KR101332336B1 - Prozone effect-preventable immunochromatographic strip and a kit comprising the same - Google Patents

Abstract

The present invention relates to an immunochromatography analysis strip and a kit containing the strip. The immunochromatography analysis strip and the kit containing the strip comprise: a) an adhesive plastic support member; b) a sample pad arranged on the upper surface of the adhesive plastic support member from an upper region to a lower region and equipped with a buffer inlet part in the upper region and a sample inlet part in the lower region to receive a liquid sample to be analyzed; c) a conjugate pad, for containing a conjugate to be specifically bonded to the analytes contained in the liquid sample of the sample pad, which is placed in a lower direction of the lower region of the sample pad; d) a signal detection pad, placed adjacent to the conjugate pad in the lower end of the conjugate pad, which contains a signal detection part for detecting analytes in the liquid sample and a control part to confirm the transfer of the liquid sample to the absorbing pad; and e) an immunochromatographic strip containing the absorbing pad placed adjacent to the signal detection pad in the lower end of the signal detection pad; and further comprises a control tape placed between the sample inlet part of the sample pad and the conjugate pad based on a material which prevents the pass of the liquid sample. Furthermore, the immunochromatography analysis strip and the kit containing the strip are provided to control the amount of analytes for analysis after transferring the sample injected according to the overlapped length of the conjugate pad and the control tape to the conjugate pad.

Description

Prozone effect-preventable immunochromatographic strip and a kit comprising the same}

The present invention relates to an immunochromatography strip and a kit including the same, and specifically, controls an amount of an analyte used or reacts with a conjugate of a conjugate pad in an analyte applied to an immunochromatography strip. The present invention relates to an immunochromatography strip and a kit comprising the same, which enables the conjugate in the pad to be shifted in time, thereby avoiding the prozone phenomenon.

Rapid diagnostic test (DRT) is called various names such as immunochromatography analysis, rapid kit analysis, etc., and its main composition is immunochromatography including support, sample pad, conjugate pad, signal detection pad, and absorption pad. As an analytical method by strip, the user can simply detect analyte from a biological or chemical sample in 2-30 minutes with a sample of 1-100 microliters without special skills or equipment. Rapid diagnostics is a method that allows you to qualitatively and quantitatively test analytes in a short time by utilizing the properties of biological or chemical substances that are specifically attached to each other. An immunochromatography kit in which the same immunochromatography strip is mounted inside a plastic housing is used. When using an immunochromatography strip, a container containing a sample is required separately, but the immunochromatography kit embedded in the housing is easy to use because it does not need a separate experimental container by directly inserting the sample into the inlet prepared in the housing.

The rapid diagnostic test is one of the most advanced assay kits developed in recent years in terms of simplicity and promptness, and is useful for diagnosing various disease agents such as antigens or antibodies of infectious pathogens, cancer factors, and heart markers.

Through analysis using an immunochromatography strip or an immunochromatography kit including the same, hepatitis C, using samples such as whole blood, plasma, serum, tears, saliva, urine, runny nose, and body fluids of humans or animals, Hepatitis A Hepatitis B, Influenza Virus, Avian Influenza Virus, Rota Virus, AIDS, Syphilis, Chlamydia, Malaria, Typhoid, Gastric Ulcer, Tuberculosis, SARS, Dengue, Leprosy etc. have. In addition, they can be used to confirm the presence of pregnancy, ovulation, cancer markers, heart disease indicators, etc., and can be used to confirm the administration of drugs such as cannabis methamphetamine, hiropon, opium, amphetamines, morphine, cocaine, etc. And furthermore can be used to identify bioterrorism by detection of lysine, anthrax, burcellella, botulinum, bexonia, salmonella, cholera, Staphylococcal enterotoxin B, tullaemia, etc., from foods such as Salmonella, Vibrio campylobacter, enterohemorrhagic It can be used for rapid testing and diagnosis in various fields, such as those that can be used to identify food poisoning bacteria such as E. coli and Yexinia.

1 is a cross-sectional view showing a representative example of an immunochromatography kit according to the prior art (Patent Documents 1 and 2).

The immunochromatography kit 10A of FIG. 1 includes an immunochromatography strip 10, wherein the immunochromatography strip 10 comprises an adhesive plastic support 11; And a sample pad 12a on the upper surface of the adhesive plastic support, receiving a liquid sample to be analyzed, and having a buffer and a sample single injection portion 15a on an upstream side thereof. Conjugate pad 12b, which is located in contact with the sample pad at a downstream end of the sample pad, contains a conjugate specifically binding to the analyte contained in the liquid sample introduced from The conjugate pad includes a signal detector 13b for detecting whether a substance is present and a control unit 13a for confirming whether a liquid sample has moved to the absorbent pad regardless of the presence of an analyte. The signal detection pad absorbs the signal detection pad 12c positioned in contact with the conjugate pad at the downstream end, and the liquid sample in which the signal detection reaction is completed. And an absorbent pad 12d positioned at a downstream end of the exit pad, the absorbent pad being located in contact with the signal detection pad.

The sample pad 12a absorbs a liquid sample (or analyte) to be analyzed and ensures a uniform flow of the liquid sample. The conjugate pad 12b includes a fluid conjugate that specifically binds to an analyte contained in the liquid sample, so that the liquid sample introduced through the sample pad 12a is used to connect the conjugate pad 12b. As it passes, specific binding between the analyte and the flow conjugate occurs. The signal detection pad 12c disposed at a position next to the sample pad 12a and the conjugate pad 12b includes a signal detection unit 13b and a matching unit 13a, which are spaced apart from each other at some distance from each other. . The signal detector 13b is an area for checking whether an analyte is present in the liquid sample, and the control unit 13a is an area for checking whether the liquid sample has normally passed through the signal detector 13b. The absorbent pad 12d absorbs the liquid sample passed through the signal detection pad 23 and assists in capillary flow of the liquid sample on the immunochromatography strip. In summary, the immunochromatography strip 10 has a sample pad 12a, a conjugate pad 12b, a signal detection pad 12c, and an absorption pad 12d attached to the support 11 from an upstream side to a downstream side. The liquid sample is moved from the sample pad 12a through the signal detection pad 12c to the absorption pad 12d, and an immunoassay is performed through signal detection on the signal detection pad 12c.

 If an appropriate amount of analyte is present in the liquid sample, a complex in which the conjugate and the analyte are specifically bound flows, and a specific substance attached to the signal detector 13b reacts specifically with the analyte and is fixed in the detection region. In this case, if the conjugate is a gold particle, the color may be identified by the manufacturer's method in purple, red, or black, and visually confirmed by forming a band. If no analyte is present in the sample, the detector displays no indication. The ultimately expressed result is a complex of (gold particle-specific reactant conjugate) + analyte + (specific reactant in signal detector), each analyte combined with a specific reactant to bridge the reaction. . However, if the analyte is present in a high concentration in the sample and reacts with the specific reactants of all the conjugates, but remains and saturates all the specific reactants in the detection region, the analyte fails to bridge and the conjugate is not connected to the detector. In spite of the presence of the substance, no indication or weakness appears in the detector. This phenomenon is called the prozone or hook effect.

Therefore, the immunochromatographic strip shown in FIG. 1 is applied to a sample having a relatively low concentration of analyte, and in order to apply a high concentration sample that causes prozone, an excessive amount of specific reactant and conjugate in the detection region must be added, but the cost is high. Not only does this increase, but the structural addition of the strip cannot be raised indefinitely. Alternatively, buffers, dilution vessels, pipettes, etc. must be provided separately, and the user is cumbersome because the sample must be diluted by adding several experiments and the dilution concentration and method must be remembered. In order to avoid such a prozone effect, the immunochromatography kits shown in FIGS. 2 and 3 have been developed.

2 and 3 are cross-sectional views showing representative examples of immunochromatography kits for blocking prozone effects in the related art (SD BIOLINE Dengue IgG / IgM, Standard diagnostic, inc, Korea).

In the immunochromatography kit of FIG. 2, the sample injection unit 25a "is positioned after the downstream side of the conjugate pad, so that the buffer is immediately supplied after the sample is injected into the sample injection unit 25a". When added to the sample, the sample first arrives at the signal detector 23b, after which the conjugate arrives, and an immune response can be made. In this case, the sample is first introduced into the sample input unit 25a ", and the analyte of the sample is present in the free state by spreading on the signal detection pad before contacting the conjugate, among which the analyte reaches the signal detection unit 23b. Is fixed in response to a specific reactant in the detection region and waits for a bridge reaction with the conjugate.When the buffer is introduced into the buffer input unit 25a ', the conjugate is fluidized by the buffer and moved to the signal detection pad 22c. At this time, a large amount of the conjugate reacts with and flows with many free analytes present between the conjugate pad 22b and the signal detector 23b, but passes through and disappears without reacting with the specific reactant of the signal detector 23b. Because only the conjugate that did not react with the analyte reacts with the analyte bound to the specific reactant of the signal detector 23b, thereby generating a signal. The color development time is late and is still not free of prozone phenomena due to excessive free analyte, so that the color is not developed or weakly developed, and the immunochromatography kit of Fig. 2 can not use whole blood but only plasma and serum.

Fig. 3 shows a filter 32e for filtering blood cells on a signal detection pad located at the sample input part 35a "of the immunochromatography kit in Fig. 2. With such a filter, whole blood other than plasma and serum is mounted. There is an advantage that can be used for.

The immunochromatography kits shown in FIGS. 2 and 3 are more advanced than the immunochromatography kits shown in FIG. 1, but have the following problems.

First, the initially flowing conjugate becomes obsolete due to the reaction with free analyte spread in the signal detection pad, and only the resulting conjugate does not completely overcome the prozone effect and shows results. The time is very slow, 15-30 minutes. Second, if there are two or more analytes and a mixture of high and low analytes, the high analyte is not a problem, but the low analyte is not detected by the sensitivity enhancer in the buffer and is detected in the diagnosis. Multiple inspections are not possible because the minimum detection limit that should be detected cannot be detected. Third, when a conjugate reaches a signal detecting pad containing a high concentration of a specific analyte, the analyte and the conjugate react at a rapid rate to form a large network and become large molecules and fail to pass through the porous signal detecting pad. Failure to reach the signal detector shows results as if no analyte is present.

Accordingly, there is a need for the development of immunochromatographic strips and kits that are capable of effectively avoiding prozone effects and are fast in assays and capable of multiple testing.

1.US 5,714,389 2.US 5,075,078

Accordingly, the present inventors have studied intensively for the development of a more preferable immunochromatography strip that can avoid the prozone effect, and as a result, it is possible to effectively control the amount of the analyte used, thereby avoiding the prozone effect, The present invention has been completed by focusing on immunochromatography strips that can be simultaneously tested in one immunochromatography kit in samples in which both high and low concentrations of analytes are present.

Accordingly, an object of the present invention is to provide an immunochromatography strip and an immunochromatography kit comprising the same, which can block the prozone effect by controlling the amount of analyte used to react with the conjugate in the conjugate pad in a sample. will be.

Another object of the present invention is not only to block the prozone effect by controlling the amount of analyte used to react with the conjugate in the conjugate pad in the sample, but also to enable two-stage immunodetection reactions in high to low concentrations. It is to provide an immunochromatography strip and a kit comprising the same or two or more different substances that need to detect a wide range over one possible immunochromatography strip.

Another object of the present invention is to provide a cost-competitive immunochromatography strip and a kit including the same, by reducing the amount of specific reactants used as raw materials for the test, thereby avoiding the prozone effect.

Definitions of terms used in the present specification are as follows.

Positioning the pads “fold” means that they are located adjacent to each other, but the distal ends of each pad are located overlapped.

The "upstream side" refers to a site that arrives first in time in the movement of the buffer, and means a site near the buffer input section.

The term "downstream" refers to a part which arrives later in time in the movement of the buffer, and means a part farther from the buffer input part.

"Midstream side" means an intermediate portion between the upstream side and the downstream side.

"Analyte used" means the analyte actually used in the experimental reaction among the analytes injected into the sample inlet.

"Insoluble analyte" means an analyte that is not used for an experimental reaction and is isolated in a specific area among the analytes injected at the sample inlet.

In order to achieve the above object,

a) an adhesive plastic support, and

Arranged from an upstream side to a downstream side on an upper surface of the adhesive plastic support body,

b) a sample pad containing a liquid sample to be analyzed and having a buffer input portion upstream and a sample input portion downstream;

c) a conjugate pad located downstream of the sample pad, the conjugate pad containing a conjugate specifically binding to an analyte contained in the liquid sample introduced from the sample pad;

d) downstream of the conjugate pad, comprising a signal detector for detecting whether an analyte is present in the liquid sample and a control for confirming whether the liquid sample has moved to the absorbent pad regardless of the presence or absence of the analyte A signal detection pad positioned at a side end with a conjugate pad, and

e) an immunochromatography strip comprising an absorbent pad positioned at a downstream end of the signal detecting pad, the absorbing pad being positioned to absorb the liquid sample from which the signal detecting reaction is completed,

f) further comprising a control tape of a material that does not pass through the liquid sample, located between the sample injection portion and the conjugate pad of the sample pad,

According to the overlapping length of the conjugate pad and the control tape provides an immunochromatographic analysis strip, which can be controlled by the amount of the sample injected into the conjugate pad to be used as an analyte.

The liquid sample introduced into the sample input unit by the introduction of the control tape becomes an insoluble analyte present in the downstream side of the sample pad in which a predetermined amount is present at the upper end of the control tape, and the remaining amount of the conjugate pad and the control tape are It spreads through the non-overlapping part to the conjugate pad and becomes an analyte used. In addition, if the buffer is immediately added to the buffer input unit after the liquid sample is added, the buffer is moved to the downstream side of the sample pad through the sample pad, and the insoluble analyte present in the downstream side is fixed to the downstream end of the sample pad. The buffer passed through the sample pad to the conjugate pad facilitates the movement of the analyte used in the conjugate pad to the signal detection pad. By such a principle, the control tape can control the amount of the analyte used in the sample introduced into the sample input unit, thus avoiding the frozen effect.

In another aspect of the present invention,

a) an adhesive plastic support, and

Attached to an upper surface of the adhesive plastic support body from an upstream side to a downstream side,

b) a first sample pad containing a liquid sample to be analyzed and having a buffer inlet upstream and a sample inlet downstream;

c) a second sample pad present below the middle stream side of the first sample pad and having a slower flow rate of the buffer than the first sample pad;

d) starting by folding with the second sample pad at the downstream end of the second sample pad, the conjugate containing a conjugate specifically binding to the analyte contained in the liquid sample introduced from the first sample pad; 1 conjugate pad located downstream of the sample pad,

e) downstream of the conjugate pad, comprising a signal detector for detecting whether an analyte is present in the liquid sample and a control for confirming whether the liquid sample has moved to the absorbent pad regardless of the presence or absence of the analyte A signal detection pad positioned at a side end with a conjugate pad, and

f) an immunochromatographic strip comprising an absorbent pad located downstream of said signal detection pad, said liquid absorbing sample having completed the signal detection reaction;

g) a first adjusting tape of a material which is located upstream of the conjugate pad in contact with the first sample pad and does not pass the liquid sample;

The first control tape serves to block the conjugate present on the upstream side of the conjugate pad from reacting with the liquid sample introduced into the sample input unit.

The immunodetection reaction of the first low analyte present in the injected sample is carried out first, followed by the detection reaction of the second high analyte high, and the first analyte and the second analyte are the same or different from each other. Another material, immunochromatography assay strip, is provided.

The immunochromatographic assay strip is an immunochromatographic assay strip required when all liquid samples used in the primary immunodetection reaction need to be used without controlling the amount of the sample. In the liquid sample introduced into the sample input unit, the entire sample is diffused toward the conjugate pad as an analyte used. A portion of the buffer immediately added after the sample is added to the sample input unit moves the used analyte and the conjugate to the signal detector, and the first immunodetection reaction proceeds to detect the first analyte. In this case, the second analyte is in a state in which the second analyte is coupled to the specific reactant of the signal detection unit in a form not bound to the conjugate. Then, due to the remaining buffer spreading slowly due to the presence of the sample pad 2, the conjugates that existed below the control tape and were protected in the conjugate pad proceed to the signal detection pad, and thus the specific reactants of the signal detection unit are already present. The secondary immunodetection reaction bridged by the second analyte coupled to proceeds to detect the second analyte in the signal detector. The first analyte and the second analyte may be the same or different from each other. Although the second analyte is higher in concentration than the first analyte, even if the immunodetection reaction is not expressed by the prozone effect during the immunodetection reaction of the first analyte, the second reaction is a conjugate that does not react with the analyte. Since the immune detection reaction is performed by moving to the signal detector, the expression of the second analyte may appear in the signal detector.

The immunochromatographic analysis strip further comprises a second control tape of a material which is positioned between the sample injection portion of the first sample pad and the downstream side of the conjugate pad, and which does not pass the liquid sample. 2 According to the overlapping length of the control tape, the injected sample is moved to the conjugate pad to control the amount of analyte used, and the immunodetection reaction of the first analyte having a low concentration in the injected sample is performed first. The immunodetection reaction of the second analyte with high is performed later, and the first analyte and the second analyte may be immunochromatographic strips that are the same or different from each other.

The liquid sample introduced into the sample input unit is partially spread by the second control tape toward the conjugate pad as an analyte used. Then, the buffer immediately added after the sample is added to the sample input part of the moving part of the analyte and conjugate used to the signal detector, the first immunodetection reaction proceeds to detect the first analyte. In this case, the second analyte is in a state in which the second analyte is not bound to the conjugate and is coupled to the specific reactant of the signal detection unit. Then, by the remaining portion of the buffer spreading slowly due to the presence of the second sample pad, the conjugate that is under the first control tape and protected in the conjugate pad proceeds toward the signal detection pad, and thus the signal detection unit The second analyte coupled with the specific reactant of the second immunodetection reaction proceeds to detect the second analyte. The first analyte and the second analyte may be the same or different from each other. Although the second analyte is higher in concentration than the first analyte and is not expressed by the prozone effect in the immunodetection reaction of the first analyte, the secondary reaction is later moved to the signal detection unit where the conjugate does not react with the analyte. Since the secondary immunodetection reaction is already bridged by the second analyte coupled with the specific reactant of the signal detector, the expression of the second analyte may appear in the signal detector.

The immunochromatographic analysis strip may be formed by passing the liquid sample through the conjugate pad located under the first control tape and below the first conjugate pad and the second control tape where the liquid sample does not reach. It is separated into two conjugate pads, and the separation is performed by inserting a conjugate separation pad between the first conjugate pad and the second conjugate pad, and the conjugate separation pad has a flow rate of the liquid sample compared to the conjugate pad. May be a strip for immunochromatography, a significantly late pad. By introducing the conjugate separation pad, the introduction of the liquid sample into the sample inlet can prevent the possibility that the liquid sample may diffuse to the first conjugate pad and react with the conjugate present in the first conjugate pad. .

The sample may be used, such as whole blood, plasma, serum, tears, saliva, urine, feces, runny nose, body fluids such as human and animal-derived materials, plant-derived materials, environmental substances, culture and culture-derived materials. The analyte may be a protein such as peptides, antibodies, antigens, carbohydrates, chemicals, and the like.

The first sample pad and the second sample pad may be polymers, peptides, proteins, carbohydrates, soaps, or the like, to further improve the selectivity of the analyte or to minimize the influence of interference materials that may be included in the liquid sample. It can be processed as, and has the function of filtering additionally. For example, when whole blood is used as a liquid sample, a more compact pad may be used to filter out red blood cells, or may contain additional aids to help filter them out. If necessary, an auxiliary pad may be further provided on the upstream side of the sample pad, which may include a material that can increase the reaction between the analyte and the conjugate or eliminate the influence by the interference material. The sample pad may be made of cellulose, acrylic fiber, rayon, polyester fiber, glass fiber, wool fiber, silk fiber, cotton yarn, flax fiber or pulp, or one or more kinds thereof may be mixed.

The second sample pad should be able to slow down the flow rate of the buffer compared to the first sample pad. For this purpose, the second sample pad may have a higher density, a finer particle size, stronger hydrophobicity, or a thinner thickness. Can be. In addition to this selection, the first sample pad and the second sample pad may be made of the same material, and the second sample pad may slow down the flow rate of the buffer compared to the first sample pad, for example, bovine serum albumin, orb. Protein selected from the group consisting of alumina, amino acids, peptides, polyethylene glycol, polyvinylalcohol, polyvinylpyrrolidone, glycerol, polysorbate The above substances can be prepared by mixing and treating the eluting solution substance. The second sample pad may be replaced with an extended conjugate pad. However, the conjugate should be contained only on the downstream side of the conjugate pad.

The sample may be used, such as whole blood, plasma, serum, tears, saliva, urine, feces, runny nose, body fluids such as human and animal-derived materials, plant-derived materials, environmental substances, culture and culture-derived materials. The analyte may be a protein such as peptides, antibodies, antigens, carbohydrates, chemicals, and the like.

The conjugate pad may be made of glass fiber, polyethylene fiber, or the like.

In the immunochromatographic assay strip, the conjugate separation pad may be made of any material that can slow the diffusion through the conjugate pad of the liquid sample, for example, cellulose, acrylic fiber, rayon, polyester fiber, It may be made of glass fiber, wool fiber, silk fiber, cotton yarn, flax fiber or pulp, or one or more kinds thereof may be mixed. Also, these materials include proteins such as bovine serum albumin and ovalbamine, amino acids, peptides, polyethylene glycol, polyvinylalcohol, polyvinylpyrrolidone, glycerol, polysorb One or more materials selected from the group consisting of bait surfactants (Polysorbate detergents) may be prepared by mixing with the eluent material, but are not limited thereto.

The signal detection pad is composed of a porous membrane pad, and may be made of nitrocellulose, cellulose, polyethylene, polyethersulfone, nylon, or the like. One or more signal detectors may be included.

The absorbent pad may be any pad capable of promoting the movement of the buffer, and may be made of, for example, pulp, cellulose, cotton fibers, or the like.

In the immunochromatographic analysis strip, the control tape including the first control tape and the second control tape may be made of any material that does not penetrate the liquid sample. For example, acetate, polyester ), Polyolefin, polypropylene, polystyrene, vinyl, polyimide, aluminum foil, or retro-reflective film, one side Or both sides may be a rubber, acrylic, or silicone-based adhesive is applied, but is not limited thereto.

The immunochromatographic analysis strip may be used as an immunochromatography strip as a form in which a front portion except for a sample inlet and a signal detection region is attached with a protective tape. The protective tape may be any tape that is transparent, translucent, or opaque. The protective tape can stably maintain the structure of the immunochromatographic analysis strip. In addition, the signal detection area may attach a signal protection tape. The signal protection tape serves to protect a certain time without damaging the signal shown in the signal detection area, and it is preferable to use a transparent tape to check the signal.

Another aspect of the present invention is

a) an adhesive plastic support, and

Attached to an upper surface of the adhesive plastic support body from an upstream side to a downstream side,

b) a sample pad containing a liquid sample to be analyzed and having a buffer and a single input portion upstream;

c) a conjugate pad containing a conjugate that specifically binds to an analyte contained in the liquid sample introduced from the sample pad, the conjugate pad being located in contact with the sample pad at a downstream end of the sample pad;

d) downstream of the conjugate pad, comprising a signal detector for detecting whether an analyte is present in the liquid sample and a control for confirming whether the liquid sample has moved to the absorbent pad regardless of the presence or absence of the analyte A signal detection pad positioned at a side end with a conjugate pad, and

e) an immunochromatography strip comprising an absorbent pad positioned at a downstream end of the signal detecting pad, the absorbing pad being positioned to absorb the liquid sample from which the signal detecting reaction is completed,

f) the sample pad has a form in which the downstream side is branched into an upper layer and a lower layer,

g) further comprising a control tape of a material which is located between branched gaps of the upper and lower layers of the sample pad and does not pass the liquid sample;

The control tape serves to block the liquid sample introduced into the branched upper layer of the sample pad from entering the conjugate pad, so that the injected sample is moved to the conjugate pad to control the amount of analyte used. Provided are strips for chromatographic analysis.

The immunochromatographic assay strip may be added with a liquid sample in a single input and then a buffer, or a mixture of a liquid sample and a buffer, and the liquid sample may be dispersed in both the branched upper and lower layers of the sample pad. The liquid sample dispersed in the upper layer is blocked from entering the conjugate pad by the control tape, so that only the liquid sample that has moved to the lower pad is used for the immunodetection reaction, thereby preventing the prozone effect.

Another aspect of the present invention, in the immunochromatography kit consisting of a lower housing for receiving the immunochromatographic analysis strip, and an upper housing complementary to the lower housing, immunochromatography housed in the kit An immunochromatography kit is provided wherein the strip is one of the immunochromatographic strips according to all aspects of the invention enumerated above.

The selection of the conjugate of the immunochromatography strip and kit comprising the same according to the present invention may be appropriately selected according to the type of analyte. The conjugate may be labeled with metal particles, in particular gold particles, latex particles, fluorescent materials, radioactive materials or enzymes.

The detection of signal detection of the immunochromatography strip according to the present invention and the kit including the same may be performed by visual reading or optical, electrochemical, or electrical conductivity according to the type of conjugate labeled.

The immunochromatography strip and kit including the same according to the present invention are prepared by using a sample of human or animal whole blood, plasma, serum, tear, saliva, urine, runny nose, body fluid, hepatitis C, hepatitis A, Hepatitis B, Influenza Virus, Avian Influenza Virus, Rota Virus, AIDS, Syphilis, Chlamydia, Malaria, Typhoid, Gastric Ulcer, Tuberculosis, SARS, Dengue Fever, Leprosy, Parvo, Distemper, Foot and Mouth Disease, Brussela, Newcastle, Heartworm, Sight It can be applied to the presence of a pathogen and an antibody, such as a disease. They also identify the presence of pregnancy, ovulation, diabetes markers, enzymes, cancer markers, heart disease markers, disease markers, C-reactive proteins, metabolites, lipids, carbohydrates, or proteins, and cannabis methamphetamine, hiropan, opiates, Whether or not drugs such as amphetamines, morphine, and cocaine are administered, lysine, anthracite, burcellella, botulinum, bexonia, Salmonella, cholera, Staphylococcal enterotoxin B, Tularemia, etc. It can be used for a variety of rapid diagnosis, such as identifying food poisoning bacteria, viruses, antibiotics, food additives such as campylobacter, intestinal hemorrhagic Escherichia coli, and cycinia.

As described above, the immunochromatography and kit according to the present invention introduces a control tape of a material through which a liquid sample does not pass between a sample input portion and a conjugate pad in a sample pad, and moves to a conjugate pad to use an analyte. This can be controlled to block the Pro Zone effect. Further, further comprising a first control tape and a second control tape of a material that does not pass the liquid sample on the downstream side of the conjugate pad and the upstream side of the conjugate pad, and the second sample pad on the upstream side of the first sample pad. By introducing a small amount of a specific reactant used as a raw material of the test kit, it is possible to prevent the prozone effect and at the same time enable two-step immunodetection reaction of two substances having different concentration ranges.

1 is a cross-sectional view showing a representative example of an immunochromatography kit according to the prior art.
2 is a cross-sectional view showing a representative example of an immunochromatography kit for blocking prozone effects according to the prior art.
Figure 3 is a cross-sectional view showing a representative example of an immunochromatography kit that can be applied whole blood while blocking the prozone effect according to the prior art.
4 is a cross-sectional view of an immunochromatographic assay strip that can prevent a prozone effect further comprising a control tape, in accordance with an embodiment of the present invention.
5A is a cross-sectional view of an immunochromatographic assay strip capable of preventing a prozone effect further comprising a first conditioning tape and a second sample pad, in accordance with an embodiment of the present invention.
FIG. 5B is a cross-sectional view of an immunochromatographic assay strip capable of preventing a prozone effect further comprising a first conditioning tape, a second conditioning tape, and a second sample pad, in accordance with an embodiment of the invention.
FIG. 5C is a cross-sectional view schematically illustrating a transfer pattern of a sample and a buffer during the rapid test in the immunochromatographic assay strip of FIG. 5B. FIG.
FIG. 5D illustrates an immunochromatographic analysis strip capable of preventing a prozone effect further comprising a first control tape, a second control tape, a third control tape, and a second sample pad, according to an embodiment of the present invention. It is a cross section.
FIG. 5E is a cross-sectional view of an immunochromatography kit containing the immunochromatographic assay strip of FIG. 5B in an upper housing and a lower housing. FIG.
FIG. 5F illustrates a method in which an immunochromatographic assay strip in which a front portion except for the sample input portion and the signal detection region of the immunochromatographic assay strip of FIG. It is a cross section.
6A is a cross-sectional view of an immunochromatographic assay strip further comprising a conjugate separation pad in the immunochromatographic assay strip of FIG. 5A according to one embodiment of the present invention.
FIG. 6B is a cross-sectional view of an immunochromatographic assay strip further comprising a conjugate separation pad in the immunochromatographic assay strip of FIG. 5B according to one embodiment of the present invention. FIG.
6C is a cross-sectional view of an immunochromatographic assay strip further comprising a conjugate separation pad in the immunochromatographic assay strip of FIG. 5C according to one embodiment of the present invention.
FIG. 7A is an immunochromatographic analysis method having a form in which a downstream side of a specimen pad is divided into an upper layer and a lower layer according to an embodiment of the present invention, and further comprising a control tape between the branched gaps. Cross section of the strip.
FIG. 7B is a cross-sectional view of the immunochromatography kit containing the immunochromatographic assay strip of FIG. 8A in the upper housing and the lower housing. FIG.
8 is a concentration of hemoglobin Alc in the liquid sample according to the length overlapping with the downstream end of the conjugate pad of the control tape in the kit comprising an immunochromatography strip corresponding to FIG. 4 according to an embodiment of the present invention ( w / v%) is a graph showing the result of detection.
Description of the Related Art
10A, 20A, 30A, 50A, 70A: Immunochromatography Kit
40, 50a, 50b, 50d, 60a, 60b, 60c, 70: immunochromatography strip
11, 21, 31, 41, 51, 61, 71: adhesive plastic support
12a, 22a, 32a, 42a: sample pad
52a ', 62a', 72a ': first sample pad
52a ", 62a", 72a ": second sample pad
12b, 22b, 32b, 42b, 52b, 72b: conjugate pad
62b ': First conjugate pad 62b ": Second conjugate pad
62g: Conjugate Separation Pad
12c, 22c, 32c, 42c, 52c, 62c, 72c: signal detection pad
12d, 22d, 32d, 42d, 52d, 62d, 72d: Absorbent pad
32e: filter
42f, 72f: adjusting tape
52f ', 62f': First adjusting tape
52f ", 62f": 2nd adjustment tape
52f "', 62f"': Third Adjustment Tape
13a, 23a, 33a, 43a, 53a, 63a, 73a: control part
13b, 23b, 33b, 43b, 53b, 63b, 73b: signal detector
14a, 24a, 34a, 54a, 74a: lower housing
14b, 24b, 34b, 54b, 74b: upper housing
15a, 75a: buffer and sample single input
25a ', 35a', 45a ', 55a', 65a ': buffer input
25a ", 35a", 45a ", 55a", 65a ": Sample input part
15b, 25b, 35b, 45b, 55b, 65b, 75b: signal detection area
α: upstream side of the specimen pad
β: midstream of the sample pad
γ: downstream of sample pad

Hereinafter, an embodiment of the present invention will be described in detail with reference to the accompanying drawings.

4 is a cross-sectional view of an immunochromatographic assay strip capable of preventing a prozone effect further comprising a control tape, according to one embodiment of the invention.

The immunochromatographic strip of FIG. 4 contains an adhesive plastic support 41 and a liquid sample to be analyzed, which is arranged on the upper surface of the adhesive plastic support from the upstream side to the downstream side, and on the upstream side α. A sample pad 42a having a sample injection section 45a "at the buffer injection section 45a 'and a downstream side gamma; and specifically binding to an analyte contained in the liquid sample introduced from the sample pad. Conjugate pad 42b located below the downstream side γ of the sample pad containing the conjugate, and a signal detector 43b for detecting whether an analyte is present in the liquid sample and the presence of the analyte. A shin positioned with the conjugate pad at the downstream end of the conjugate pad, including a control portion 43a for confirming whether or not the liquid sample has moved to the absorbent pad, with or without a sample. Immunochromatography strip 40 comprising a detection pad 42c and an absorption pad 42d positioned to be in contact with the signal detection pad at a downstream end of the signal detection pad to absorb the liquid sample from which the signal detection reaction is completed. The apparatus further includes a control tape 42f of a material which does not pass the liquid sample, which is located between the sample injection part 45a "and the conjugate pad 42b of the sample pad.

According to the length in which the conjugate pad 42b and the control tape 42f overlap, the amount of the injected sample is moved to the conjugate pad to be used as an analyte, thereby preventing a prozone effect. The longer the length of the conjugate pad 42b and the control tape 42f overlaps, the smaller the amount of the analyte used. The shorter the length, the larger the amount of the analyte used. Therefore, when the amount of the analyte used is large and the prozone effect is displayed, the prozone effect can be prevented by increasing the length of the conjugate pad 42b and the control tape 42f.

The liquid sample introduced into the sample input portion by introduction of the adjusting tape becomes an insoluble analyte present in the downstream side γ of the sample pad 42a in which a predetermined amount is present at the upper end of the adjusting tape 42f, and the remaining predetermined The amount is diffused into the conjugate pad through the portion where the conjugate pad 42b and the control tape 42f do not overlap to form an analyte for use. In addition, when the buffer is immediately added to the buffer input part 45a 'after the liquid sample is added, the buffer moves to the downstream side γ of the sample pad through the sample pad 42a, and the insoluble analysis present in the downstream side is performed. The material is fixed to the downstream end of the sample pad, and the buffer passed through the sample pad to the conjugate pad 42b facilitates the movement of the analyte used to the signal detection pad 42c diffused into the conjugate pad. . By such a principle, the adjusting tape 42f can adjust the amount of the analyte to be used in the sample introduced into the sample input unit, and thus the prozone effect can be avoided.

Looking at the process of the immunodetection reaction of the liquid sample using the immunochromatography strip of Figure 4 as follows.

First, when a liquid sample containing a high concentration of analyte is introduced into the sample input part 45a ", it penetrates into the sample input part 45a", and at this time, the upstream end of the control tape 42f immediately below At the boundary, the liquid sample reacts specifically with the conjugate and moves to the signal detection pad, which is used as an "analyte" used for the test and is not used in the test and is isolated and insoluble at the downstream end of the sample pad 42a. Insoluble analytes. If a predetermined amount of buffer is introduced into the buffer input section 45a 'by the continuous test operation after the liquid sample is added, the injected buffer is the downstream side (γ) end of the sample pad 42a and the conjugate pad ( 42b). Thus, the buffer is divided and moved in the direction of the signal detection pad while passing through the sample pad downstream end and the conjugate pad by the control tape 1, and the buffer moving downstream of the sample pad moves the insoluble analyte downstream of the sample pad. Push to the side end to enhance isolation and stop movement. Most of the remaining buffer is transferred to the conjugate pad and mixed with the analyte used in the conjugate pad to dissolve the conjugate, to help the reaction between the specific substance attached to the conjugate and the analyte used, and to the signal detection pad 42c. It moves to react with the specific substance attached to the signal detector 43b.

Based on this principle, the prozone effect can be obtained by simply diluting the sample with the control tape 42f by disabling only a part of the analyte for the immunodetection reaction and not using the rest of the analyte. Can be controlled. As described above, the longer the overlapping length of the conjugate pad 42b and the control tape 42f may reduce the amount of analyte used, and the shorter the amount of analyte used, the greater the amount of analyte. In order to avoid the prozone effect depending on the severity of the prozone effect depending on the type of, an appropriate adjustment tape length capable of avoiding the prozone effect can be selected by adjusting the overlapping length of the conjugate pad and the control tape.

In the immunochromatography strip of FIG. 4, when hemoglobin as an analyte is present in high concentrations in blood, such as hemoglobin A1c analysis, high and low concentrations are relatively constant, so that the dilution effect can be blocked by the above method alone to block the prozone effect. Can be used for

However, certain analytes, such as anti-IgM and IgG, varying in concentration from high to low in certain samples, are not complete for immunodetection with the immunochromatographic assay strip in FIG. 4. Because adjusting the overlap length of the conjugate pad and the control tape to reduce the amount of analyte used prevents the prozone effect for high concentration analytes, but uses too little analyte to show results for low concentration analytes. It can be used to give false negative results. Conversely, increasing the amount of analyte used would result in low-concentration samples, but high-concentration samples may show false negative results due to the prozone effect. Therefore, the immunochromatographic assay strip of FIG. 4 may be inappropriate to use when the concentration range to be detected is wide. Complementary to this an immunochromatographic analysis strip according to an embodiment of the present invention is shown in Figures 5a, 5b and 5d.

FIG. 5A is an immunochromatin which can further prevent a prozone effect, further comprising a first control tape and a second sample pad positioned upstream of the conjugate pad in contact with the first sample pad, according to an embodiment of the present invention. A cross-sectional view of a strip for graphic analysis.

The immunochromatographic analysis strip of FIG. 5A contains an adhesive plastic support 51 and a liquid sample to be analyzed attached to an upstream surface of the adhesive plastic support, and a buffer injection portion upstream (α). And a first sample pad 52a ′ having a sample input portion on the downstream side γ and below the midstream side β of the first sample pad 52 a ′. At the second sample pad downstream end, the second sample pad 52a "having a slow flow rate, and a conjugate specifically binding to an analyte contained in the liquid sample introduced from the first sample pad; A conjugate pad 52b starting with the second sample pad and being located below the downstream side γ of the first sample pad, and a signal detector 53b for detecting whether an analyte is present in the liquid sample. And presence of analytes A signal detection pad, which is positioned in contact with the conjugate pad at a downstream end of the conjugate pad 52b, includes a control portion 53a for checking whether the liquid sample has moved to the absorption pad, with or without a liquid sample ( 52c) and an immunochromatography strip 50a comprising an absorbent pad 52d positioned downstream of the signal detection pad 52c, which absorbs the liquid sample from which the signal detection reaction is completed, wherein the conjugate pad is provided. It further comprises a first control tape (52f ') of the material located in the upstream side of the contact with the first sample pad does not pass the liquid sample.

The first control tape 52f 'serves to block the conjugate present upstream of the conjugate pad 52b from reacting with the liquid sample introduced into the sample injection section 55a ", thereby present in the injected sample. The immunodetection reaction of the first analyte having a low concentration may be performed first, followed by the detection reaction of the second analyte having a high concentration, and the first analyte and the second analyte may be the same or different. have.

Looking at the process of the immunodetection reaction of the liquid sample using the immunochromatography strip of Figure 5a as follows.

The immunodetection reaction in the immunochromatographic assay strip of FIG. 5A is performed by separating the first and second immunodetection reactions. The principle of the primary immunodetection reaction and the secondary immunodetection reaction is that in the primary immunodetection reaction, the amount of the liquid sample is not controlled by the control tape, and the entire liquid sample injected into the sample injection unit 55a "is first immunized. This is used when the amount of analyte to be detected in the primary immunodetection reaction is so small that the entire liquid sample should be used without controlling the amount of the liquid sample.

In more detail, the primary immune detection reaction, the sample introduced through the sample injection unit 55a "enters the conjugate pad without interfering with the control tape, reacts with the specific reactant of the conjugate, and passes through the first sample pad. It is mixed with the introduced buffer and moved to the signal detection pad 53c to perform an immunodetection reaction with a specific reactant coupled to the signal detection unit 53b, where a low concentration of analyte is detected but shows a prozone effect. The high concentration of the analyte is combined with the specific reactant of the signal detector and the first reaction is completed.

In the secondary immunodetection reaction, when the buffer moving at a delayed speed through the second sample pad 52a "reaches the conjugate pad 52b which is located in contact with the second sample pad 52a", the first control tape The conjugate protected from the buffer moving to the first sample pad 52a 'by 52f' is dissolved, and the analyte and the conjugate used in the primary immunodetection reaction move to the signal detection pad, followed by signal detection. Move to the pad. Thus, the conjugate, which was protected by the buffer and control tape moved to the signal detection pad, undergoes a secondary immunodetection reaction with the analyte previously bound to the reaction specific material of the signal detection unit. In the second reaction, the analyte does not react with the conjugate in the conjugate pad 52b in advance, and only reacts with the specific reactant in the signal detection pad, so that the prozone phenomenon occurs even if the concentration is very high in the liquid sample. Does not appear Therefore, an unfinished reaction that is not signaled by the prozone phenomenon in the first immunodetection reaction is completed by the conjugate of the secondary reaction, thereby overcoming the prozone effect. By this two-step immunodetection reaction, the first analyte, which is present at a very low concentration and is not concerned with prozone effects, is first detected, and the second, which must be present at a relatively high concentration to block prozone effects. The analyte can be detected.

FIG. 5B is a cross-sectional view of an immunochromatographic analysis strip capable of preventing a prozone effect including a first control tape, a second control tape, and a second sample pad according to an embodiment of the present invention.

The immunochromatographic strip 50 of FIG. 5B contains a plastic support 51 and a liquid sample to be analyzed, arranged from an upstream side to a downstream side on an upper surface of the adhesive plastic support and buffered upstream. A first sample pad 52a 'having a sample input portion at the lower and downstream sides, and a second sample pad below the midstream side β of the first sample pad, and having a slower flow rate than the first sample pad. A second sample pad 52a "downstream side (γ) containing a sample pad 52a" and a conjugate specifically binding to an analyte contained in the liquid sample introduced from said first sample pad Detects whether an analyte is present in the liquid sample, starting with the second sample pad, the conjugate pad 52b positioned below the downstream side γ of the first sample pad 52a '. Signal detection unit 53b A signal detection pad which is positioned in contact with the conjugate pad at the downstream end of the conjugate pad, including a control unit 53a for checking whether the liquid sample has moved to the absorption pad regardless of the presence of an analyte. (52c) and an immunochromatography strip (50) comprising an absorbent pad (52d) which is located in a position adjacent to the signal detection pad at a downstream end of the signal detection pad, which absorbs the liquid sample from which the signal detection reaction is completed. A first adjusting tape 52f 'of a material which is located in a portion where the upstream side of the conjugate pad is in contact with the first sample pad and does not pass the liquid sample, and a sample injection portion 55a "of the first sample pad; And further comprising a second adjusting tape 52f " of material located between the downstream side of the conjugate pad 52b and not allowing the liquid sample to pass therethrough. That is, in the immunochromatographic analysis strip 50 of FIG. 5A, the material is positioned between the sample injection portion 55a ″ of the first sample pad and the downstream side of the conjugate pad 52b and does not pass the liquid sample. The difference is that it further includes a second adjusting tape 52f ". The second control tape is additionally included to control the amount of the analyte used in the first immunodetection reaction, thereby preventing the prozone phenomenon from the first immunodetection reaction.

According to the overlapping length of the conjugate pad 52b and the second control tape 52f ″, the amount of the injected sample is moved to the conjugate pad to be used as an analyte, and the first control tape 52f 'is adjusted. The first analyte having a low concentration present in the injected sample serves to block the conjugate present on the upstream side of the conjugate pad 52b to react with the liquid sample introduced into the sample injection section 55a ". The immunodetection reaction of is performed first, followed by the immunodetection reaction of the second analyte with high concentration, and the first analyte and the second analyte may be the same or different.

Using the immunochromatographic strip 50b of FIG. 5b, a liquid sample undergoes an immunodetection reaction as follows.

The immunochromatographic analysis strip 50b of FIG. 5B further includes a second sample pad and a second control tape on the immunochromatographic analysis strip of FIG. 4, and uses the second control tape to relatively use the analyte. After the input is adjusted to a large amount, the conjugate pad 50b is separated and protected by the first adjusting tape and the second adjusting tape, and then the conjugate and the sample protected by the second adjusting tape react with the signal detecting pad. After the primary immunodetection reaction is performed, the conjugate protected on the first control tape is mixed with the buffer to move to the signal detection pad to perform the secondary immunodetection reaction.

For better understanding, the arrangement of the analyte and the insoluble analyte and the moving direction of the buffer in the immunochromatographic assay strip of FIG. 5b are shown in FIG. 5c.

More specifically, when the liquid sample containing the analyte is introduced through the sample input unit, the liquid sample penetrates into the sample input unit 55a "of the first sample pad, and at this time, the second control tape 52f" directly below At the boundary, the liquid sample reacts specifically with the conjugate and moves to the signal detection pad to move to the signal detecting pad, and to be used at the end of the first sample pad 52a ', which is not used in the test and is not used in the test. Divided into "insoluble analytes". The liquid sample is blocked from entering the conjugate pad existing under the first control tape by the presence of the first control tape 52f '. Therefore, the analyte used does not react with the conjugate in the conjugate pad existing under the first control tape 52f ', and the conjugate in the conjugate pad under the second control tape 52f ". You can only react with.

When a predetermined amount of buffer is introduced into the buffer input unit 55a 'by the continuous test operation after the liquid sample is added, the injected buffer moves in the direction of the conjugate pad by capillary reaction. The first sample pad 52a 'and the second sample pad 52a' are moved in two directions. The buffer moving along the downstream (γ) direction of the first sample pad is an analyte used in the injected analyte. Are mixed with each other and divided by the first control tape in the direction of the conjugate pad 52b between the first sample tape downstream (γ) end and the first control tape 52f 'and the first control tape 52f'. The buffer that moves to the downstream end of the first sample pad to move intensifies the isolation of the insoluble analyte and stops moving at the downstream end of the first sample pad. Adjuster Move to the gap between the tape and the second control tape to help the reaction with the conjugate protected by the second control tape and move to the signal detection pad 52c to react with the specific substance of the signal detection unit 53b. An immunodetection reaction takes place.

By adjusting Control Tape 2 so that a relatively large amount of analyte in the liquid sample is used, relatively low concentrations of the analyte in the liquid sample may show results in the primary immunodetection reaction, but relatively high concentrations of the analyte still have a prozone effect. It does not show the detection result and waits for the second immunodetection reaction in which the specific reaction material and the analyte are combined.

In the secondary immunodetection reaction, the flow rate of the buffer moved toward the second sample pad 52a "in the administered buffer is significantly slower than that of the first sample pad 52a '. Second sample pad 52a" When the buffer moving at a delayed speed reaches the conjugate pad 52b, which is located at the second sample pad, the conjugate protects the conjugate protected from the buffer moving to the first sample pad by the adjusting tape 1 52f '. After thawing, the analyte and conjugate used in the primary immunodetection reaction move to the signal detection pad and then to the signal detection pad 52c. Thus, the conjugate, which was protected by the buffer and the control tape 1 moved to the signal detection pad, was subjected to the secondary immunodetection reaction by the bridge reaction by the analyte, which had previously been combined with the specific reaction material of the signal detection part, Even at very high concentrations, Prozone does not appear. Therefore, an unfinished reaction that is not signaled by the prozone phenomenon in the first immunodetection reaction is completed by the conjugate of the secondary reaction, thereby overcoming the prozone effect. By this two-step immunodetection reaction, relatively high concentrations of analytes can be detected first, and relatively high concentrations of analytes can be detected later.

FIG. 5D is a cross-sectional view of an immunochromatographic analysis strip capable of preventing a prozone effect including a first control tape, a second control tape, and a third control tape, and a second sample pad according to an embodiment of the present invention. FIG. .

The immunochromatographic strip 50d of FIG. 5D further includes a third conditioning tape 52f '′ in the immunochromatographic strip 50b of FIG. 5B, and the first conditioning tape 52f'. ) And the second adjusting tape 52f "'are connected by the third adjusting tape 52f"', and the third adjusting tape includes a hole or a gap through which the liquid sample can pass. The amount to be made can be further controlled.

The process of immunodetection of the liquid sample using the immunochromatography strip 50d of FIG. 5d is performed by the immunoassay reaction of the liquid sample using the immunochromatography strip 50b of FIG. 5b. Same as the process. However, the liquid sample passes through the third control tape including a hole or a gap, the amount of the analyte to be used is further adjusted, thereby blocking the prozone effect. The amount of analyte used may be adjusted by adjusting the size or density of the hole or gap included in the third control tape. The third adjustment tape may be connected to the first adjustment tape and the second adjustment tape by folding, but may also be present as a single piece.

The immunochromatographic assay strips of FIGS. 5A, 5B, and 5D may be used for rapid diagnosis, and are immunochromatography kits containing the immunochromatographic assay strips in upper and lower housings. Can also be used. A cross-sectional view of the immunochromatography kit containing the immunochromatographic analysis strip of FIG. 5B in the upper housing 54b and the lower housing 54a is shown in FIG. 5E. 5A, 5B, and 5D without the housing for accommodating the immunochromatographic strip, except for the sample input portion 55a "and the signal detection area 55b of the immunochromatographic strip when used as a strip The front part may be attached with a protective tape, and / or the signal detection area 55b may be attached with a signal protection tape, and the front part except the sample input part and the signal detection area of the immunochromatographic analysis strip of FIG. 5F is a cross-sectional view of the immunochromatographic assay strip in which the immunochromatographic assay strip is attached to the buffer container in a buffer container to perform a sample assay reaction. Immediately after the liquid sample is added to the sample, a buffer input unit is immersed in the buffer in the buffer container to perform an immunodetection reaction. The protective tape and the signal protective tape can be used in the conventional immunochromatography strips, for example, acetate (acetate), polyester (polyester), polyolefin (polyolefin), polypropylene (polypropylene), polystyrene ( polystyrene, vinyl, polyimide, aluminum foil, or retro-reflective film, wherein a tape coated with an adhesive such as rubber, acrylic, or silicone It may be used, but is not limited thereto.

In the immunochromatographic assay strips of FIGS. 5A, 5B and 5D, the analyte used in the liquid sample introduced into the sample input portion of the first sample pad is diffused to the conjugate pad portion protected by the first control tape. It may occur that the conjugate that is introduced and protected by the first conditioning tape is not protected as a result. To alleviate this concern, immunochromatographic assay strips according to one embodiment of the present invention with conjugated connection pads are shown in FIGS. 6A, 6B and 6C, respectively.

In the immunochromatographic assay strip 60a of FIG. 6A, the conjugate pad 62b is positioned below the first control tape 62f 'and the first conjugate pad 62b' and the sample through which the liquid sample does not pass. Located below the input portion 65a ", it is separated into the second conjugate pad 62b" through which the liquid sample passes, and the separation is performed by the first conjugate pad 62b 'and the second conjugate pad 62b ". Conjugate separation pad (62g) is inserted between the), the conjugate separation pad (62g) is a immunochromatography analysis strip that is a pad with a slower buffer flow rate than the conjugate pad.

In the immunochromatographic strip 60b of FIG. 6B, the conjugate pad 62b is positioned below the first control tape 62f ', and the first conjugate pad 62b' and the liquid sample do not pass. 2 is separated into a second conjugate pad 62b "positioned below the control tape 62f" and through which the liquid sample passes, and the separation is performed by the first conjugate pad 62b 'and the second conjugate pad 62b. Conjugate separation pad 62g is inserted between "), and the conjugate separation pad 62g is an immunochromatographic analysis strip that is a pad having a slower buffer flow rate than the conjugate pad.

6C shows that the immunochromatographic strip 60c of FIG. 6B further includes a third conditioning tape 62f ″ ′ in the immunochromatographic assay strip 60b of FIG. 6B, and the first conditioning tape 62f 'and the second adjusting tape 62f "are connected by the third adjusting tape 62f"', and the third adjusting tape includes holes or gaps through which the liquid sample can pass. The amount of material can be further controlled.

The process of immunodetection of the liquid sample using the immunochromatography strip 60c of FIG. 6c is performed by the immunoassay reaction of the liquid sample using the immunochromatography strip 60b of FIG. 6b. Same as the process. However, the liquid sample passes through the third control tape including a hole or a gap, the amount of the analyte to be used is further adjusted, thereby blocking the prozone effect. The amount of analyte used may be adjusted by adjusting the size or density of the hole or gap included in the third control tape. The third adjustment tape may be connected to the first adjustment tape and the second adjustment tape by folding, but may also be present as a single piece.

7A is an immunochromatographic analysis capable of preventing a prozone effect in which a downstream side of a sample pad is branched into an upper layer and a lower layer according to an embodiment of the present invention, and further comprising a control tape between the branched gaps. Sectional view of the strip.

The immunochromatographic strip 70 of FIG. 7A contains an adhesive plastic support 71 and a liquid sample to be analyzed attached to an upper surface of the adhesive plastic support and is single upstream (α). A sample pad 72a having an inlet area and a conjugate that specifically binds to an analyte contained in the liquid sample introduced from the sample pad, and is folded with the sample pad at a downstream end of the sample pad. The conjugate pad 72b is positioned, a signal detector 73b that detects whether an analyte is present in the liquid sample, and whether the liquid sample has moved to the absorbent pad regardless of the presence or absence of an analyte. A signal detection pad 72c, which is located in contact with the conjugate pad at a downstream end of the conjugate pad, including a control portion 73a, and a signal detection An immunochromatography strip 70 comprising an absorbent pad 72d positioned at a downstream end of the signal detecting pad, the absorbing pad 72 d absorbing a liquid sample after completion of the reaction, wherein the sample pad 72a. The downstream side has a form branched into the upper layer (72a ') and the lower layer (72a "), located between the branched gap of the upper layer (72a') and lower layer (72a") of the sample pad 72a It further includes a control tape 72f of the material that does not pass through. The control tape 72f serves to block the liquid sample introduced into the branched upper layer 72a 'of the sample pad from entering the conjugate pad 72b, and the injected sample is moved to the conjugate pad 72b. The amount of analyte used can be controlled.

Looking at the process of the immunodetection reaction of the liquid sample using the immunochromatography strip of Figure 7a as follows.

A mixture of the liquid sample and the buffer is first introduced into the buffer and the single input portion 75a of the sample in the sample pad 72a of the immunochromatographic assay strip 70 of FIG. 7A. The injected liquid sample is divided into an upper layer basin and a lower layer basin of the sample pad 72a, and the liquid sample transferred to the upper layer basin 72a 'is controlled by an adjustment tape 72f located between the upper layer basin and the lower layer basin. At the downstream end of the sample pad 72a, the sample pad 72a does not flow into the conjugate pad 72b which is located in contact with the sample pad 72a, and stays in the branched upper layer 72a 'of the sample pad. Instead, the liquid sample moved to the lower layer basin 72a "is introduced into the conjugate pad 72b which is located in contact with the end of the sample pad, thereby performing an immunodetection reaction. Thus, by the branched sample pad 72a, Substantially the amount of the sample to be used analyte is reduced compared to the amount of the liquid sample injected into the sample input, so that the high concentration of the analyte shows the effect of dilution, the prozone effect can be blocked.

A cross-sectional view of the immunochromatography kit containing the immunochromatographic assay strip of FIG. 7A in the upper housing 74b and the lower housing 74a is shown in FIG. 7B.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited by the following Examples.

Example 1

Hemoglobin A1c Testing Using Immunochromatography Strips and Kits

(A) Antibody Immobilized Nitrocellulose Pads (42c)

Mouse anti-hemoglobin A1c monoclonal antibody, mouse anti-hemoglobin monoclonal antibody, and goat anti-rabbit IgG antibody were diluted with phosphate buffer containing trehalose and then injected into the signal detection unit of the nitrocellulose pad (signal detection pad). It sprayed in the test | inspection line 1, the test | inspection line 2, and a control part position, respectively, and it fixed and dried in a 37 degreeC thermostat.

(B) Preparation of Antibody-Gold Conjugate Pad 42b

To 1 ml of an aqueous solution of colloidal gold, monoclonal antibodies directed against hemoglobin were added for surface adsorption, and then blocked with 1% by weight bovine serum albumin. The conjugate solution was centrifuged at 12,000 rpm in a centrifuge to remove supernatant and resuspended in phosphate buffer containing 0.7 wt% bovine serum albumin and 5 wt% sucrose to have an absorbance of 1. Rabbit IgG antibodies were conjugated in the same manner, mixed with hemoglobin antibodies, and then dispensed at 80 μl / 25 mm ² on glass fiber pads. The pad containing the gold colloidal antibody conjugate was dried for 5 hours in a dehumidification chamber, and the dried pad was cut into a size of 7 x 300 mm.

(C) Preparation of Immunochromatography Strip 40 and Kit Adjusted with Control Tape 42f

The signal detection pad (25X300 mm) 42c prepared in step A was attached and absorbed pad (18X300) 42d at a distance of 15 mm from the downstream end of the adhesive-coated polypropylene plate support (60X300 mm) 41. ) Is attached to the downstream direction to overlap with the signal detection pad 42c, and the conjugate pad 42b prepared in step B is attached to the upstream direction to overlap 2 mm with the signal detection pad 42c and the adjustment tape (Scotch Magic Tape, 3M) cut into 5X300, 6X300, 7X300, 8X300, 9X300 mm, 10X300 mm, 11X300 mm and cut them downstream of the conjugate pad 42b and 0 mm, 1 mm, 2 mm, 3 5 mm, 4 mm, 5 mm, 6 mm and 5 mm upstream of the signal detection pad are covered, and a sample pad (0.5X22X300 mm) is attached from the upstream end of the support to the downstream end of the conjugate pad 42b. An immunochromatographic strip was prepared. An immunochromatography kit was prepared by cutting the laminated sheet to 4 × 6 mm and mounting it in a housing having respective openings at the portions corresponding to the buffer loading section 45a 'and the sample loading section 45a ". Test kits adjusted to 0 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, and 6 mm were prepared using 5 microliters of standard blood containing 8% hemoglobin A1c. After administration to 100 microliters of buffer was added to the buffer input unit (45a ') 20 minutes after the results were read with a rapid diagnostic test reader, the results are shown in FIG.

Figure 9 shows the concentration (w / v%) of hemoglobin Alc in the liquid sample according to the length overlapping with the downstream end of the conjugate pad of the control tape in the kit comprising an immunochromatography strip according to an embodiment of the present invention It is a graph showing the result of detection.

(D) Check the usefulness according to the length of the adjusting tape

The rapid diagnostic test reader was used to quantify the density of the band formed as a result of the experiment, and was adjusted to indicate the relative percentage of the hemoglobin A1c band based on the band density of normal hemoglobin 100%.

According to Fig. 9, the portion where the adjusting tape overlaps the downstream side of the conjugate pad 42b (i.e., the length of the portion overlapping with the sample insert of the adjusting tape) is 0 mm, 1 mm 2 mm, 3 mm 4 In kits adjusted to mm, 5 mm and 6 mm, respectively, the concentrations of Alc were found to be 150%, 60.4%, 27.6%, 17.3% 13.7%, 9.5% and 8.2%. In kits in which the conjugate pad 42b was not superimposed by the control tape 1 (0 mm), the normal hemoglobin was overdosed so that the normal band density was weaker than the band density of hemoglomine A1c due to the prozone effect. However, as the blood dose was controlled and decreased, the value was lowered, and when the conjugate was protected and controlled at 6 mm, 8.2% was similar to 8% of the standard product.

(E) Preparation of Immunochromatography Strip 40 and Kit Adjusted with Control Tape 42f

The signal detection pad (25X300 mm) 42c prepared in step A was attached at a downstream end of the adhesive-coated polypropylene plate support (60X300 mm) at a distance of 15 mm, and the absorbent pads 18X300 and 42d were signaled. Attached in the downstream direction to overlap with the detection pad 42c, the conjugate pad 42d prepared in step B was attached in the upstream direction so as to overlap 2 mm with the signal detection pad 42c, and the adjustment tape 11X300 mm. , Scotch Magic Tape, 3M), cover the conjugate pad downstream and 6 mm and the upstream side of the signal detection pad 5 mm, respectively, and the sample pad (0.5X22X300 mm) from the upstream end to the conjugate pad 42b. Attached to the downstream end, an immunochromatography strip was made. Immunochromatography kit was prepared by cutting the finished strip to 4 × 6 mm and mounted in a housing having a respective inlet to the portion corresponding to the buffer inlet (45a ') and the sample inlet (45a ").

(F) Experiment and Results

5 microliters of whole blood standard samples with hemoglobin A1c of 5%, 6%, 8%, and 10% 12% were added to the sample inlet and buffered (PBS, 0.5% Triton X100, 0.12% EDTA, 1% BSA 0.8% tween). 0.1 ml was added to the perfume inlet, and the band concentration of the detection area shown after about 20 minutes was read with a rapid diagnostic test reader. The test was conducted 5 times each and the average of the test results was 5.1% and 6.2%, respectively. It was found that 7.9%, 10.2% and 11.8%, and CV (variation coefficient) values were all within 10%, indicating that the prepared hemoglobin A1c rapid diagnostic kit was effective as a test kit.

Example 2

Dengue IgG / IgM Test Kit Preparation Using Immunochromatography Strips and Kits

A conjugate pad was prepared by dispensing mouse monoclonal anti-human IgG, IgM antibody and goat anti-rabbit IgG antibody into nitrocellulose pads and conjugating inactivated dengue virus to gold particles in the same manner as in Example 1. As in Example 1, the signal detecting pad 52c, the absorbing pad 52d, and the conjugate pad 52b are attached to the support 51, and the conjugate pad is formed with a second adjusting tape 52f " Cover the downstream end 2 mm of 52b and the upstream end 5 mm of the signal detection pad 52c, and attach the second sample pad of 0.2 X 9 X 300 mm 1 mm overlapping the upstream end of the conjugate. 3 mm of conjugate pad 52b and 5 mm downstream end of second sample pad 52a "are covered with 7 X 300 1st adjustment tape 52f 'by which the adhesive was apply | coated on both surfaces, and the 1st sample pad 0.5 X 22 X 300 mm was covered from the upstream end of the support to the downstream end of the conjugate pad 52b. Immunochromatography kit was prepared by cutting the finished strip to 4 × 6 mm and mounted in a housing having a respective inlet to the portion corresponding to the buffer inlet (55a ') and the sample inlet (55a ").

Twenty dengue IgG and IgM serum samples, which were not detected by the conventional rapid diagnostic method or had low detection strength, were administered to the sample input unit 55a "by 5 microliters and 120 microliters of the buffer input unit 55a '. The buffer was added and the results were observed: It was confirmed visually that IgG and IgM were detected from 3 to 10 minutes in all 20 samples, therefore, two analytes were identified in Prozone in one immunochromatography kit. The effect was avoided and a relatively low concentration of analyte was also detected.

Example 3

Heartworm testing using immunochromatography strips and kits

In the same manner as in Example 1, a mouse monoclonal anti-heartworm antiantibody antibody and a goat anti-rabbit IgG antibody were dispensed onto nitrocellulose pads, and another mouse monoclonal anti-heartworm antiantibody antibody was conjugated to gold particles to prepare a conjugate pad. It was. As in Example 1, the signal detecting pad 52c, the absorbing pad 52d, and the conjugate pad 52b are attached to the support 51, and the second sample pad 52a "of 0.2 X 9 X 300 is conjugated. 3 mm of conjugate pad 52b and the downstream end of the second sample pad 52a "with a 7 X 300 first adjusting tape 52f 'pasted with an overlap of 1 mm at the upstream end of 5 mm was covered and the first sample pad 0.5 X 22 X 300 mm was covered from the upstream end of the support 51 to the downstream end of the conjugate pad 52b. Immunochromatography kit (50a) was prepared by cutting the completed strip to 4 X 6 mm and mounted in a housing having respective inlets at the portions corresponding to the buffer inlet (55a ') and the sample inlet (55a "). .

15 microliters of blood samples from 30 heartworm-infected dogs and one heartworm-infected dog that could not be obtained by the conventional method due to the Prozone effect were put into the sample input unit 55a ", respectively. 120 microliters of buffer was added to the input unit 55a 'and the results were observed, and the results were visually confirmed from 7 minutes to 10 minutes in both samples.

The technical scope of the present invention is not limited to the contents described in the above embodiments, and the equivalent structure modified or changed by those skilled in the art can be applied to the technical It is clear that the present invention does not depart from the scope of thought.

Claims (21)

a) an adhesive plastic support, and
Arranged from an upstream side to a downstream side on an upper surface of the adhesive plastic support body,
b) a sample pad containing a liquid sample to be analyzed and having a buffer input portion upstream and a sample input portion downstream;
c) a conjugate pad located downstream of the sample pad, the conjugate pad containing a conjugate specifically binding to an analyte contained in the liquid sample introduced from the sample pad;
d) downstream of the conjugate pad, comprising a signal detector for detecting whether an analyte is present in the liquid sample and a control for confirming whether the liquid sample has moved to the absorbent pad regardless of the presence or absence of the analyte A signal detection pad positioned at a side end with a conjugate pad, and
e) an immunochromatography strip comprising an absorbent pad positioned at a downstream end of the signal detecting pad, the absorbing pad being positioned to absorb the liquid sample from which the signal detecting reaction is completed,
f) further comprising a control tape of a material that does not pass through the liquid sample, located between the sample injection portion and the conjugate pad of the sample pad,
According to the length of the conjugate pad and the control tape overlap, the injected sample is moved to the conjugate pad, the amount of the analyte used can be controlled, immunochromatography strip. a) an adhesive plastic support, and
Attached to an upper surface of the adhesive plastic support body from an upstream side to a downstream side,
b) a first sample pad containing a liquid sample to be analyzed and having a buffer inlet upstream and a sample inlet downstream;
c) a second sample pad present below the middle stream side of the first sample pad and having a slower flow rate of the buffer than the first sample pad;
d) starting by folding with the second sample pad at the downstream end of the second sample pad, the conjugate containing a conjugate specifically binding to the analyte contained in the liquid sample introduced from the first sample pad; 1 conjugate pad located downstream of the sample pad,
e) downstream of the conjugate pad, comprising a signal detector for detecting whether an analyte is present in the liquid sample and a control for confirming whether the liquid sample has moved to the absorbent pad regardless of the presence or absence of the analyte A signal detection pad positioned at a side end with a conjugate pad, and
f) an immunochromatographic strip comprising an absorbent pad located downstream of said signal detection pad, said liquid absorbing sample having completed the signal detection reaction;
g) a first adjusting tape of a material which is located upstream of the conjugate pad in contact with the first sample pad and does not pass the liquid sample;
The control tape serves to block the conjugate present on the upstream side of the conjugate pad from reacting with the liquid sample introduced into the sample input unit.
The immunodetection reaction of the first low analyte present in the injected sample is carried out first, followed by the detection reaction of the second high analyte high, and the first analyte and the second analyte are the same or different from each other. Another material, immunochromatography strip. The method of claim 2, further comprising a second control tape of the material which is located between the sample injection portion and the conjugate pad downstream side of the first sample pad and does not pass the liquid sample,
According to the overlapping length of the conjugate pad and the second control tape, the injected sample is moved to the conjugate pad to adjust the amount of the analyte to be used.
The immunodetection reaction of the first low analyte in the injected sample is carried out first, followed by the immunodetection reaction of the second high analyte, and the first analyte and the second analyte are the same. Or other material immunochromatographic strip. The method of claim 3, wherein the first control tape and the second control tape is connected by a third control tape, the third control tape includes a hole or gap that can penetrate the liquid sample, An immunochromatographic assay strip, wherein the amount can be further controlled. The immunochromatographic assay strip of claim 4, wherein the first conditioning tape, the second conditioning tape, and the third conditioning tape are integral. The method of claim 2, wherein the conjugate pad is separated into a first conjugate pad located below the first control tape and a second conjugate pad through which the liquid sample passes through the sample inlet. An conjugate separation pad is inserted between the first conjugate pad and the second conjugate pad, and the conjugate separation pad is a pad having a slower buffer flow rate than the conjugate pad. The method of claim 3, wherein the conjugate pad is separated into a first conjugate pad positioned below the first control tape and a second conjugate pad positioned below the second control tape and through which a liquid sample passes. Separation is performed by inserting a conjugate separation pad between the first conjugate pad and the second conjugate pad, the conjugate separation pad is a pad for immunochromatographic analysis slower buffer flow rate than the conjugate pad. 8. The method of claim 7, wherein the first control tape and the second control tape is connected by a third control tape, the third control tape includes a hole or gap that can penetrate the liquid sample, An immunochromatographic assay strip, wherein the amount can be further controlled. 9. The immunochromatographic assay strip of claim 8, wherein the first conditioning tape, the second conditioning tape, and the third conditioning tape are integral. 10. The method according to any one of claims 1 to 9, wherein the control tape, the first control tape, the second control tape, or the third control tape are each independently acetate, polyester, polyolefin ( Polyolefin, polypropylene, polystyrene, vinyl, polyimide, aluminum foil, or retro-reflective film tape applied on one or both sides with adhesive Tape for immunochromatographic analysis. The immunochromatographic analysis strip of claim 10, wherein the pressure-sensitive adhesive is an acrylic, rubber, or silicone-based pressure-sensitive adhesive. The method according to any one of claims 2 to 9, wherein the second sample pad is cellulose, acrylic fiber, polyester fiber, rayon, glass fiber, wool fiber, silk fiber, cotton yarn, flax fiber or pulp. Treatment with one or more substances selected from the group consisting of amino acids, peptides, polyethylene glycol, polyvinylalcohol, polyvinylpyrrolidone, glycerol, and polysorbate The prepared immunochromatographic strip. 10. The method of claim 6, wherein the conjugate separation pad is polyethylene, rayon, glass fiber, cellulose, or pulp, proteins, amino acids, peptides, polyethylene glycol, polyvinyl alcohol ( An immunochromatographic analysis strip prepared by treating with one or more substances selected from the group consisting of Polyvinylalcohol, Polyvinylpyrrolidone, Glycerol, and polysorbate. 10. The immunochromatographic assay strip according to any one of claims 1 to 9, wherein the front portion except for the sample injection portion and the signal detection region is attached with a protective tape. a) an adhesive plastic support, and
Attached to an upper surface of the adhesive plastic support body from an upstream side to a downstream side,
b) a sample pad containing a liquid sample to be analyzed and having a buffer and a single input portion upstream;
c) a conjugate pad containing a conjugate that specifically binds to an analyte contained in the liquid sample introduced from the sample pad, the conjugate pad being located in contact with the sample pad at a downstream end of the sample pad;
d) downstream of the conjugate pad, comprising a signal detector for detecting whether an analyte is present in the liquid sample and a control for confirming whether the liquid sample has moved to the absorbent pad regardless of the presence or absence of the analyte A signal detection pad positioned at a side end with a conjugate pad, and
e) an immunochromatography strip comprising an absorbent pad positioned at a downstream end of the signal detecting pad, the absorbing pad being positioned to absorb the liquid sample from which the signal detecting reaction is completed,
f) the sample pad has a form in which the downstream side is branched into an upper layer and a lower layer,
g) further comprising a control tape of a material which is located between branched gaps of the upper and lower layers of the sample pad and does not pass the liquid sample;
The control tape serves to block the liquid sample introduced into the branched upper layer of the sample pad from entering the conjugate pad, so that the injected sample is moved to the conjugate pad to control the amount of analyte used. Chromatographic analysis strips. 16. The immunochromatographic assay strip according to any one of claims 1 to 9 and 15, wherein the conjugate is labeled with metal particles, latex particles, fluorescent substances or enzymes. 16. The immunochromatographic strip according to any one of claims 1 to 9 and 15, wherein the signal detection is read by visual, optical, electrochemical, or electrical conductivity. An immunochromatography kit comprising a lower housing accommodating an immunochromatographic analysis strip and an upper housing complementarily coupled to the lower housing, wherein the immunochromatographic strip housed in the kit is selected from claim 1. And an immunochromatography strip according to any one of claims 15 to 17. The method of claim 18, wherein hepatitis C, hepatitis A, hepatitis B, influenza, avian influenza, rotavirus, AIDS, syphilis, chlamydia, malaria, typhoid, gastric ulcer, tuberculosis, SARS, dengue, leprosy, parvo, distemper , Foot and mouth disease, brussels, newcastle, heartworm, speculum, antigen, antibody, pregnancy, ovulation, diabetes markers, enzymes, cancer markers, heart disease markers, C-reactive protein, metabolites, lipids, carbohydrates, proteins, drug dosing, Immunochromatography kit for the detection of lysine, anthrax, burcellella, botulinum, vessinia, salmonella, cholera, Staphylococcal enterotoxin B, tullaremia, salmonella, vibrio campylobacter, enterohemorrative Escherichia coli, or cycinia. 19. The immunochromatography kit of claim 18, wherein said signal detection is read by visual, optical, electrochemical, or electrical conductivity. The hepatitis C, hepatitis A, hepatitis B, influenza, avian influenza, rotavirus, AIDS, syphilis, chlamydia, malaria, typhoid, gastric ulcer causative agent according to any one of claims 1 to 9 and 15. , Tuberculosis, SARS, dengue, leprosy, parvo, distemper, foot and mouth disease, burcellella, newcastle, heartworm, sight disease, antigen, antibody, pregnancy, ovulation, diabetic marker, enzyme, cancer marker, heart disease marker, C response protein, Detection of metabolites, lipids, carbohydrates, proteins, drug dosing, lysine, anthrax, burcellella, botulinum, vessinia, salmonella, cholera, Staphylococcal enterotoxin B, tullaemia, salmonella, vibrio campylobacter, enterocoliform coli, or chickini For immunochromatographic analysis.

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