KR20230094506A - Method of enhancing the signal intensity in immunochromatographic assay - Google Patents

Abstract

Disclosed herein are methods for improving the sensitivity of immunochromatographic assays. In the method according to the present disclosure, the detection sensitivity of the target analyte included in the sample is improved by sequentially applying a sample to be detected and a buffer for chromatography development at separate locations. The method according to the present disclosure can greatly reduce false negatives in antigen-antibody binding-based in vitro detection.

Description

Method for enhancing the sensitivity of immunochromatographic test {Method of enhancing the signal intensity in immunochromatographic assay}

It is related to a technique capable of improving the sensitivity of the biological material test of the present immunochromatography method.

Immunochromatography-based diagnostic reagents, which are generally implemented in the form of lateral flow or dipsticks, have been used in a wide range of fields, such as diagnosis of human and animal infectious diseases, food testing, and environmental sample analysis, since they were first developed in the 1980s. It is widely used (Wong & Tse, Lateral Flow Immunoassay, 2009). The best-known example is a pregnancy test reagent. These reagents are convenient and portable. If urine is soaked in the sample pad, the result can be judged by looking at the color band of the test line and control line through the result window within 5 minutes. Also, in the diagnosis of infectious diseases, samples such as blood, serum, plasma, nasopharynx, and nasal specimens are used.

This immunochromatographic technique is based on the principle of antigen-antibody reaction. For example, a detector antibody is conjugated to gold particles (20-40nm) or latex particles (100-300nm), and depending on the type of analyte, the antibody or antigen is deposited on a nitrocellulose membrane (10-15um pores) in a certain amount. It is divided and fixed. The analyte may be a protein of a pathogen in a biological sample (eg, a protein or antigen derived from COVID-19) or an antibody (IgM, IgA, IgG, etc.) produced by an immune response in the body after infection with a pathogen.

Recently, the SARS-CoV-2 virus, which is the cause of COVID-19 infection, is being detected by a nucleic acid-based PCR test. On the other hand, immunochromatographic antigen-antibody-based tests, despite their speed and convenience, are limited in use due to false negatives due to relatively low sensitivity compared to PCR tests.

Therefore, it is necessary to develop a technique to increase the sensitivity of such an immunochromatography-based test.

Patent Application Publication No. 2003-0065341 (published on August 6, 2003)

An object of the present application is to provide a method for improving the sensitivity of detecting a target analyte in a sample using immunochromatography based on an antigen-antibody reaction.

In one aspect, the present invention provides a method for improving the detection sensitivity of the target analyte in an immunochromatographic method for detecting a target analyte in a sample, wherein the method is a conventional method by mixing a sample and a developing buffer and dropping them on a sample pad Instead, it is characterized in that the sample and the development buffer are sequentially dropped onto the development medium and the sample pad, respectively, without mixing.

In one embodiment, the method according to the present disclosure includes providing a test strip used for immunochromatography, wherein the strip includes a developing medium, a sample pad, and an absorbent pad, and both ends of the developing medium are respectively One end of the sample pad and one end of the absorption pad are in contact with each other, so that the development medium is located between the sample pad and the absorption pad, and the development medium can bind specifically to the target analyte. attaching a capturer and a control material at regular intervals in order, the capturer being attached in a direction close to the sample pad, and dropping the sample into a development medium in front of the capturer attachment site; and dropping a development buffer for chromatography development on the strip onto the sample pad after loading the sample.

In one embodiment, in the method according to the present disclosure, the strip further comprises a conjugation pad, and the conjugation pad is positioned between the sample pad and the developing medium.

In another embodiment, a position on the development medium onto which the sample used in the method of the present disclosure is loaded may be marked with a dye, and the dye moves along with the chromatographic development.

In another embodiment, the strip used in the method of the present disclosure further includes a support, and the sample pad, the development medium, and the absorbent pad are positioned on the support.

In another aspect, the present application provides a strip for use in the method disclosed herein, wherein the strip includes a developing medium, a sample pad, and an absorbent pad, wherein both ends of the developing medium are at one end of the sample pad and the other, respectively. Adjacent to one end of the absorbent pad, the development medium is located between the sample pad and the absorbent pad, and a capture agent and a control material capable of specific binding to the target analyte are constant in the development medium. It is attached in order at intervals, the capturer is located in a direction close to the sample pad, and includes a sample dropping site indicated by a dye in front of the capturer attachment site on the development medium, and the dye is chromatographically developed. It is a water-soluble pigment that can move in one direction.

In one embodiment, the strip on which the method according to the present disclosure is used further comprises a conjugation pad, and the conjugation pad is located between the sample pad and the developing medium.

In another aspect, the present disclosure also provides a housing for accommodating a strip according to the present disclosure, the housing comprising a first window for loading a buffer solution into the sample pad of the strip, a second window for loading a sample, and a capturer and control material. and a third window for detecting a reaction at the attached site.

In the method for improving the sensitivity of an immunochromatographic test according to the present disclosure, the detection sensitivity of a target analyte included in the sample is improved by sequentially applying a sample to be detected and a buffer for chromatographic development at separate locations. The method according to the present disclosure can greatly reduce false negatives, which is the biggest disadvantage of antigen-antibody binding-based in vitro detection.

Figure 1a schematically shows the structure of a strip that can be used for immunochromatography of a method according to an embodiment of the present invention, and indicates a developing buffer and a sample loading site according to the method of the present invention.
FIG. 1B schematically shows the structure of a strip that can be used for immunochromatography of a method according to an embodiment of the present disclosure, and a developing buffer and a sample loading site according to an existing method are indicated here.
Figure 2 shows the results of examining Japanese encephalitis virus IgM antibody as an analyte by immunochromatography using the sample loading method (①) according to the present application and the conventional loading method (②). Low concentrations of the analyte were detected as positive with the loading method according to the present application, whereas negative with the conventional method. This indicates that the result by the existing method is a false negative due to relatively low sensitivity.
Figure 3 is one embodiment of a script that can be used in a method according to the present disclosure where the sample drop site is marked with a dye.
Figure 4 is an embodiment of a housing capable of accommodating a strip that can be used in the method according to the present invention, and the housing is separately provided with a window for loading a chromatographic development buffer and a window for dropping a sample.
5 schematically shows a specific example of a strip in which the method according to the invention is used.
Figure 6 schematically shows a strip in the form of a dipstick and an example of its use in which the method according to the present invention can be used.

Disclosure of the present invention: In chromatographic analysis for the detection of a specific target analyte in a sample, the discovery that the detection sensitivity of a specific target analyte can be improved by changing the loading site of the sample, whether or not it is mixed with the developing buffer, and the loading time. is based on

Accordingly, in one aspect, the present disclosure relates to a method for improving detection sensitivity of a target analyte in an immunochromatographic method for detecting the target analyte in a sample.

In one embodiment, the method according to the present disclosure includes providing a strip for lateral flow analysis used in immunochromatography; dropping a sample including a target analyte to be analyzed onto a first position of the strip; and dropping a developing buffer for chromatography development on a second position of the strip after the sample is loaded, wherein the strip includes a developing medium, a sample pad, and an absorption pad, wherein the amount of the developing medium The end portions are in contact with one end of the sample pad and one end of the absorbent pad, respectively, so that the development medium is located between the sample pad and the absorbent pad, and the development medium contains a specific amount of the target analyte. A captor capable of binding and a control material are attached in order at regular intervals, the captor is located in a direction close to the sample pad, and the first position is in front of the captor on the development medium at a regular interval. , and the second position is located on the sample pad.

As used herein, the term "sample" or specimen refers to a material to be analysed including a target analyte, and a sample that can be used in the present invention is a liquid or liquid-like material, for example, various solid tissues/cells, It includes blood, saliva, urine, sweat, body hair, or substances derived therefrom, or substances collected from the surrounding environment (eg, air, soil, water, etc.). Examples include, but are not limited to, blood, urine, saliva, etc., and blood may include whole blood, plasma, serum, or blood that has undergone a predetermined treatment (eg, anticoagulation), plasma, serum, etc. . Extracts of tissues or cells are selected from, for example, carbohydrates, lipids, nucleic acids, proteins and the like. The analyte is, for example, but not limited to, markers associated with various diseases, for example, proteins of viral pathogens (HIV, HCV, SARS-CoV-2, dengue virus, Japanese encephalitis virus, etc.) or corresponding These include human antibodies produced, inflammatory disease markers (CRP, PCT), tumor markers (PSA, AFP CA-125, etc.), hormones (Cortisol, progesterone, etc.).

As used herein, the term "analyte" refers to a compound to be detected or analyzed in a sample, also referred to as a target analyte or target, including proteins or nucleic acids. Proteins herein include polypeptides and peptides, and functionally include antibodies, antigens, specific proteins derived from pathogenic viruses or bacteria. In one embodiment, it comprises an antibody due to SARS-CoV-2 infection or a protein such as a spike derived from SARS-CoV-2. Also herein, the term "nucleic acid" includes chemosynthetic DNA and RNA, including genomic DNA, cDNA and oligonucleotides.

The reagent herein is a material suitable for the detection and analysis of the above-described analyte, and is different depending on the specific analyte. It may be a buffer solution for development of graphics, and the like, but is not limited thereto.

Immunochromatography detects a target analyte in a sample by chromatography based on an antigen/antibody reaction, and includes lateral flow analysis according to a chromatography method. The lateral flow analysis is a method for quantitatively or qualitatively detecting a target analyte, such as a specific protein or nucleic acid, contained in a sample. A method of detecting a target analyte in a sample through a sequence-specific hybridization reaction or an antigen-antibody reaction by moving a sample by chromatography on a strip containing a nitrocellulose membrane (development medium) bound to a specific position. For example, reference may be made to those described in Korean Patent Publication Nos. 2003-0065341, 2011-0007699, 2011-0127386, and Registration No. 1149357.

For example, in one embodiment, a lateral flow strip to which the method of the present invention can be applied includes a developing medium, a sample pad, and an absorbent pad, and the developing medium is interposed between the sample pad and the absorbent pad. Both ends of the development medium are in contact with one end of the absorbent pad and one end of the sample pad to form a flow of liquid, and the capture agent and control material are disposed at regular intervals in the development medium. are attached in order, and the capturer is located close to the sample pad. The developing medium included in the strip itself may serve as a support.

For example, in another embodiment, a lateral flow strip to which the method of the present invention can be applied includes a support, a development medium, a sample pad, and an absorbent pad, and the support supports the development medium, the sample pad, and the absorbent pad. The sample pad and the absorption pad are positioned at both ends of the support so as not to overlap each other, the development medium is located between the sample pad and the absorption pad, and both ends of the development medium are One end of the absorbent pad and one end of the sample pad are in contact with each other so that a liquid flow can be formed, and a capturer and a control material are sequentially attached to the development medium at regular intervals, and the capturer is the sample located close to the pad.

In addition, as a chromatographic analysis means, a strip used for lateral flow and each component included therein may refer to a strip for lateral flow analysis as disclosed in, for example, Korean Patent Publication No. 2011-0046833. For example, the development medium usually refers to a chromatography medium, and preferably a liquid sample, an analyte, especially a protein or nucleic acid contained in a biological sample moves rapidly by capillary force to reach a capturer attached thereon. Anything that can be used can be used. Chromatographic media for development are generally porous materials that migrate with an aqueous medium in response to capillary forces. Examples of developing media include, but are not limited to, cellulose, nitrocellulose, polyethersulfone, polyvinylidine, fluoride, nylon, charged nylon, and polytetrafluoroethylene, which can be used for lateral transfer. In one embodiment the developing medium is nitrocellulose. When using nitrocellulose, the diameter of the pores is typically between 5 μm and 15 μm. In another embodiment, when a particulate label is used, the size of the pores should be about 10 times or more than the particle diameter. The materials exemplified above may be used alone or in combination with other materials. Another example is a ceramic material. Chromatographic media may also be multifunctional or modified to be multifunctional to allow covalent binding to the capturer.

Referring to FIGS. 1 to 4 herein, the strip used in the method according to the present invention comprises a capturer (T). As used herein, the term "capturer" refers to a substance capable of specific binding to a target analyte in a sample, and when the target analyte is a protein such as an antigen or an antibody, it may be a corresponding antibody or antigen, respectively, and the target analyte In the case of this nucleic acid, it includes a complementary nucleic acid capable of sequence-specific binding. The capturer can selectively capture the target analyte moving through the development medium by this specific binding reaction, and is also referred to as a test line. The capturer may be attached to the development medium through a covalent or non-covalent bond, and the attachment refers to a state in which it does not move along with the mobile phase of the development medium and is positioned in a fixed position. In one embodiment, the capturer is non-covalently attached to the developing medium. Methods for attaching proteins or nucleic acids, including antigens or antibodies, to porous membranes, such as, for example, nitrocellulose, are known. Such a trapper may be dispensed, for example, into a nitrocellulose membrane with a line width of about 1 mm or less, but it is not excluded that it is outside this range. Specific values may vary depending on the type of development medium used. At least one capturer is positioned at a predetermined location on the developing medium used as the strip, and may include a capturer for a positive or negative control for the assay.

Referring to FIGS. 1 to 4, the strip used in the method according to the present invention includes a control or control material, also called a control line (C), at a point spaced apart from the capturer attachment site in the direction of chromatography development. . A substance (antibody or antigen) unrelated to the analyte and capture material is attached to the control line, and a substance (antibody or antigen) that binds to the material attached to the control line is attached to the gold particle on the conjugation pad.

The test line and the control line of the above-described strip may be divided to a line width of about 1 mm or less, but it is not excluded that they exceed this range.

Referring to FIGS. 1 to 6, in the method according to the present invention, a sample such as serum is loaded at a predetermined position on a development medium in front of the capturer attachment site, not on the sample pad of the strip, unlike conventional methods.

Referring to FIGS. 1 to 6 of the present application, a predetermined position where a sample is dropped in the present application is a position spaced apart from the capturer attachment site in front of the capturer attachment site by a predetermined distance. The position at which the sample is dropped should be adjusted in consideration of the width of the test strip (usually about 4 mm to 5 mm), the air gap of the developing medium (10 μm to 15 μm), and the amount of the sample to be dropped on the developing medium. When the sample is dropped in front of the capturer attachment site, the sample dropped in the development medium moves and reaches the site (T, test line) where the capturer (Japanese encephalitis virus protein, for example in this example) is attached Antigen-antibody reaction (binding reaction between human anti-Japanese encephalitis virus antibody and viral protein) occurs. This is the biggest difference from the traditional method of reacting the sample with the capturer-gold particle conjugate first, and this improves the sensitivity. Then, when the development buffer is dropped from the sample pad side, the detector antibody (goat anti-human IgM antibody)-gold particle conjugate contained in the conjugation pad, for example, moves toward the development medium, forming an antigen-antibody conjugate arrives at the formed capturer line. In one embodiment, as disclosed in Example 1, when the width of the test strip is 4 mm, the opening of the developing medium is 12 μm, and the amount of sample is to be applied in an amount of about 3 μl to 5 μl, the sample dropping site is the captor attachment site. It was confirmed that it is the optimal condition for sensitivity improvement when it is located about 4-5 mm away from (test line, T).

In addition, when an excessive amount of sample (10 μl or more) is dropped on the development medium, the gold particle-detector antibody conjugate does not flow well through the development film when the development buffer is dropped on the sample pad because the development medium is too wet. may occur and the inspection may not be performed. Therefore, the amount of sample to be applied should be determined considering the width of the strip and the air gap of the developing film.

Therefore, those skilled in the art will determine the sample loading site of the method according to the present invention by appropriately considering the width of the test strip, the gap of the development medium, and the amount of the sample to be loaded into the development medium based on the disclosed contents including the embodiments of the present invention. You will be able to.

In one embodiment, the sample dropping site according to the present disclosure may be marked with food coloring having a line width of about 1 mm or less on the developing medium of the strip. Red No. 2, Red No. 3, Red No. 40, Red No. 102, Yellow No. 4, Yellow No. 5, Green No. 3, Blue No. 1, Blue No. 2, etc. may be used.

A strip used in a method according to the present disclosure may further comprise a conjugation pad. As an example, referring to FIG. 5 , the conjugation pad includes detection particles labeled with a material capable of specifically binding to a target analyte such as an antibody or an antigen. Examples of detection particles include, but are not limited to, various beads such as polystyrene microspheres, latex particles, nano gold particles, colloidal gold particles, metal particles, magnetic particles, fluorescent particles, and semiconductor nanocrystals. The detection particle labeled with a substance capable of specifically binding to the target analyte binds to the analyte by a specific reaction, which in turn binds to the capturer of the development medium, and this complex is detected through the detection device. can For example, for sandwich detection, an antibody capable of detecting the antigen of the sample as a capturer is attached to the test line of the unfolding membrane, and an antibody capable of detecting the antigen of the sample is conjugated to the conjugate pad. contains beads. Alternatively, for indirect detection, when the target to be analyzed in the sample is an antibody, antigen is attached to the unfolding membrane and beads conjugated with anti-species antibodies (IgA, IgG, IgM, IgE) are placed on the conjugation pad. include These beads included in the conjugation pad can move according to the capillary force due to the buffer solution applied to the sample pad and bind to the material of the capturer (test line) and the control line.

In one embodiment, the method according to the present disclosure can also be applied to immunochromatography in which a developing buffer is applied in the form of a dipstick as shown in FIG. 6 . In this case, the sample is applied as described above, and when one end of the sample pad of the strip is immersed in a buffer solution, capillary force is generated in the direction of the absorbent pad.

In another aspect the present invention relates to a strip in which a method according to the present invention is used. In one embodiment, the strip according to the present disclosure includes a development medium, a sample pad, and an absorption pad, and both ends of the development medium are in contact with one end of the sample pad and one end of the absorption pad, respectively. A development medium is located between the sample pad and the absorbent pad, and a capture agent capable of specific binding to a target analyte and a control material are sequentially attached to the development medium at regular intervals. A ruler is located in a direction proximal to the sample pad, and a sample loading site indicated by a dye is included in front of the capturer attachment site on the development medium, and the dye is a water-soluble dye that can move in the direction of chromatography development.

In one embodiment, the strip according to the present disclosure further includes a conjugation pad, and the conjugation pad is positioned between the sample pad and the developing medium.

In another aspect the present invention relates to a housing for receiving a strip to which a method according to the present invention may be applied. Referring to FIG. 4, in one embodiment, such a housing has a first window for dropping a buffer on the sample pad of the strip, a second window for dropping a sample, and a reaction at the site where the capturer and control materials are attached. and a third window for detection. The second window is a position corresponding to the sample drop site according to the method according to the present invention. In one embodiment, it can be particularly used for strips in which the developing buffer is applied using a tool such as a pipette.

Hereinafter, examples are presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, but the present invention is not limited to the following examples.

Example 1 - Production of Japanese encephalitis IgM antibody test kit

1.1 Preparation of Goat anti-Human IgM antibody and gold particle conjugate

To prepare antibody-gold particle conjugates, goat anti-Human IgM antibody was diluted in tertiary distilled water to a concentration of 2 mg/mL. 6.0 ug to 12.0 ug per 1 mL of 40 nm diameter gold particle solution (pH 8.0 to pH 8.8) was added to the antibody solution, and reacted with stirring at room temperature for 10 to 30 minutes. Blocker solution (10% BSA or 1% PEG 6,000) was added to this solution to a final concentration of 0.02% to 0.1% and reacted for 5 to 10 minutes. After completion of the reaction, the antibody-gold particle conjugate was precipitated by centrifugation, suspended in 2 mM borate buffer (pH 8.0 to pH 8.8, including 0.01% NaN3), and stored in a refrigerator.

1.2 Preparation of antibody-gold particle conjugation pad

The [Goat anti-Human IgM antibody-gold particle conjugate] prepared in Example 1 was mixed with a Tris-based solution (20-100 mM Tris) containing sucrose (4% to 10%) and TritonX-100 (0.1% to 0.5%). -Cl, pH 8.0 ~ 8.8) to make the final OD _526-532 value 5.0 ~ 6.0, evenly wet the polyester fiber (10mm X 300mm), and then dry at 40 ℃ ~ 50 ℃ temperature to make a dry pad manufactured.

1.3 Protein immobilization on nitrocellulose membrane (development medium)

1.5 mg/mL to 2.0 mg/mL of Japanese encephalitis virus envelope protein (test line) and 0.8 mg/mL to 1.2 mg/mL of rabbit anti-goat IgG on the surface of a nitrocellulose membrane (pore 12μm) using a dispenser The antibody (control line) was dispensed in the form of a 1.0 mm wide line. After dispensing, the nitrocellulose membrane was fixed by drying at 37°C to 50°C for 2 to 3 hours.

1.4 Sample Pad Pretreatment

After the sample loaded on the sample pad reacts with the antibody-gold particle conjugate, the sample pad is placed in a Tris-based buffer solution (20 -100 mM Tris-Cl, pH 7.5 ~ 8.5) and then completely dried at 30 ℃ ~ 40 ℃ temperature.

1.5 Preparation of developing buffer

The developing buffer to aid in sample development is based on Tris (20mM to 100mM, pH 7.5 to 8.5) and contains Triton X-100 (0.1% to 1%), Tween-20 (0.1% to 1.0%) and preservative (azide). 0.01% to 0.1% of sodium chloride).

1.6 Assembly of diagnostic kit

A conjugation pad containing [antibody-gold particle conjugate] and a sample pad are attached to the nitrocellulose membrane so as to overlap, and an absorption pad is placed on the nitrocellulose membrane opposite the sample pad so that the complex can be smoothly developed through capillary action. overlapped and attached. The prepared diagnostic strip (60 mm x 4.0 mm) was placed in a plastic case marked with the test line (T) and the control line (C) and assembled to prepare a diagnostic kit.

Example 2. Comparison of detection efficiency of the method according to the present invention and the existing method

The principle of the existing method is explained as follows. 10 μl of human serum (plasma) for detection of Japanese encephalitis is dropped into the sample inlet, and then 100 μl of the developing buffer is added into the same inlet. The sample solution then moves towards the gold conjugate pad. Here, the human IgM antibody in the sample binds to the goat anti-human IgM antibody dried on the gold conjugate pad. Subsequently, the gold particle-antibody complex continues to migrate to the developing membrane. If the anti-Japanese encephalitis virus IgM antibody is present in the sample, when the complex passes the test line (T) to which the capturer (Japanese encephalitis virus antigen) is fixed, [gold particle + goat anti-human IgM + human IgM + Japanese encephalitis Viral antigen] complexes are generated and the complexes continuously accumulated on the test line are shown in red. The remaining complex continues to reach the control line (C, anti-goat IgG antibody is fixed) along the developing membrane. Here, a [gold particle + goat anti-human IgM + anti-goat IgG antibody] complex is generated, and the complex continuously accumulated in the control line is shown in red. Therefore, if red is seen in both the test line and the control line, it is judged as positive, and if red is seen only in the control line, it is judged as negative.

In order to compare the performance of the existing method and the method according to the present application, Japanese encephalitis patient serum was directly dropped onto the nitrocellulose membrane, which is the method of the present application, in the diagnostic kit prepared in Example 1, and then the developing buffer was applied to the sample pad. It was dropped into the hole, and as in the previous method, 2) the sample was dropped into the hole on the sample pad side and allowed to permeate the pad, and then the developing buffer was dropped into the same hole. did

The sample was a Japanese encephalitis virus-positive sample, and a sample confirmed positive by the ELISA method was used. After dividing positive samples into high concentration, medium concentration, and low concentration, the two methods described above were applied to confirm the difference in sensitivity according to color development.

To apply the method according to the present invention, 3 μl of the sample was taken with an experimental pipette and dropped directly in front of the test line of the nitrocellulose membrane, and 100 μl of the development buffer was dropped into the hole on the sample pad side. Results were visually read after 10 minutes. In the case of the conventional method, 10 μl of the sample was taken with a laboratory pipette and dropped into the hole on the sample pad side, and when the sample permeated the pad, 100 μl of the developing buffer was dropped into the same hole. The test result was determined visually after 10 minutes. If both the test line (T) and the control line (C) turned red, it was judged positive, and if only the control line (C) turned red, it was judged negative.

As shown in FIG. 2, as a result of the experiment, the test method proposed in the present invention showed significantly higher detection sensitivity than the traditional method despite using a smaller amount of sample. In particular, when a positive low-concentration sample was tested, the method according to the present invention gave a positive result, while the traditional method gave a negative result. This indicates that the method according to the present invention can solve the false negative problem by improving the sensitivity.

Although the exemplary embodiments of the present application have been described in detail above, the scope of the present application is not limited thereto, and various modifications and improvements of those skilled in the art using the basic concepts of the present application defined in the following claims are also within the scope of the present application. that belongs to

All technical terms used in the present invention, unless defined otherwise, are used with the same meaning as commonly understood by one of ordinary skill in the art related to the present invention. The contents of all publications incorporated herein by reference are incorporated herein by reference.

Claims (7)

In an immunochromatographic method for detecting a target analyte in a sample, a method for improving detection sensitivity of the target analyte, the method comprising:
A step of providing a test strip used in the immunochromatography, wherein the strip includes a development medium, a sample pad, and an absorption pad, and both ends of the development medium are one end of the sample pad and the absorption pad, respectively. In contact with one end of the pad, the development medium is located between the sample pad and the absorbent pad, and the development medium includes a capture agent capable of specific binding to the target analyte and a control material. It is attached in order at regular intervals, and the catcher is attached in a direction close to the sample pad,
dropping the sample into a developing medium in front of the capturer attachment site; and
After loading the sample, dropping a developing buffer for chromatography development on the strip onto the sample pad.
According to claim 1,
The method of claim 1 , wherein the strip further comprises a conjugation pad, the conjugation pad positioned between the sample pad and the developing medium.
According to claim 1,
Wherein the position on the development medium onto which the sample is dropped is indicated by a dye, and the dye moves along with the chromatographic development.
According to claim 1,
The method of claim 1 , wherein the strip further comprises a support, and the sample pad, the development medium, and the absorbent pad are positioned on the support.
A strip used in the method according to any one of claims 1 to 4,
The strip includes a development medium, a sample pad, and an absorption pad, and both ends of the development medium are in contact with one end of the sample pad and one end of the absorption pad, respectively, so that the development medium is in contact with the sample pad. positioned between the pad and the absorbent pad;
A capture agent capable of specific binding to a target analyte and a control material are sequentially attached to the development medium at regular intervals, the capture agent is located in a direction close to the sample pad, and on the development medium The strip includes a sample dropping site marked with a dye in front of the capturer attachment site, wherein the dye is a water-soluble dye capable of moving in a chromatographic development direction.
According to claim 5,
The strip further comprises a conjugation pad, wherein the conjugation pad is positioned between the sample pad and the development medium.
A housing for accommodating the strip according to claim 5,
The housing includes a first window for dropping a buffer on the sample pad of the strip, a second window for dropping a sample, and a third window for detecting a reaction at a site where the capturer and control substances are attached. that is, the housing.

Images