm from the end of the chromatographic development starting point side of the chromatographic development membrane carrier 3, and this was dried at 50° C. for 30 minutes and then dried at room temperature overnight. This was used as the capture site 4 for the complex of E. coli ribosomal protein L7 / L12 antigen and gold colloid labeling antibody.

[0180] (e) Preparation of immunochromatography device In addition to the labeling antibody-attached member 1, the bacteriolytic agent-attached member 2, and the chromatographic development membrane carrier 3, cotton cloth was prepared as the sample contact member 5, and filter paper was prepared as the absorption member 7. These members were then attached to a base material 8 (254 μm thick, made of polystyrene with an adhesive for attaching the members), and the upper part of the figure (the surface opposite to the base material 8) except for the immersion region 9 was covered with a cover film (water-impermeable member). This was cut to a width of 5 mm to prepare the immunochromatographic devices whose cross-sectional views are shown in FIGS. 1 to 10. The upstream 10 mm of each immunochromatographic device was designated as the immersion region 9.

[0181] (f) Preparation of reaction mixture In the immunochromatography devices of FIGS. 1 to 9, 0.1% Tween 20 was added as a surfactant for liquid development to 0.1 M MOPSO buffer solution (pH 7.4) to prepare a reaction solution for immunochromatography. Since the immunochromatography device in Figure 10 does not contain a lytic agent, 9 mg / mL lysozyme was added as a lytic agent and 0.1% Tween 20 was added as a surfactant for liquid development to 0.1 M MOPSO buffer solution (pH 7.4) to prepare the reaction solution for immunochromatography.

[0182] (g) Sample fluid holding container As the sample liquid holding container 20, an Eppendorf microtube with a capacity of 2 mL was used.

[0183] (2) Detection of Escherichia coli by immunochromatography In a microtube, add the sample solution, i.e., 1x10 final concentration 4 cfu / mL, 1×10 5 cfu / mL, 1×10 6 300 μL of physiological saline containing E. coli (ATCC25922 strain) cfu / mL or physiological saline without bacteria was taken, to which 300 μL of the reaction solution was added and mixed at room temperature. For the immunochromatographic device of FIG. 10, the device was left to stand for 30 minutes before being immersed. The immunochromatographic device was immersed in the mixed solution from the sample contact member 5 so that the entire immersion area was immersed in the sample solution. After that, the sample solution was stirred in the immunochromatographic device, and the device was left to stand at room temperature for 60 minutes for development, and the presence or absence of the capture of the complex between the ribosomal protein L7 / L12 antigen and the gold colloid labeling antibody captured at the capture site 4 was visually determined. For the immunochromatographic devices of FIG. 4 and FIG. 10, the time from mixing the sample solution and the reaction solution to visually confirming the capture of the complex at the capture site 4 was measured, and the time was noted in parentheses in the table.

[0184] [Table 2-1] [Table 2-2]

[0185] The immunochromatographic devices having the structures shown in Figs. 1 to 6, in which the lytic agent-imparted member 2 to which the lytic enzyme used as a lytic agent is impregnated and the labeling antibody-imparted member 1 are at least partially immersed in the sample liquid, were able to detect bacterial concentrations up to 1e4cfu / mL, whereas the immunochromatographic devices having the structures shown in Figs. 7 to 10, in which the labeling antibody-imparted member 1 and / or the lytic agent-imparted member 2 are not immersed in the sample liquid, were only able to detect bacterial concentrations up to 1e5cfu / mL. It is also clear that the immunochromatographic device of Example I-10 according to the present invention is able to detect the target antigen in a shorter time than the immunochromatographic device of Comparative Example I-8, which corresponds to the prior art.

[0186] Test 3: Detection of Streptococcus uberis by immunochromatography (1) Construction of an immunochromatographic device for detecting streptococci The immunochromatography device shown in the schematic cross-sectional views of FIGS. 1 to 9 was fabricated according to the following description.

[0187] (a) Generation of ribosomal protein L7 / L12 antibodies Streptococcus ( Streptococcus uberis For the detection of Streptococcus pneumoniae, a monoclonal antibody against ribosomal protein L7 / L12 was used. According to the method described in Example 5 of WO 2000 / 006603, streptococcal L7 / L12 ribosomal protein was obtained, and a monoclonal antibody was produced using the protein. The monoclonal antibody was a combination of two types (SU-1 and SU-2) that can simultaneously bind to different sites of the L7 / L12 ribosomal protein.

[0188] (b) Labeling antibody attachment member Monoclonal antibody SU-2 was used as the antibody used for the labeling antibody that forms the first complex by antigen-antibody reaction with the antigen derived from the target bacteria. 0.9 mL of gold colloid solution (particle size 60 nm) manufactured by BBI International was mixed with 0.1 M potassium phosphate (pH 7.5), 0.1 mL of monoclonal antibody SU-2 with a concentration of 100 μg / mL for labeling gold colloid was added, and the mixture was left at room temperature for 5 minutes to bind the antibody to the surface of the gold colloid particles. Next, a 10% aqueous solution of bovine serum albumin (BSA) was added so that the final concentration in the gold colloid solution was 1%, and the remaining surface of the gold colloid particles was blocked with BSA to prepare a solution of monoclonal antibody SU-2 labeled with gold colloid (hereinafter referred to as "labeling antibody" as appropriate). This solution was centrifuged (15,000×rpm, 5 minutes) to precipitate the labeling antibody, and the supernatant was removed to obtain the labeling antibody. This labeling antibody was suspended in 20 mM Tris-HCl buffer (pH 8.2) containing 0.25% BSA and 2.5% sucrose, and adjusted so that the absorbance at a wavelength of 540 nm was 0.5 when diluted 21 times to obtain a labeling antibody solution. A 5 mm x 300 mm strip of glass fiber pad was impregnated with 1 mL of the labeling antibody solution and dried under reduced pressure at room temperature to obtain a labeling antibody-attached member 1.

[0189] (c) Bacteriolytic agent-impregnated material Labiase manufactured by Ozeki Co., Ltd. and egg white-derived lysozyme manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. were dissolved in a citrate-phosphate buffer solution (pH 4.0) containing 5% sucrose to a concentration of 100 mg / mL, and a 5 mm x 300 mm strip of glass fiber pad was impregnated with 1 mL of the enzyme solution and air-dried at room temperature to prepare a lytic agent-imparted member 2.

[0190] (d) Detection area with immobilized capture antibody A nitrocellulose membrane 25 mm wide and 300 mm long was prepared as the chromatographic development membrane carrier 3. Monoclonal antibody SU-1 was used as a capture antibody that forms a second complex by antigen-antibody reaction with the first complex. A solution containing 1.5 mg / mL of monoclonal antibody SU-1 was applied in a line shape at 1 μL / cm to a position 10 mm from the end of the chromatographic development starting point side of the chromatographic development membrane carrier 3, and this was dried at 50° C. for 30 minutes and then dried at room temperature overnight. This was used as the capture site 4 for the complex of streptococcal ribosomal protein L7 / L12 antigen and gold colloid labeling antibody.

[0191] (e) Preparation of immunochromatography device In addition to the labeling antibody-attached member 1, the bacteriolytic agent-attached member 2, and the chromatographic development membrane carrier 3, cotton cloth was prepared as the sample contact member 5, and filter paper was prepared as the absorption member 7. These members were then attached to a base material 8 (254 μm thick, made of polystyrene with an adhesive for attaching the members), and the upper part of the figure (the surface opposite to the base material 8) except for the immersion region 9 was covered with a cover film (water-impermeable member). This was cut to a width of 5 mm to prepare the immunochromatographic devices whose cross-sectional views are shown in FIGS. 1 to 9. The upstream 10 mm of each immunochromatographic device was designated as the immersion region 9.

[0192] (f) Preparation of reaction mixture A reaction solution for immunochromatography was prepared by adding 0.1% Tween 20 as a surfactant for liquid development to 0.1 M MOPSO buffer solution (pH 6.3).

[0193] (g) Sample fluid holding container As the sample liquid holding container 20, an Eppendorf microtube with a capacity of 2 mL was used.

[0194] (2) Detection of Streptococcus by Immunochromatography Final concentration in a microtube: 1x10 4 cfu / mL, 1×10 5 cfu / mL, 1×10 6300 μL of saline containing Streptococcus pyogenes (ATCC19436 strain) at 100 cfu / mL or saline without bacteria was taken, and 300 μL of the reaction solution was added and mixed at room temperature. The immunochromatography device was immersed in the mixed solution from the sample contact member 5 so that the entire immersion area was immersed in the sample liquid. After that, the sample liquid was stirred with the immunochromatography device, and then left to stand at room temperature for 60 minutes to develop, and the presence or absence of the complex between the ribosomal protein L7 / L12 antigen captured at the capture site 4 and the gold colloid labeling antibody was visually determined.

[0195] [Table 3-1] [Table 3-2]

[0196] The immunochromatographic devices having the structures shown in Figures 1 to 6, in which the lytic agent-applied member 2 to which the lytic enzyme used as a lytic agent is applied, and at least a portion of the labeling antibody-applied member 1 are immersed in the sample liquid, were able to detect bacterial concentrations up to 1e4 cfu / mL, whereas the immunochromatographic devices having the structures shown in Figures 7 to 9, in which the labeling antibody-applied member 1 and / or the lytic agent-applied member 2 are not immersed in the sample liquid, were only able to detect bacterial concentrations up to 1e5 cfu / mL.

[0197] Test 4: Detection of Staphylococcus aureus in milk by immunochromatography (1) Construction of an immunochromatographic device for detecting Staphylococcus aureus in milk The immunochromatography devices shown in the schematic cross-sectional views of FIGS. 11 to 13 were fabricated according to the following description.

[0198] (a) Generation of ribosomal protein L7 / L12 antibodies The combination of SA-1 and SA-2 selected in Test 1 was used.

[0199] (b) Labeling antibody attachment member The labeling antibody-attached member 1 prepared in Test 1 was used.

[0200] (c) Bacteriolytic agent attachment In Example I-19, the bacteriolytic agent-impregnated member 2 prepared in Test 1 was used. In Example I-20, recombinant lysostaphin manufactured by Fujifilm Wako Pure Chemical Industries was dissolved in 20 mM sodium acetate buffer (pH 4.5) containing 5% casein to a concentration of 50 μg / mL, and a 10 mm x 300 mm strip of glass fiber pad was impregnated with 2 mL of the enzyme solution and air-dried at room temperature to prepare a lytic agent-applied member 2.

[0201] (d) Detection area with immobilized capture antibody A chromatographic development membrane carrier 3 and a capture site 4 were prepared in the same manner as in Test 1, except that 1% sucrose and 0.15% casein were added to the solution containing 1.5 mg / mL of monoclonal antibody SA-1 to be applied to the chromatographic development membrane carrier.

[0202] (e) Preparation of immunochromatography device In Example I-19, in addition to the above-mentioned labeling antibody-attached member 1, the above-mentioned bacteriolytic agent-attached member 2, and the above-mentioned chromatographic development membrane carrier 3, 25 mm GF / DVA (Cytiva: a filter made of glass fiber, 776 μm thick, with a retention particle size of 3.5 μm) as the sample contact member 5, 15 mm GF / AVA (Cytiva: a filter made of glass fiber, 299 μm thick, with a retention particle size of 1.7 μm) as the filter member 6, and filter paper were prepared as the absorption member 7. Then, these members were attached to a base material 8 (254 μm thick, made of polystyrene with an adhesive for attaching members), and then the upper part of the figure (the surface opposite to the base material 8) other than the immersion region 9 was covered with a cover film (water-impermeable member). This was cut to a width of 5 mm to prepare an immunochromatographic device whose cross-sectional view is shown in FIG. 11. The upstream side of the immunochromatography device, 10 mm in length, was defined as an immersion region 9 .

[0203] In Example I-20, in addition to the above-mentioned labeling antibody-attached member 1, the above-mentioned bacteriolytic agent-attached member 2, and the above-mentioned membrane carrier for chromatographic development 3, 25 mm GF / DVA (manufactured by Cytiva: a filter made of glass fiber, with a thickness of 776 μm and a retention particle size of 3.5 μm) was prepared as the sample contact member 5, 15 mm GF / AVA (manufactured by Cytiva: a filter made of glass fiber, with a thickness of 299 μm and a retention particle size of 1.7 μm) was prepared as the filter member 6, and filter paper was prepared as the absorption member 7. Then, the same procedure as in Example I-19 was carried out except that the above-mentioned bacteriolytic agent-attached member 2 was attached onto the above-mentioned labeling antibody-attached member 1, and an immunochromatography kit shown in the cross-sectional view in FIG. 12 was produced. The upstream 10 mm of the immunochromatography device was set as the immersion area 9.

[0204] In Comparative Example I-12, in addition to the above-mentioned labeling antibody-attached member 1 and the above-mentioned chromatographic development membrane carrier 3, a 20 mm GF / DVA (manufactured by Cytiva: a filter made of glass fiber, 776 μm thick, with a retention particle size of 3.5 μm) was prepared as the sample contact member 5, a 15 mm GF / AVA (manufactured by Cytiva: a filter made of glass fiber, 299 μm thick, with a retention particle size of 1.7 μm) was prepared as the filter member 6, and filter paper was prepared as the absorption member 7. An immunochromatography kit shown in the cross-sectional view of FIG. 13 was prepared. An area 10 mm upstream of the immunochromatography device was set as the immersion region 9.

[0205] (f) Preparation of reaction mixture In Examples I-19 and I-20, 1% Tween 20 was added as a surfactant for liquid development to 0.1 M MOPSO buffer solution (pH 7.4) to prepare a reaction solution for immunochromatography. Since no lytic agent was applied to the immunochromatography device of Comparative Example I-12, 16 μg / mL (75 units / mL) lysostaphin was added as a lytic agent to 0.1 M MOPSO buffer (pH 7.4) and 11% Tween 20 was added as a surfactant for liquid development to prepare a reaction solution for immunochromatography.

[0206] (g) Sample fluid holding container As the sample liquid holding container 20, an Eppendorf microtube with a capacity of 2 mL was used.

[0207] (2) Detection of Staphylococcus aureus in milk by immunochromatography Final concentration in a microtube: 1x10 4 cfu / mL, 1×10 5 cfu / mL, 1×10 6 300 μL of saline containing Staphylococcus aureus (ATCC25923 strain) at 100 cfu / mL or milk containing saline containing no bacteria was taken, and 300 μL of the reaction solution was added and mixed at room temperature. A commercially available milk was used. For the immunochromatographic device in FIG. 13, the device was left to stand for 30 minutes before immersion. The immunochromatographic device was immersed in the mixed solution from the sample contact member 5 so that the entire immersion area was immersed in the sample liquid. After that, the sample liquid was stirred with the immunochromatographic device, and then the samples were left to stand for 60 minutes at room temperature for Examples I-19 and I-20, and for 30 minutes at room temperature for Comparative Example I-12, and developed. The presence or absence of the complex between the ribosomal protein L7 / L12 antigen and the gold colloid labeling antibody captured at the capture site 4 was visually determined. 12 and 13, the time from mixing the sample solution and the reaction solution until the capture of the complex at the capture site 4 was visually confirmed was measured.

[0208] [Table 4]

[0209] The immunochromatographic device having the structure shown in Figures 11 and 12 was able to detect Staphylococcus aureus at 1e4cfu / mL in milk with high sensitivity. It was also found that the immunochromatographic device of Example I-20 according to the present invention was able to detect the target antigen in a shorter time than the immunochromatographic device of Comparative Example I-12, which corresponds to the prior art.

[0210] Test 5: Detection of Escherichia coli in milk by immunochromatography (1) Construction of an immunochromatographic device for detecting E. coli in milk The immunochromatographic devices shown in the schematic cross-sectional views of Figures 10, 14, and 13 were fabricated according to the following description.

[0211] (a) Generation of ribosomal protein L7 / L12 antibodies The combination of EC-1 and EC-2 selected in Test 2 was used.

[0212] (b) Labeling antibody attachment member In Example I-21, the labeling antibody-attached member 1 prepared in Test 2 was used. In Example I-22, monoclonal antibody EC-2 was used as the antibody used for the labeling antibody that forms the first complex by antigen-antibody reaction with the antigen derived from the target bacteria. 0.9 mL of gold colloid solution (particle size 60 nm) manufactured by BBI International was mixed with 0.1 M potassium phosphate (pH 7.5), 0.1 mL of monoclonal antibody EC-2 with a concentration of 100 μg / mL for labeling gold colloid was added, and the mixture was left at room temperature for 5 minutes to bind the antibody to the surface of the gold colloid particles. Next, a 10% aqueous solution of bovine serum albumin (BSA) was added so that the final concentration in the gold colloid solution was 1%, and the remaining surface of the gold colloid particles was blocked with BSA to prepare a solution of monoclonal antibody EC-2 labeled with gold colloid (hereinafter referred to as "labeling antibody" as appropriate). This solution was centrifuged (15000×rpm, 5 minutes) to precipitate the labeling antibody, and the supernatant was removed to obtain the labeling antibody. This labeling antibody was suspended in 20 mM Tris-HCl buffer (pH 8.2) containing 0.25% BSA and 2.5% sucrose, and adjusted so that the absorbance at a wavelength of 540 nm was 0.25 when diluted 21 times to obtain a labeling antibody solution. A 10 mm x 300 mm belt-shaped glass fiber pad was impregnated with 2 mL of the labeling antibody solution and dried under reduced pressure at room temperature to obtain a labeling antibody-attached member 1.

[0213] (c) Bacteriolytic agent attachment In Examples I-21 and I-22, the lytic agent-impregnated member 2 prepared in Test 2 was used.

[0214] (d) Detection area with immobilized capture antibody A chromatographic development membrane carrier 3 and a capture site 4 were prepared in the same manner as in Test 2, except that 2% sucrose and 0.1% casein were added to the solution containing 1.5 mg / mL of monoclonal antibody EC-1 to be applied to the chromatographic development membrane carrier.

[0215] (e) Preparation of immunochromatography device In Example I-21, in addition to the above-mentioned labeling antibody-attached member 1, the above-mentioned bacteriolytic agent-attached member 2, and the above-mentioned chromatographic development membrane carrier 3, 25 mm GF / DVA (Cytiva: a filter made of glass fiber, 776 μm thick, with a retention particle size of 3.5 μm) was prepared as the sample contact member 5, 15 mm GF / AVA (Cytiva: a filter made of glass fiber, 299 μm thick, with a retention particle size of 1.7 μm) was prepared as the filter member 6, and filter paper was prepared as the absorption member 7. Then, these members were attached to a base material 8 (254 μm thick, made of polystyrene with an adhesive for attaching members), and then the upper part of the figure other than the immersion region 9 (the surface opposite to the base material 8) was covered with a cover film (water-impermeable member). This was cut into a width of 5 mm to prepare an immunochromatographic device whose cross-sectional view is shown in FIG. 11. The upstream side of the immunochromatography device, 10 mm in length, was defined as an immersion region 9 .

[0216] In Example I-22, in addition to the above-mentioned labeling antibody-attached member 1, the above-mentioned bacteriolytic agent-attached member 2, and the above-mentioned membrane carrier for chromatographic development 3, 25 mm GF / DVA (Cytiva: a filter made of glass fiber, 776 μm thick, with a retention particle size of 3.5 μm) was prepared as the sample contact member 5, 15 mm GF / AVA (Cytiva: a filter made of glass fiber, 299 μm thick, with a retention particle size of 1.7 μm) was prepared as the filter member 6, and filter paper was prepared as the absorption member 7. Then, the same procedure as in Example I-19 was carried out except that the above-mentioned bacteriolytic agent-attached member 2 was attached onto the above-mentioned labeling antibody-attached member 1, and an immunochromatography kit shown in the cross-sectional view in FIG. 14 was produced. The upstream 10 mm of the immunochromatography device was set as the immersion area 9.

[0217] In Comparative Example I-13, in addition to the above-mentioned labeling antibody-attached member 1 and the above-mentioned chromatographic development membrane carrier 3, a 20 mm GF / DVA (manufactured by Cytiva: a filter made of glass fiber, 776 μm thick, with a retention particle size of 3.5 μm) was prepared as the sample contact member 5, a 15 mm GF / AVA (manufactured by Cytiva: a filter made of glass fiber, 299 μm thick, with a retention particle size of 1.7 μm) was prepared as the filter member 6, and filter paper was prepared as the absorption member 7. An immunochromatography kit shown in the cross-sectional view of FIG. 13 was prepared. An area 10 mm upstream of the immunochromatography device was set as the immersion region 9.

[0218] (f) Preparation of reaction mixture In Examples I-21 and I-22, 1% Tween 20 was added as a surfactant for liquid development to 0.1 M MOPSO buffer solution (pH 7.4) to prepare a reaction solution for immunochromatography. Since the immunochromatography device of Comparative Example I-13 was not loaded with a lytic agent, 9 mg / mL lysozyme was added as a lytic agent to 0.1 M MOPSO buffer (pH 7.4) and 1% Tween 20 was added as a surfactant for liquid development to prepare a reaction solution for immunochromatography.

[0219] (g) Sample fluid holding container As the sample liquid holding container 20, an Eppendorf microtube with a capacity of 2 mL was used.

[0220] (2) Immunochromatography of E. coli in milk ( Escherichia coli ) detection Final concentration in a microtube: 1x10 4 cfu / mL, 1×10 5 cfu / mL, 1×10 6300 μL of saline containing E. coli (ATCC25922 strain) at cfu / mL or milk containing saline without bacteria was taken, and 300 μL of the reaction solution was added and mixed at room temperature. A commercially available milk was used. For the immunochromatographic device in FIG. 13, the device was left to stand for 30 minutes before immersion. The immunochromatographic device was immersed in the mixed solution from the sample contact member 5 so that the entire immersion area was immersed in the sample liquid. After that, the sample liquid was stirred with the immunochromatographic device, and then the samples were left to stand for 60 minutes at room temperature for Examples I-21 and I-22, and for 30 minutes at room temperature for Comparative Example I-13, and then developed. The presence or absence of the complex between the ribosomal protein L7 / L12 antigen captured at the capture site 4 and the gold colloid labeling antibody was visually determined. For the immunochromatography devices of FIG. 14 and FIG. 13, the time from mixing the sample solution and the reaction solution until the capture of the complex at the capture site 4 was visually confirmed was measured.

[0221] [Table 5]

[0222] The immunochromatographic devices having the structures shown in Figures 11 and 14 were able to detect 1e4cfu / mL of E. coli in milk with high sensitivity. It was also found that the immunochromatographic device of Example I-22 according to the present invention was able to detect the target antigen in a shorter time than the immunochromatographic device of Comparative Example I-13, which corresponds to the prior art.

[0223] Test 6: Detection of Staphylococcus aureus, Escherichia coli, and Streptococcus in milk by immunochromatography (1) Construction of an immunochromatographic device for detecting Staphylococcus aureus in milk Example I-23 used the members prepared in Example I-19 of Test 4 to prepare an immunochromatographic device having the cross-sectional view of Fig. 15, Example I-24 used the immunochromatographic device prepared and used in Example I-19 of Test 4, and Example I-25 used the immunochromatographic device prepared and used in Example I-20 of Test 4. The reaction solution and sample holding containers used were those prepared and used in Test 4.

[0224] (2) Detection of Staphylococcus aureus in milk by immunochromatography Final concentration in a microtube: 1x10 4 300 μL of saline containing Staphylococcus aureus (ATCC25923 strain) at 100 cfu / mL or saline containing no bacteria was taken, and 300 μL of the reaction solution was added and mixed at room temperature. A commercially available product was used for the milk. The immunochromatographic device was immersed in the mixed solution from the sample contact member 5 so that the entire immersion area was immersed in the sample liquid. Thereafter, for each of Examples I-23, I-24, and I-25, two conditions were carried out, one in which the sample liquid was stirred and the other in which it was not stirred, and the sample was left to stand at room temperature for 60 minutes to develop, and then the presence or absence of the complex between the ribosomal protein L7 / L12 antigen captured at the capture site 4 and the antibody for labeling with gold colloid was visually determined.

[0225] [Table 6-1]

[0226] (3) Construction of an immunochromatographic device for detecting streptococci in milk The immunochromatographic device shown in the schematic cross-sectional views of Figures 16, 17, and 18 was fabricated according to the following description.

[0227] (a) Generation of ribosomal protein L7 / L12 antibodies The combination of SU-1 and SU-2 selected in Test 3 was used.

[0228] (b) Labeling antibody attachment member In Example I-26, the labeling antibody-attached member 1 prepared in Test 3 was used. The labeling antibody-attached member 1 used in Examples I-27 and I-28 was prepared as follows. The monoclonal antibody SU-2 was used as the labeling antibody that forms the first complex by antigen-antibody reaction with the antigen derived from the target bacteria. 0.9 mL of gold colloid solution (particle size 60 nm) manufactured by BBI International was mixed with 0.1 M potassium phosphate (pH 7.5), 0.1 mL of monoclonal antibody SU-2 with a concentration of 100 μg / mL for labeling gold colloid was added, and the mixture was left at room temperature for 5 minutes to bind the antibody to the surface of the gold colloid particles. Next, a 10% aqueous solution of bovine serum albumin (BSA) was added so that the final concentration in the gold colloid solution was 1%, and the remaining surface of the gold colloid particles was blocked with BSA to prepare a solution of monoclonal antibody SU-2 labeled with gold colloid (hereinafter referred to as "labeling antibody" as appropriate). This solution was centrifuged (15000×rpm, 5 minutes) to precipitate the labeling antibody, and the supernatant was removed to obtain the labeling antibody. This labeling antibody was suspended in 20 mM Tris-HCl buffer (pH 8.2) containing 0.25% BSA and 2.5% sucrose, and adjusted so that the absorbance at a wavelength of 540 nm was 0.25 when diluted 21 times to obtain a labeling antibody solution. A 10 mm x 300 mm strip of glass fiber pad was impregnated with 2 mL of the labeling antibody solution and dried under reduced pressure at room temperature to obtain the labeling antibody attachment member 1-2.

[0229] (c) Bacteriolytic agent-impregnated material Labiase manufactured by Ozeki Co., Ltd. was dissolved in McIlvaine buffer (pH 4.0) containing 5% sucrose to a concentration of 30 mg / mL, and a 5 mm x 300 mm strip-shaped glass fiber pad was impregnated with 1 mL of the enzyme solution and air-dried at room temperature to obtain a lytic enzyme-imparted member 2-1. Lysozyme manufactured by Creative Enzymes was dissolved in 20 mM Tris-HCl buffer (pH 8.0) containing 5% sucrose to a concentration of 50 mg / mL, and a 5 mm x 300 mm strip-shaped glass fiber pad was impregnated with 1 mL of the enzyme solution and air-dried at room temperature to obtain a lytic enzyme-imparted member 2-2.

[0230] (d) Detection area with immobilized capture antibody A nitrocellulose membrane 25 mm wide and 300 mm long was prepared as the chromatographic development membrane carrier 3. Monoclonal antibody SU-1 was used as a capture antibody that forms a second complex by antigen-antibody reaction with the first complex. A solution containing 1% sucrose, 0.1% casein, and 1.5 mg / mL of monoclonal antibody SU-1 was applied in a line shape at 1 μL / cm to a position 10 mm from the end of the chromatographic development starting point side of the chromatographic development membrane carrier 3, and this was dried at 50° C. for 30 minutes and then dried at room temperature overnight. This was used as the capture site 4 for the complex of streptococcal ribosomal protein L7 / L12 antigen and gold colloid labeling antibody.

[0231] (e) Preparation of immunochromatography device In Example I-26, in addition to the above-mentioned labeling antibody-attached member 1, the above-mentioned bacteriolytic agent-attached members 2-1 and 2-2, and the above-mentioned chromatographic development membrane carrier 3, 32 mm GF / DVA (manufactured by Cytiva: a filter made of glass fiber, 776 μm thick, with a retention particle size of 3.5 μm) as the sample contact member 5, 16 mm GF / AVA (manufactured by Cytiva: a filter made of glass fiber, 299 μm thick, with a retention particle size of 1.7 μm) as the filter member 6, and filter paper were prepared as the absorption member 7. Then, these members were attached to a base material 8 (254 μm thick, made of polystyrene with an adhesive for attaching members), and then the upper part of the figure other than the immersion region 9 (the surface opposite to the base material 8) was covered with a cover film (water-impermeable member). This was cut to a width of 5 mm to prepare an immunochromatographic device whose cross-sectional view is shown in FIG. 16. The upstream side of the immunochromatography device, 10 mm in length, was defined as an immersion region 9 .

[0232] In Example I-27, an immunochromatography device the cross-sectional view of which is shown in FIG. 17 was produced in the same manner as in Example I-26, except that the labeling antibody-attached member 1-2 was used.

[0233] In Example I-28, an immunochromatography device the cross-sectional view of which is shown in FIG. 18 was produced in the same manner as in Example I-26, except that the labeling antibody-attached member 1-2 was used.

[0234] The upstream 10 mm area of ​​each of the immunochromatography devices prepared in Examples I-26, I-27, and I-28 was used as an immersion region 9.

[0235] (f) Preparation of reaction mixture A reaction solution for immunochromatography was prepared by adding 1% Tween 20 as a liquid development surfactant to 0.1 M MOPSO buffer solution (pH 6.3).

[0236] (g) Sample fluid holding container As the sample liquid holding container 20, an Eppendorf microtube with a capacity of 2 mL was used.

[0237] (4) Detection of Streptococcus in Milk by Immunochromatography Final concentration in a microtube: 1x10 4 300 μL of saline containing Streptococcus pyogenes (ATCC19436 strain) at 100 cfu / mL or saline without bacteria was taken, and 300 μL of the reaction solution was added and mixed at room temperature. The immunochromatographic device was immersed in the mixed solution from the sample contact member 5 so that the entire immersion area was immersed in the sample liquid. Thereafter, for each of Examples I-26, I-27, and I-28, two conditions were carried out, one in which the sample liquid was stirred and the other in which it was not stirred, and the sample was left to stand at room temperature for 60 minutes to develop, and then the presence or absence of the complex between the ribosomal protein L7 / L12 antigen captured at the capture site 4 and the gold colloid labeling antibody was visually determined.

[0238] [Table 6-2]

[0239] (1) Construction of an immunochromatographic device for detecting E. coli in milk In Example I-29, an immunochromatographic device was used that was fabricated to have the cross-sectional view of Fig. 15 using the members fabricated in Example I-21 of Test 5, and in Example I-30, an immunochromatographic device was used that was fabricated to have the cross-sectional view of Fig. 19 using the members fabricated and used in Example I-22 of Test 5. The reaction solution and sample holding containers used were those fabricated and used in Test 5.

[0240] (2) Immunochromatography of E. coli in milk ( Escherichia coli ) detection Final concentration in a microtube: 1x10 4 300 μL of saline containing E. coli (ATCC25922 strain) cfu / mL or milk containing saline without bacteria was taken, and 300 μL of the reaction solution was added and mixed at room temperature. A commercially available milk was used. The immunochromatography device was immersed in the mixed solution from the sample contact member 5 so that the entire immersion area was immersed in the sample liquid. Thereafter, for each of Examples I-29 and I-30, two conditions were carried out, one in which the sample liquid was stirred with the immunochromatography device and the other in which it was not stirred, and after leaving it to stand at room temperature for 60 minutes to develop, the presence or absence of the capture of the complex between the ribosomal protein L7 / L12 antigen captured at the capture site 4 and the antibody for labeling with gold colloid was visually determined.

[0241] [Table 6-3]

[0242] It can be seen that when the labeling antibody-attached member 1 and the lytic agent-attached member 2 are arranged so that their positions contact or overlap when projected in a direction perpendicular to the chromatographic development direction, the detection performance can be maintained regardless of the stirring conditions during the test. Specifically, in order to obtain higher detection sensitivity, it is preferable that at least a part of the labeling antibody-attached member 1 and at least a part of the lytic agent-attached member 2 are in close proximity to each other. More specifically, when the immersion area side or the sample contact member side of the chromatographic device is called the upstream side and the detection area side is called the downstream side, and the direction from the upstream to the downstream is called the chromatographic development direction, it is preferable that the labeling antibody-attached member 1 and the lytic agent-attached member 2 are arranged so that their positions contact or overlap when projected in a direction perpendicular to the chromatographic development direction (i.e., the labeling antibody-attached member 1 and the lytic agent-attached member 2 include the same position in at least a part of the chromatographic development direction). The "overlapping" arrangement referred to here is not limited to the case where the labeling antibody-applied member 1 and the lytic agent-applied member 2 are in contact with or overlapping each other on the same surface of the chromatography device, but also includes a state in which one is placed on the back of the chromatography device and separated from the other. This is thought to be because if the labeling antibody-applied member 1 and the lytic agent-applied member 2 are close to each other, they will be present in the vicinity of each other in the sample liquid when the labeling antibody and the lytic agent are eluted, and the lytic reaction and the antigen-antibody reaction will proceed more efficiently.

[0243] [Example Group II: Immunochromatography Device According to the Second Embodiment]

[0244] Test 1: Detection of Staphylococcus aureus in milk by immunochromatography (1) Construction of an immunochromatographic device for detecting Staphylococcus aureus in milk An immunochromatography device shown in the schematic cross-sectional view of FIG. 22 was fabricated according to the following description.

[0245] (a) Generation of ribosomal protein L7 / L12 antibodies Staphylococcus aureus ( Staphylococcus aureusFor the detection of Staphylococcus aureus, a monoclonal antibody against the ribosomal protein L7 / L12 was used. The L7 / L12 ribosomal protein of Staphylococcus aureus was obtained according to the method described in Example 5 of WO 2000 / 006603, and a monoclonal antibody was produced using the protein. The monoclonal antibodies were a combination of two types (SA-1 and SA-2) that can simultaneously bind to different sites of the L7 / L12 ribosomal protein (capable of forming a sandwich-type complex with the L7 / L12 antigen).

[0246] (b) Labeling antibody attachment member Monoclonal antibody SA-2 was used as the antibody for labeling, which forms the first complex by antigen-antibody reaction with the antigen derived from the target bacteria. 0.9 mL of colloidal gold solution (particle size 60 nm) manufactured by BBI International was mixed with 0.1 M potassium phosphate (pH 7.5), 0.1 mL of monoclonal antibody SA-2 with a concentration of 100 μg / mL for labeling colloidal gold was added, and the mixture was left at room temperature for 5 minutes to bind the antibody to the surface of colloidal gold particles. Next, a 10% aqueous solution of bovine serum albumin (BSA) was added so that the final concentration in the colloidal gold solution was 1%, and the remaining surface of the colloidal gold particles was blocked with BSA to prepare a solution of monoclonal antibody SA-2 labeled with colloidal gold (hereinafter referred to as "labeling antibody" as appropriate). This solution was centrifuged (15,000×rpm, 5 minutes) to precipitate the labeling antibody, and the supernatant was removed to obtain the labeling antibody. This labeling antibody was suspended in 20 mM Tris-HCl buffer (pH 8.2) containing 0.25% BSA and 2.5% sucrose, and adjusted so that the absorbance at a wavelength of 540 nm was 0.5 when diluted 21 times to obtain a labeling antibody solution. A 5 mm x 300 mm strip of glass fiber pad was impregnated with 1 mL of the labeling antibody solution and dried under reduced pressure at room temperature to obtain a labeling antibody-attached member 1.

[0247] (c) Bacteriolytic agent-impregnated material Recombinant lysostaphin manufactured by Fujifilm Wako Pure Chemical Industries was dissolved in 20 mM sodium acetate buffer (pH 4.5) to a concentration of 100 μg / mL, and a 5 mm x 300 mm strip of glass fiber pad was impregnated with 1 mL of the enzyme solution and air-dried at room temperature to prepare a lytic agent-applied member 2.

[0248] (d) Detection area with immobilized capture antibody A nitrocellulose membrane with a width of 25 mm and a length of 300 mm was prepared as the membrane carrier 3 for chromatographic development. Monoclonal antibody SA-1 was used as a capture antibody that forms a second complex by antigen-antibody reaction with the first complex. An aqueous solution of pH 6.2 containing 3.0 mg / mL monoclonal antibody SA-1, 25 mM sodium phosphate, 1% sucrose, and 0.15% casein was applied in a line shape at 1 μL / cm to a position 10 mm from the end of the chromatographic development starting point side of the membrane carrier 3 for chromatographic development, and this was dried under the conditions described in Table 7. This was used as the capture site 4 for the complex of Staphylococcus aureus ribosomal protein L7 / L12 antigen and gold colloid labeling antibody.

[0249] (e) Preparation of immunochromatography device In addition to the labeling antibody-attached member 1, the bacteriolytic agent-attached member 2, and the chromatographic development membrane carrier 3, 25 mm GF / DVA (Cytiva: a filter made of glass fiber, 776 μm thick, with a retention particle size of 3.5 μm) was prepared as the sample contact member 5, 15 mm GF / AVA (Cytiva: a filter made of glass fiber, 299 μm thick, with a retention particle size of 1.7 μm) was prepared as the filter member 6, and filter paper was prepared as the absorption member 7. These members were then attached to a base material 8 (254 μm thick, made of polystyrene with an adhesive for attaching members), and the upper part of the figure other than the immersion region 9 (the surface opposite to the base material 8) was covered with a cover film (water-impermeable member) as a cover member 11. This was cut to a width of 5 mm to prepare an immunochromatographic device whose cross section is shown in FIG. 22. The upstream side of the immunochromatography device, 10 mm in length, was defined as an immersion region 9 .

[0250] (f) Accelerated heating test The immunochromatography device was divided into Group A and Group B. Group A was left standing in a room temperature, low humidity environment for one week, while Group B was placed in an aluminum pouch together with a desiccant and then left standing in a 50°C environment for one week in order to understand the change in performance during storage at room temperature. According to the inventors' investigation, storage at 50°C for one week under the same conditions is equivalent to storage at room temperature for approximately 3 to 6 months.

[0251] (g) Preparation of reaction mixture 1.6% Tween 20 and 0.16% Zwittergent 3-12 were added to 0.16 M MOPSO buffer (pH 7.4) to prepare a reaction solution for immunochromatography.

[0252] (h) Sample fluid holding container As the sample liquid holding container 20, an Eppendorf microtube with a capacity of 2 mL was used.

[0253] (2) Detection of Staphylococcus aureus in milk by immunochromatography Final concentration in a microtube: 2 x 10 3 cfu / mL, 4 × 10 3 300 μL of commercially available milk to which 100 cfu / mL Staphylococcus aureus (ATCC25923 strain) or physiological saline had been added was taken, and 300 μL of the above reaction solution was added and mixed at room temperature. The immunochromatography devices divided into two groups in the (f) accelerated heating test were immersed in the mixed solution (sample solution) starting from the sample contact member 5 so that the entire immersion area was immersed in the sample solution. The state when the immunochromatography device shown in FIG. 22 was used is shown in FIG. 23.

[0254] The sample liquid was then stirred using the immunochromatography device and allowed to stand at room temperature for 60 minutes for development, after which the presence or absence of a complex between the ribosomal protein L7 / L12 antigen captured at capture site 4 and the gold colloid labeling antibody was visually determined.

[0255] [Table 7]

[0256] The immunochromatographic device prepared by applying a capture antibody to the chromatographic development membrane carrier 3 and then drying it at 60°C for 17 hours was able to detect a bacterial concentration of up to 2e3cfu / mL regardless of whether or not a heat acceleration test was performed, whereas the immunochromatographic device prepared by applying a capture antibody to the chromatographic development membrane carrier 3 and then drying it at 50°C for 0.5 hours was only able to detect a bacterial concentration of 4e3cfu / mL without a heat acceleration test. From this result, it is expected that when dried at 50°C for 0.5 hours, the detection performance will increase by about 2 times when stored at room temperature for a long period after production.

[0257] Test 2: Detection of Escherichia coli in milk by immunochromatography (1) Construction of an immunochromatographic device for detecting E. coli in milk An immunochromatography device shown in the schematic cross-sectional view of FIG. 22 was fabricated according to the following description.

[0258] (a) Generation of ribosomal protein L7 / L12 antibodies To detect Escherichia coli, a monoclonal antibody against the ribosomal protein L7 / L12 of Escherichia coli was used. According to the method described in Example 5 of WO 2000 / 006603, the L7 / L12 ribosomal protein of Escherichia coli was obtained, and a monoclonal antibody was produced using the protein. The monoclonal antibody was a combination of two types (EC-1 and EC-2) that can simultaneously bind to different sites of the L7 / L12 ribosomal protein.

[0259] (b) Labeling antibody attachment member Monoclonal antibody EC-2 was used as the antibody for labeling, which forms the first complex by antigen-antibody reaction with the antigen derived from the target bacteria. 0.9 mL of colloidal gold solution (particle size 60 nm) manufactured by BBI International was mixed with 0.1 M potassium phosphate, pH 7.5, and 0.1 mL of monoclonal antibody EC-2 with a concentration of 100 μg / mL for labeling colloidal gold was added and left at room temperature for 5 minutes to bind the antibody to the surface of colloidal gold particles. Next, a 10% aqueous solution of bovine serum albumin (BSA) was added so that the final concentration in the colloidal gold solution was 1%, and the remaining surface of the colloidal gold particles was blocked with BSA to prepare a solution of monoclonal antibody EC-2 labeled with colloidal gold (hereinafter referred to as "labeling antibody" as appropriate). This solution was centrifuged (15,000×rpm, 5 minutes) to precipitate the labeling antibody, and the supernatant was removed to obtain the labeling antibody. This labeling antibody was suspended in 20 mM Tris-HCl buffer (pH 8.2) containing 0.25% BSA and 2.5% sucrose, and adjusted so that the absorbance at a wavelength of 540 nm was 0.5 when diluted 21 times to obtain a labeling antibody solution. A 5 mm x 300 mm strip of glass fiber pad was impregnated with 1 mL of the labeling antibody solution and dried under reduced pressure at room temperature to obtain a labeling antibody-attached member 1.

[0260] (c) Bacteriolytic agent-impregnated material Egg white lysozyme manufactured by Fujifilm Wako Pure Chemical Industries was dissolved in 20 mM Tris-HCl buffer (pH 8.0) to a concentration of 10 mg / mL, and a 5 mm x 300 mm strip of glass fiber pad was impregnated with 1 mL of the enzyme solution and air-dried at room temperature to obtain a lytic agent-imparted member 2.

[0261] (d) Detection area with immobilized capture antibody A nitrocellulose membrane 25 mm wide and 300 mm long was prepared as the chromatographic development membrane carrier 3. Monoclonal antibody EC-1 was used as a capture antibody that forms a second complex by antigen-antibody reaction with the first complex. An aqueous solution of pH 10.5 containing 1.0 mg / mL monoclonal antibody EC-1, 10 mM CAPSO, 2% sucrose, and 0.1% casein was applied in a line shape at 1 μL / cm to a position 10 mm from the end of the chromatographic development starting point side of the chromatographic development membrane carrier 3, and dried under the conditions described in Table 8. This was used as the capture site 4 for the complex of the E. coli ribosomal protein L7 / L12 antigen and the gold colloid labeling antibody.

[0262] (e) Preparation of immunochromatography device In addition to the labeling antibody-attached member 1, the bacteriolytic agent-attached member 2, and the chromatographic development membrane carrier 3, 25 mm GF / DVA (Cytiva: a filter made of glass fiber, 776 μm thick, with a retention particle size of 3.5 μm) was prepared as the sample contact member 5, 15 mm GF / AVA (Cytiva: a filter made of glass fiber, 299 μm thick, with a retention particle size of 1.7 μm) was prepared as the filter member 6, and filter paper was prepared as the absorption member 7. These members were then attached to a base material 8 (254 μm thick, made of polystyrene with an adhesive for attaching members), and the upper part of the figure other than the immersion region 9 (the surface opposite to the base material 8) was covered with a cover film (water-impermeable member) as a cover member 11. This was cut to a width of 5 mm to prepare an immunochromatographic device whose cross section is shown in FIG. 22. The upstream side of the immunochromatography device, 10 mm in length, was defined as an immersion region 9 .

[0263] (f) Accelerated heating test The above immunochromatography device was divided into Group A and Group B. Group A was left standing in a room temperature, low humidity environment for one week, while Group B was placed in an aluminum pouch with a desiccant and then left standing in a 50°C environment for one week in order to understand any changes in performance when stored at room temperature.

[0264] (g) Preparation of reaction mixture A reaction solution for immunochromatography was prepared by adding 1.8% TritonX100, 0.48% sodium N-dodecanoylsarcosinate, and 0.32 M sodium chloride to 0.08 M MOPSO buffer (pH 7.4).

[0265] (h) Sample fluid holding container As the sample liquid holding container 20, an Eppendorf microtube with a capacity of 2 mL was used.

[0266] (2) Detection of Escherichia coli in milk by immunochromatography Final concentration in a microtube: 5x10 4 cfu / mL, 1×10 5 300 μL of commercially available milk to which cfu / mL of E. coli (ATCC25922 strain) or physiological saline had been added was taken, and 300 μL of the above reaction solution was added and mixed at room temperature. The immunochromatography devices divided into two groups in the (f) accelerated heating test were immersed in the mixed solution from the sample contact member 5 so that the entire immersion area was immersed in the sample liquid. After that, the sample liquid was stirred in the immunochromatography device, and then left to stand at room temperature for 60 minutes to develop, and the presence or absence of capture of the complex between the ribosomal protein L7 / L12 antigen captured at the capture site 4 and the gold colloid labeling antibody was visually determined.

[0267] [Table 8]

[0268] The immunochromatographic device prepared by applying a capture antibody to the chromatographic development membrane carrier 3 and then drying it at 60°C for 17 hours was able to detect a bacterial concentration of up to 5e4cfu / mL regardless of whether or not a heating acceleration test was performed, whereas the immunochromatographic device prepared by applying a capture antibody to the chromatographic development membrane carrier 3 and then drying it at 50°C for 0.5 hours was only able to detect a bacterial concentration of 1e5cfu / mL without a heating acceleration test. From this result, it is expected that when dried at 50°C for 0.5 hours, the detection performance will increase by about 2 times when stored at room temperature for a long period after production.

[0269] Test 3: Detection of Streptococcus uberis in milk by immunochromatography (1) Construction of an immunochromatographic device for detecting streptococci in milk An immunochromatographic device, the cross-sectional schematic of which is shown in FIG. 22, was prepared according to the following description.

[0270] (a) Generation of ribosomal protein L7 / L12 antibodies Streptococcus ( Streptococcus uberis For the detection of Streptococcus pneumoniae, a monoclonal antibody against ribosomal protein L7 / L12 was used. According to the method described in Example 5 of WO 2000 / 006603, streptococcal L7 / L12 ribosomal protein was obtained, and a monoclonal antibody was produced using the protein. The monoclonal antibody was a combination of two types (SU-1 and SU-2) that can simultaneously bind to different sites of the L7 / L12 ribosomal protein.

[0271] (b) Labeling antibody attachment member Monoclonal antibody SU-2 was used as the antibody used for the labeling antibody that forms the first complex by antigen-antibody reaction with the antigen derived from the target bacteria. 0.9 mL of gold colloid solution (particle size 60 nm) manufactured by BBI International was mixed with 0.1 M potassium phosphate (pH 7.5), 0.1 mL of monoclonal antibody SU-2 with a concentration of 100 μg / mL for labeling gold colloid was added, and the mixture was left at room temperature for 5 minutes to bind the antibody to the surface of the gold colloid particles. Next, a 10% by mass aqueous solution of bovine serum albumin (BSA) was added so that the final concentration in the gold colloid solution was 1% by mass, and the remaining surface of the gold colloid particles was blocked with BSA to prepare a solution of monoclonal antibody SU-2 labeled with gold colloid (hereinafter referred to as "labeling antibody" as appropriate). This solution was centrifuged (15,000×rpm, 5 minutes) to precipitate the labeling antibody, and the supernatant was removed to obtain the labeling antibody. This labeling antibody was suspended in 20 mM Tris-HCl buffer (pH 8.2) containing 0.25% by mass BSA and 2.5% by mass sucrose, and adjusted so that the absorbance at a wavelength of 540 nm was 0.5 when diluted 21-fold to obtain a labeling antibody solution. A 5 mm x 300 mm strip of glass fiber pad was impregnated with 1 mL of the labeling antibody solution and dried under reduced pressure at room temperature to obtain a labeling antibody-attached member 1.

[0272] (c) Bacteriolytic agent-impregnated material Labiase manufactured by Ozeki Co., Ltd. and egg white-derived lysozyme manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. were dissolved in citrate-phosphate buffer (pH 4.0) to a concentration of 100 mg / mL, and a 5 mm x 300 mm strip of glass fiber pad was impregnated with 1 mL of the enzyme solution and air-dried at room temperature to obtain a lytic agent-imparted member 2.

[0273] (d) Detection area with immobilized capture antibody A nitrocellulose membrane 25 mm wide and 300 mm long was prepared as the membrane carrier 3 for chromatographic development. Monoclonal antibody SU-1 was used as a capture antibody that forms a second complex by antigen-antibody reaction with the first complex. An aqueous solution containing 1.0 mg / mL of monoclonal antibody SU-1, 10 mM potassium phosphate, and 1% by mass sucrose at pH 8 was applied in a line shape at 1 μL / cm to a position 10 mm from the end of the chromatographic development starting point side of the membrane carrier 3 for chromatographic development, and dried under the conditions described in Table 9. This was used as the capture site 4 for the complex of streptococcal ribosomal protein L7 / L12 antigen and gold colloid labeling antibody.

[0274] (e) Preparation of immunochromatography device In addition to the labeling antibody-attached member 1, the bacteriolytic agent-attached member 2, and the chromatographic development membrane carrier 3, 25 mm GF / DVA (Cytiva: a filter made of glass fiber, 776 μm thick, with a retention particle size of 3.5 μm) was prepared as the sample contact member 5, 15 mm GF / AVA (Cytiva: a filter made of glass fiber, 299 μm thick, with a retention particle size of 1.7 μm) was prepared as the filter member 6, and filter paper was prepared as the absorption member 7. These members were then attached to a base material 8 (254 μm thick, made of polystyrene with an adhesive for attaching members), and the upper part of the figure other than the immersion region 9 (the surface opposite to the base material 8) was covered with a cover film (water-impermeable member) as a cover member 11. This was cut to a width of 5 mm to prepare an immunochromatographic device whose cross section is shown in FIG. 22. The upstream side of the immunochromatography device, 10 mm in length, was defined as an immersion region 9 .

[0275] (f) Accelerated heat stability test The above immunochromatography device was divided into Group A and Group B. Group A was left standing in a room temperature, low humidity environment for one week, while Group B was placed in an aluminum pouch with a desiccant and then left standing in a 50°C environment for one week in order to understand any changes in performance when stored at room temperature.

[0276] (g) Preparation of reaction mixture A reaction solution for immunochromatography was prepared by adding 1.8% by mass of TritonX100, 1.2% by mass of Tween20, 0.48% by mass of sodium N-dodecanoylsarcosinate, 0.12 M sodium chloride, and 0.001 M EGTA to 0.16 M MOPSO buffer solution (pH 6.3).

[0277] (h) Sample fluid holding container As the sample liquid holding container 20, an Eppendorf microtube with a capacity of 2 mL was used.

[0278] (2) Detection of Streptococcus in Milk by Immunochromatography Final concentration in a microtube: 2 x 10 4 cfu / mL, 4 × 10 4 300 μL of commercially available milk to which cfu / mL streptococci (ATCC19436 strain) or physiological saline had been added was dispensed, and 300 μL of the above reaction solution was added and mixed at room temperature.

[0279] The immunochromatographic devices divided into two groups in the (f) accelerated heating test were immersed in the same mixed solution, starting from the sample contact member 5, so that the entire immersion area was immersed in the sample liquid. After that, the sample liquid was stirred in the immunochromatographic device, and then left to stand at room temperature for 60 minutes to develop, after which the presence or absence of capture of the complex between the ribosomal protein L7 / L12 antigen and the gold colloid labeling antibody captured at the capture site 4 was visually determined.

[0280] [Table 9]

[0281] The immunochromatographic device prepared by applying a capture antibody to the chromatographic development membrane carrier 3 and then drying it at 60°C for 17 hours was able to detect a bacterial concentration of up to 2e4cfu / mL regardless of whether or not a heating acceleration test was performed, whereas the immunochromatographic device prepared by applying a capture antibody to the chromatographic development membrane carrier 3 and then drying it at 50°C for 0.5 hours was only able to detect a bacterial concentration of 4e4cfu / mL without a heating acceleration test. From this result, it is expected that when dried at 50°C for 0.5 hours, the detection performance will increase by about 2 times when stored at room temperature for a long period after production.

[0282] Test 4: Comparison of drying conditions after application of capture antibody (1) Preparation of an immunochromatographic device for detecting E. coli An immunochromatographic device, the cross-sectional schematic of which is shown in FIG. 22, was prepared according to the following description.

[0283] (a) Generation of ribosomal protein L7 / L12 antibodies The combination of EC-1 and EC-2 selected in Test 2 was used.

[0284] (b) Labeling antibody attachment member The labeling antibody-attached member 1 prepared in Test 2 was used.

[0285] (c) Bacteriolytic agent-impregnated material The bacteriolytic agent-impregnated member 2 prepared in Test 2 was used.

[0286] (d) Detection area with immobilized capture antibody A nitrocellulose membrane with a width of 25 mm and a length of 300 mm was prepared as the membrane carrier 3 for chromatographic development. Monoclonal antibody EC-1 was used as a capture antibody that forms a second complex by antigen-antibody reaction with the first complex. An aqueous solution of pH 10.5 containing 1.0 mg / mL monoclonal antibody EC-1, 10 mM CAPSO, 2 mass % sucrose, and 0.1 mass % casein was applied in a line shape at 1 μL / cm to a position 10 mm from the end of the chromatographic development starting point side of the membrane carrier 3 for chromatographic development, and dried under the conditions described in Table 10. This was used as the capture site 4 for the complex of the E. coli ribosomal protein L7 / L12 antigen and the antibody for gold colloid labeling.

[0287] (e) Preparation of immunochromatography device In addition to the labeling antibody-attached member 1, the bacteriolytic agent-attached member 2, and the chromatographic development membrane carrier 3, 25 mm GF / DVA (Cytiva: a filter made of glass fiber, 776 μm thick, with a retention particle size of 3.5 μm) was prepared as the sample contact member 5, 15 mm GF / AVA (Cytiva: a filter made of glass fiber, 299 μm thick, with a retention particle size of 1.7 μm) was prepared as the filter member 6, and filter paper was prepared as the absorption member 7. These members were then attached to a base material 8 (254 μm thick, made of polystyrene with an adhesive for attaching members), and the upper part of the figure other than the immersion region 9 (the surface opposite to the base material 8) was covered with a cover film (water-impermeable member) as a cover member 11. This was cut to a width of 5 mm to prepare an immunochromatographic device whose cross section is shown in FIG. 22. The upstream side of the immunochromatography device, 10 mm in length, was defined as an immersion region 9 .

[0288] (f) Storage of the kit The immunochromatography device was allowed to stand for one week in a room temperature, low humidity environment.

[0289] (g) Preparation of reaction mixture The reaction solution prepared in Test 2 was used.

[0290] (h) Sample fluid holding container As the sample liquid holding container 20, an Eppendorf microtube with a capacity of 2 mL was used.

[0291] (2) Detection of Escherichia coli in milk by immunochromatography Final concentration in a microtube: 5x10 4 cfu / mL, 1×10 5 300 μL of commercially available milk to which cfu / mL of E. coli (ATCC25922 strain) or physiological saline had been added was taken, and 300 μL of the above reaction solution was added and mixed at room temperature. Each of the above immunochromatography devices was immersed in the mixed solution from the sample contact member 5 so that the entire immersion area was immersed in the sample liquid. After leaving it to stand at room temperature for 60 minutes and developing, the presence or absence of capture of the complex between the ribosomal protein L7 / L12 antigen captured at the above capture site 4 and the gold colloid labeling antibody was visually determined.

[0292] [Table 10]

[0293] The immunochromatographic device prepared by applying a capture antibody to the chromatographic development membrane carrier 3 and drying it at 60°C for 6 hours or more was able to detect a bacterial concentration of up to 5e4cfu / mL without heating and storage, as in Example II-3 of Test 2, whereas the immunochromatographic device prepared by applying a capture antibody to the chromatographic development membrane carrier 3 and drying it at 60°C for 0.5 hours was only able to detect a bacterial concentration of up to 1e5cfu / mL. From this result, it is expected that drying at 60°C for 6 hours or more can suppress changes in detection performance during long-term storage at room temperature after production.

[0294] [Example Group III: Immunochromatography device according to the third embodiment]

[0295] An immunochromatography device shown in the schematic cross-sectional view of FIG. 25 was fabricated according to the following description.

[0296] (a) Generation of ribosomal protein L7 / L12 antibodies The antibody to be labeled with gold colloids is Staphylococcus aureus ( Staphylococcus aureus ) Ribosomal protein L7 / L12 monoclonal antibody was used. According to the method described in Example 5 of WO 2000 / 006603, L7 / L12 ribosomal protein of Staphylococcus aureus was obtained, and monoclonal antibodies were produced using the protein. The monoclonal antibodies were a combination of two types (SA-1 and SA-2) that can simultaneously bind to different sites of the L7 / L12 ribosomal protein.

[0297] (b) Labeling antibody attachment member Monoclonal antibody SA-2 was used as the antibody for labeling, which forms the first complex by antigen-antibody reaction with the antigen derived from the target bacteria. 0.9 mL of BB International gold colloid solution (particle size 60 nm) was mixed with 0.1 M potassium phosphate (pH 7.5), 100 μg / mL of monoclonal antibody SA-2 for labeling gold colloid was added, and the mixture was left at room temperature for 5 minutes to bind the antibody to the gold colloid particle surface. Next, a 10% aqueous solution of bovine serum albumin (BSA) was added so that the final concentration in the gold colloid solution was 1%, and the remaining surface of the gold colloid particles was blocked with BSA to prepare a solution of monoclonal antibody SA-2 labeled with gold colloid (hereinafter referred to as "labeling antibody" as appropriate). This solution was centrifuged (15,000×rpm, 5 minutes) to precipitate the labeling antibody, and the supernatant was removed to obtain the labeling antibody. The labeling antibody was suspended in 20 mm Tris-HCl buffer (pH 8.2) containing 0.25% BSA and 2.5% sucrose, and adjusted so that the absorbance at a wavelength of 540 nm was 0.5 when diluted 21 times to obtain a labeling antibody solution. A 5 mm x 300 mm strip of glass fiber pad was impregnated with 1 mL of the labeling antibody solution, and this was dried under reduced pressure at room temperature to obtain a labeling antibody-attached member 1.

[0298] (c) Bacteriolytic agent-impregnated material Recombinant lysostaphin manufactured by Fujifilm Wako Pure Chemical Industries was dissolved in 20 mM sodium acetate buffer (pH 4.5) to a concentration of 100 μg / mL, and a 5 mm x 300 mm strip of glass fiber pad was impregnated with 1 mL of the enzyme solution and air-dried at room temperature to prepare a lytic agent-applied member 2.

[0299] (d) Detection area with immobilized capture antibody A nitrocellulose membrane 25 mm wide and 300 mm long was prepared as the membrane member 3 for chromatographic development of the chromatographic medium. Monoclonal antibody SA-1 was used as a capture antibody that forms a second complex by antigen-antibody reaction with the first complex. A solution containing 1.5 mg / mL of monoclonal antibody SA-1 was applied in a line shape at 1 μL / cm to a position 10 mm from the end of the chromatographic development starting point side of the membrane member 3 for chromatographic development, and this was dried at 50° C. for 30 minutes and then dried at room temperature overnight. This was used as a capture region for the complex of Staphylococcus aureus ribosomal protein L7 / L12 antigen and gold colloid labeling antibody.

[0300] (e) Preparation of immunochromatography device In addition to the above-mentioned labeling antibody-attached member 1 (length 5 mm), the above-mentioned bacteriolytic agent-attached member 2 (length 5 mm), and the above-mentioned chromatographic development membrane member 3 (Sartorius CN95, length 25 mm), a filter paper (Cytiva 17CHR, length 25 mm) was prepared as the absorption member 7. Furthermore, GF / DVA (Cytiva: a filter member made of glass fiber, thickness 776 μm, retention particle size 3.5 μm) was prepared as the first filter member 6-1, and GF / AVA (Cytiva: a filter member made of glass fiber, thickness 299 μm, retention particle size 1.7 μm) was prepared as the second filter member 6-2. These members were then attached to AR Care 9020 (thickness 254 μm, length 75 mm, made of polystyrene with an adhesive for attaching members) as the substrate 8, and then cut to a width of 5 mm. The lengths of the first filter and the second filter were adjusted according to the following Table 11. The immunochromatography devices of Examples III-1, III-5, and III-6 are shown in schematic diagrams together with their dimensions (FIGS. 26(A), (B), and (C)).

[0301] [Table 11]

[0302] (f) Milk sample The milk used was prepared by mixing raw milk (not heat-sterilized or homogenized) collected from lactating Holstein cows with a 0.1 M MOPSO aqueous solution (pH 7.4) in a 1:1 ratio.

[0303] (g) Sample fluid holding container A 2 mL microtube manufactured by Eppendorf was used as the specimen liquid holding container 20. Ten immunochromatographic devices were manufactured, and 600 μL of milk sample was dispensed into the specimen holding container 20 to conduct a chromatographic development experiment, and the proportion of cases in which chromatographic development was poor was determined. The overlapping distance X of the first filter member and the second filter member was determined by measuring the longitudinal length of the overlapping portion of the first filter member and the second filter member when observing the immunochromatographic device from the longitudinal side. The total water absorption amount Y (g) was measured as follows. After the specimen liquid was developed in the immunochromatographic device, when a sufficient amount of time had passed and the weight of the entire device had not changed, the filter member, the labeling enzyme-imparted member, and the lytic agent-imparted member were removed from the device, and the weight W2 (g) of only the substrate, the chromatographic development membrane member, and the absorbing member was measured. Similarly, the filter member, labeling enzyme-imparting member, and lytic agent-imparting member were removed from an unused immunochromatography device before the sample liquid was developed, and the weight W1 (g) of the substrate, chromatographic development membrane member, and absorbing member alone was measured. At this time, W2-W1 is the amount of liquid developed by chromatography, i.e., the total amount of liquid absorbed by the chromatographic development membrane member and absorbing member Y (g).

[0304] The chromatographic development failure rate was as high as 50% when X / Y was less than 30, but was 40% or less when X / Y was 30 or more. When X / Y was 70 or more, the failure rate was 25% or less, and when X / Y was 100 or more, the failure rate was 15% or less. When X / Y was the same, the longer the first filter member, the lower the failure rate tended to be. Examples III-5 and III-6 (X / Y=112.5 mm / g) had a failure rate of 0% and did not experience any failures. [Explanation of symbols]

[0305] 1. Labeling antibody attachment member 2, 2-1, 2-2 Bacteriolytic agent-imparted material 3. Chromatographic development membrane carrier 4. Capture site, detection area 5 Sample contact member 6, 6-1, 6-2 Filter parts 7 Absorption material 8 Base material 9 Immersion area 10 Immunochromatography device 11 Cover member 20 Sample liquid holding container, milk sample holding container 100 Overlap between the first filter member and the second filter member

Claims

1. A method for detecting a target bacterium in a sample liquid by antigen-antibody reaction using an immunochromatography device, comprising: 1) a step of immersing at least a part of a labeling antibody-applied member to which a labeling antibody has been applied and at least a part of a lytic agent-applied member to which a lytic agent has been applied in the sample liquid, and at least a part of an antigen-antibody reaction between an antigen derived from the target bacterium and the labeling antibody in the sample liquid is carried out to form a first complex; 2) a step of forming a second complex by causing an antigen-antibody reaction between the capture antibody immobilized on the immunochromatography device and the first complex, and detecting bacteria based on the label of the labeling antibody; A method for detecting bacteria, comprising:

2. The method according to claim 1 , wherein at least a portion of the labeling antibody-applied member and at least a portion of the lytic agent-applied member to which the lytic agent has been applied are simultaneously immersed in the specimen liquid.

3. The method of claim 1 or 2, further comprising agitating the sample liquid after said immersion and prior to said detecting.

4. The method according to any one of claims 1 to 3, wherein the antigen of the target bacterium is an intracellular antigen.

5. The method of claim 4, wherein the intracellular antigen is a ribosomal protein.

6. The method of claim 5 , wherein the ribosomal protein is an L7 / L12 protein.

7. The method according to any one of claims 1 to 6, wherein after the immersion, at least a part of the labeling antibody attached to the labeling antibody-attached member and the lytic agent attached to the lytic agent-attached member are eluted into the specimen liquid.

8. An immunochromatographic device for detecting a target bacterium in a sample liquid by an antigen-antibody reaction, the immunochromatographic device comprising: (a) a labeling antibody-attached member having attached thereto a labeling antibody that forms a first complex through an antigen-antibody reaction with an antigen derived from a target bacterium; (b) a chromatographic development member including an immersion area to be immersed in a sample liquid and a detection area in which a capture antibody that forms a second complex by an antigen-antibody reaction with the first complex is immobilized; and (c) a bacteriolytic agent-impregnated member to which a bacteriolytic agent has been impregnated; Including, wherein, when the immunochromatography device is in use, at least a part of the labeling antibody-attached member and the immersion area of ​​the chromatographic development member are both immersed in a sample liquid, and when the immunochromatography device is in use, at least a part of the lytic agent-attached member is immersed in a sample liquid; Here, the detection of target bacteria in a sample liquid by the immunochromatographic device is performed by detecting the second complex captured in the detection area of ​​the chromatographic development member based on the label of the labeling antibody.

9. The immunochromatographic device according to claim 8 , wherein at least a portion of the labeling antibody-attached member is disposed within the immersion area.

10. 9. The immunochromatographic device according to claim 8, wherein the lytic agent-attached member and the labeling antibody-attached member are each formed as separate members.

11. The immunochromatographic device according to claim 8 , wherein the lytic agent-attached member and the labeling antibody-attached member are formed as a single member.

12. The immunochromatography device according to any one of claims 8 to 11, wherein the lysing agent-applied member is disposed so that at least a portion of the lysing agent-applied member is included in the immersion area.

13. The immunochromatography device according to any one of claims 8 to 12, wherein one or both of the lytic agent-applied member and the labeling antibody-applied member are provided as separate members from the chromatographic development member.

14. The immunochromatography device comprises: (d) Sample contact member Further comprising: The immunochromatographic device according to any one of claims 8 to 13, wherein at least a portion of the sample contact member is immersed in a sample liquid when the immunochromatographic device is in use.

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