Method for forming the sample development section of an immunochromatographic testing device and an immunochromatographic testing device

Abstract

Disclosed are: an immunochromatographic test device that allows quantification or detection of the presence of a substance to be detected to be performed more quickly and with higher sensitivity than conventional measurement methods; and a method for forming a sample-development part of the immunochromatographic test device. The method for forming the sample-development part of the immunochromatographic test device comprises applying cholic acid or a salt thereof to the sample-development part. The immunochromatographic test device contains cholic acid or a salt thereof in the sample-development part. By using a test device comprising the sample-development part formed using this method, the visibility of a detection part can be improved, resulting in quick and highly-sensitive measurement.

Description

Technical Field

[0001] The present invention relates to a method for forming a sample developing portion of an immunochromatography test device and an immunochromatography test device having a sample developing portion formed by the method.

Background Art

[0002] In recent years, simple test reagents and kits have been developed for performing various tests such as the presence or absence of pathogen infections such as viruses and bacteria, and the presence or absence of pregnancy, in a short period of time. Pathogen components, human chorionic gonadotropin, etc. are the targets of detection or quantification. Many simple test reagents have the characteristics of not requiring special equipment, being easy to operate, and being inexpensive. For example, simple test reagents for pregnancy diagnosis are sold at general pharmacies. In addition, simple test reagents for detecting pathogen infections are widely used not only in large hospitals and medical test centers but also in general hospitals and clinics other than these. These facilities are often the first medical institutions that patients visit. If the presence or absence of infection can be determined on the spot for the specimens collected from patients, treatment measures can be taken at an early stage. Therefore, the importance of simple test reagents in medicine is increasing more and more.

[0003] Currently, as a simple test method, an immunoassay method utilizing an antigen-antibody reaction, particularly the immunochromatography method, is generally known. In the immunochromatography method, a complex of a capturer (capturing substance) that specifically binds to the detected substance and a label that specifically binds to the detected substance is formed on a membrane, and the label is detected / quantified to detect (measure or quantify) the detected substance. The immunochromatography method is widely used for detecting a variety of detected substances because the measuring device is simple and it is also excellent in terms of cost.

[0004] In one form of immunochromatography, a test device is equipped with a detection unit on which an antibody that specifically binds to the substance to be detected is immobilized as a capture substance on a membrane strip such as nitrocellulose, and a labeling unit containing a label that specifically binds to the substance to be detected. A sample containing the substance to be detected is dropped onto the device, and a complex of the substance to be detected and the label is formed and unfolded, and this complex is captured by the detection unit to detect or quantify the label (for example, Patent Documents 1 and 2).

[0005] In recent years, a technique has been developed to pre-treat sample specimens on the sample addition section of an immunochromatographic testing device by pre-impregnating the sample addition section with a reagent. (Patent Document 3) [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2008-268043 [Patent Document 2] Japanese Patent Publication No. 2008-203135 [Patent Document 3] Japanese Patent Publication No. 2016-161329 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] In immunochromatography, the visibility of the detection area is a crucial factor and significantly impacts the results. However, visibility can deteriorate when sample components are present. Possible solutions include removing sample components by centrifugation or improving visibility using surfactants. However, centrifugation requires the introduction of specialized equipment and reduces handling efficiency, while the addition of surfactants can decrease sensitivity and specificity.

[0008] The object of the present invention is to improve upon the aforementioned problems and provide an immunochromatographic inspection device and a method for forming its sample development section, which enable the detection or quantification of a substance to be detected more rapidly and with higher sensitivity than conventional measurement methods. [Means for solving the problem]

[0009] As a result of diligent research, the inventors of the present invention have discovered that applying cholic acid or a salt thereof to the sample development area in immunochromatography improves the visibility of the detection area, thus completing the present invention.

[0010] In other words, the present invention provides the following: (1) A method for forming a sample development section of an immunochromatographic testing device, comprising applying cholic acid or a salt thereof to the sample development section. (2) The method according to (1), wherein the amount of cholic acid or its salt added is 0.5 μg to 10 μg. (3) The method according to (1) or (2), wherein the cholic acid or its salt is sodium cholate. (4) An immunochromatographic testing device comprising cholic acid or a salt thereof in the sample development section. [Effects of the Invention]

[0011] By using an inspection device equipped with a sample development section formed using the method of the present invention, the visibility of the detection section can be improved, and measurements can be performed quickly and with high sensitivity. [Brief explanation of the drawing]

[0012] [Figure 1] These are a top view and a cross-sectional view of an inspection device, which is one embodiment of the present invention. [Modes for carrying out the invention]

[0013] In this invention, the sample development section is the area in an immunochromatographic testing device where the sample is developed. More specifically, it is the area from the tip of the sample addition section to the detection line.

[0014] The sample development section is formed from a porous substrate such as a nitrocellulose membrane.

[0015] The sample development section can be pre-treated with reagents necessary for the antigen-antibody reaction. For example, surfactants can be applied to prevent non-specific components in the sample from participating in the antigen-antibody reaction, or hemolytic agents can be applied when measuring blood samples. In immunochromatography, the sample is often pre-suspended in a suspension solution before being developed in the sample development section. However, in this case, the sample is diluted, and the substance to be detected is also diluted, which can impair detection sensitivity. Therefore, buffers, surfactants, various proteins, salts, and sugars, which are components of the sample suspension solution, can be pre-treated.

[0016] In the method of the present invention, it is necessary to apply cholic acid or a salt thereof to the sample development section. A preferred method for applying cholic acid or a salt thereof to the sample development section is to add a solution of cholic acid or a salt thereof, preferably an aqueous solution using water or an aqueous buffer as the solvent, by impregnation or dropwise addition. For example, an aqueous solution of cholic acid or a salt thereof can be added to the labeled sample section. The concentration of cholic acid or a salt thereof in the solution to be added is usually 0.005% to 5% by mass, preferably 0.05% to 2% by mass, more preferably 0.1% to 0.5% by mass, and even more preferably 0.3% to 0.5% by mass. The amount of cholic acid or a salt thereof added to the sample development section is usually about 0.01 μg to 100 μg, preferably about 0.1 μg to 30 μg, more preferably about 0.5 μg to 10 μg, and even more preferably about 2 μg to 8 μg. In a preferred embodiment, for example, an aqueous solution of cholic acid or a salt having a concentration of 0.1% to 0.4% by mass can be added in an amount of 2 μg to 8 μg of cholic acid or a salt thereof.

[0017] The substances to be detected and the specimens to be detected using the immunochromatographic test device of the present invention are not limited. For example, specimens can be pharyngeal or nasal swabs, nasal aspirates, pharyngeal or nasal lavage fluids, saliva, serum, plasma, whole blood, fecal suspensions, urine, culture media, and these can be used diluted with buffer or used undiluted. The substances to be detected are also not limited in any way and may be any substance to be detected. Specific examples include viral antigens such as influenza virus, adenovirus, RSV, HAV, HBs, HIV, and norovirus; bacterial antigens such as MRSA, Group A Streptococcus, Group B Streptococcus, and Legionella; toxins produced by bacteria, etc.; hormones such as Mycoplasma, Chlamydia trachomatis, and human chorionic gonadotropin; C-reactive protein, myoglobin, cardiac troponin, various tumor markers, pesticides, and endocrine disruptors; and antibodies against the above-mentioned bacteria, viruses, etc.

[0018] The present invention also provides an immunochromatographic testing device that includes a sample development area formed as described above. Except for the application of cholic acid or a salt thereof to the sample development area, the configuration of the testing device itself may be well-known, such as those described in Patent Documents 1, 2, and 3. A schematic diagram of a specific example of the immunochromatographic testing device of the present invention is shown in Figure 1, but the testing device of the present invention is not limited thereto.

[0019] The top of Figure 1 is a top view, and the bottom is a cross-sectional view. In the specific example shown, a nitrocellulose membrane (1) with two detection units (3) formed thereon, an absorption pad (5) made of filter paper, a labeling unit (2) formed by the above method, and a sample addition unit (4) made of glass fiber filter are stacked on a plastic plate (6). As shown in the figure, one end region of the absorption pad (5) is superimposed on one end region of the nitrocellulose membrane (1), the other end region of the nitrocellulose membrane (1) is superimposed on one end region of the labeling unit (2), and the other end region of the labeling unit (2) is superimposed on one end region of the sample addition unit (4), thereby forming a continuous lateral flow channel.

[0020] Next, an immunoassay method using this test device will be described. First, a sample solution is prepared by suspending / extracting a sample in a buffer for sample suspension / extraction. A labeled body part (2) including a stabilized dried labeled body pad formed by the above method is provided, in which an antibody that reacts with an analyte in an antigen-antibody reaction is labeled with colored polystyrene particles on a nitrocellulose membrane (1) laminated on a plastic plate (6). Further, a detection part (3) in which an antibody that reacts with an analyte in an antigen-antibody reaction is immobilized linearly as a capture substance is provided. The sample solution is dropped onto a sample addition part (4) of the test device. The sample solution containing the analyte moves horizontally on the membrane and develops the labeled body. Therefore, if the analyte is present, a complex of the analyte-labeled body is formed. When it further reaches the detection part (3), a complex of the capture antibody-analyte-labeled body is formed on the line. The presence or absence of the analyte in the sample is determined by detecting the presence of the complex by the colored polystyrene particles in the complex. The detection part (3) is a region in which an antibody or an antigen-binding fragment thereof that reacts with the analyte substance and can bind to the analyte simultaneously with the antibody or its antigen-binding fragment on the colored polystyrene particles is immobilized linearly. Other components and the like that did not participate in the reaction are absorbed by the absorption pad part (5).

[0021] By using the test device of the present invention using colored polystyrene particles conjugated with an antibody or an antigen-binding fragment thereof that reacts with an analyte as a stabilized dried labeled body, the analyte can be measured quickly and easily.

Example

[0022] Hereinafter, the present invention will be described more specifically based on examples and comparative examples. However, the present invention is not limited to the following examples.

[0023] Example 1 Preparation of a monoclonal antibody that recognizes the N protein of SARS-CoV-2 1. Preparation of SARS-CoV-2 N protein antigen The DNA encoding the SARS-CoV-2N protein was expressed in E. coli using an expression vector, cultured for several days, and then the protein was purified before use.

[0024] 2. Production of anti-SARS-CoV-2N protein monoclonal antibodies By immunizing BALB / c mice with the SARS-CoV-2N protein antigen described in 1., and then extracting iliac lymph nodes from the mice after a certain period of rearing, several hybridoma cell lines producing anti-SARS-CoV-2N protein antibodies were obtained using the "mouse iliac lymph node method" (Sado Y et al., Acta Histochem. Cytochem. 39: 89-94 (2006)).

[0025] The acquired cell line was intraperitoneally administered to pristane-treated BALB / c mice, and approximately two weeks later, antibody-containing ascites fluid was collected. From the obtained ascites fluid, IgG was purified by affinity chromatography using a protein A column, and multiple purified anti-SARS-CoV-2N protein monoclonal antibodies (hereinafter sometimes referred to as "anti-N protein antibodies") were obtained.

[0026] In the following examples, antibodies were selected from among several obtained anti-SARS-CoV-2N protein monoclonal antibodies, taking into consideration their reactivity and specificity.

[0027] Example 2: Immunoassay device for measuring SARS-CoV-2 1. Immobilization of anti-SARS-CoV-2N protein antibodies onto nitrocellulose membranes. The anti-N protein antibodies classified in Example 1 were diluted in buffer solution, and anti-mouse IgG antibodies were prepared. The anti-N protein antibodies were linearly applied to the sample pad side and the anti-mouse IgG antibodies to the absorbent side of a nitrocellulose membrane backed with a PET film. The nitrocellulose membrane was then thoroughly dried under warm air to obtain an anti-N protein antibody-immobilized membrane.

[0028] 2. Immobilization of anti-SARS-CoV-2N protein antibodies onto colored polystyrene particles The anti-N protein antibody prepared in Example 1 was bound to colored polystyrene particles, suspended in a buffer solution, and thoroughly dispersed by sonication to obtain anti-N protein antibody-conjugated colored polystyrene particles. These are referred to as anti-N protein antibody-immobilized particles in this specification.

[0029] 3. Coating and drying of colored polystyrene particles conjugated with anti-SARS-CoV-2N protein antibody. 8.0 μg of the suspension of anti-N protein antibody-immobilized particles prepared in step 2 was coated onto a fiberglass nonwoven fabric and thoroughly dried by hot air. This is referred to as the labeled pad in this specification.

[0030] 4. Fabrication of a SARS-CoV-2 testing device The anti-N protein antibody-immobilized membrane prepared in step 1 and the labeled pads prepared in steps 2 and 3 were bonded together with other components (backing sheet, absorbent band, sample pad), cut into 5 mm wide strips, and used to create a SARS-CoV-2 testing device.

[0031] Example 3 Test 1. Visibility test using sodium cholate A testing device was fabricated by diluting the anti-SARS-CoV-2N protein antibody-conjugated colored polystyrene particles described in 3. above with a diluent, and then adding sodium cholate to the solution to achieve concentrations of 0, 0.1, 0.2, 0.4, and 0.8% by mass.

[0032] Immunochromatographic testing was performed using SARS-CoV-2 recombinant NP antigen (CerTest) with each testing device, followed by a visibility test. For the visibility test, 10 units of each testing device were used, and the visibility of the detection line was evaluated using a scale of ◎, ○, △, and ×. The results are shown in Table 1.

[0033] [Table 1]

[0034] As shown in Table 1, the addition of sodium cholate improved the visibility of the detection line. The greatest improvement in visibility was observed at 0.4% by mass, and the improvement in visibility tended to decrease at concentrations higher than 0.8% by mass.

[0035] 2. Comparison of the sites where sodium cholate is added. We investigated whether the visibility could be improved by adding two types of surfactants, sodium cholate (cholic acid) and sodium deoxycholate (DOC), to the sample addition section and sample development section. The anti-N protein antibody described in 1. above was diluted with buffer, and the concentration of cholic acid was adjusted to 0.2% by mass and the concentration of DOC to 0.1% by mass. Furthermore, the anti-SARS-CoV-2N protein antibody-conjugated colored polystyrene particles described in 3. above were diluted with a diluent, and the concentration of cholic acid was adjusted to 0.2% by mass and the concentration of DOC to 0.1% by mass, and a testing device was fabricated.

[0036] Immunochromatographic studies were performed using SARS-CoV-2 recombinant NP antigen with each testing device, followed by a visibility test. The visibility test involved using eight of each testing device to evaluate the visibility of the detection line using a scale of ○, △, and ×. The results are shown in Table 2.

[0037] [Table 2]

[0038] As shown in Table 2, adding cholic acid to both the sample development section and the sample addition section improved the visibility of the detection line. Furthermore, it was confirmed that adding cholic acid to the sample development section yielded a greater improvement in detection line visibility compared to adding it to the sample addition section. In addition, adding cholic acid to both the sample development section and the sample addition section dramatically improved the visibility of the detection line. [Explanation of Symbols]

[0039] 1. Nitrocellulose membrane 2 Sign body part 3. Detection Unit 4. Sample addition section 5 Absorbent pad section 6 Plastic sheet

Claims

1. A method for preparing a label portion of an immunochromatographic testing device, comprising applying cholic acid or a salt thereof to the label portion, wherein the label portion contained in the label portion is colored polystyrene particles, and an aqueous solution of cholic acid or a salt thereof with a concentration of 0.1% to 0.4% by mass is added in an amount of 0.5 μg to 10 μg.

2. The method according to claim 1, wherein the cholic acid or its salt is sodium cholate.

3. An immunochromatographic testing device comprising cholic acid or a salt thereof in the label portion, and colored polystyrene particles as the label, wherein the amount of cholic acid or a salt thereof is 0.5 μg to 10 μg.

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