WO2014196803A1

WO2014196803A1 - A strip for rapid testing having a variable control line and a diagnosis kit using the same

Info

Publication number: WO2014196803A1
Application number: PCT/KR2014/004961
Authority: WO
Inventors: Kook Jin Lim; Yu Sam Kim; Myung Sook CHUNG; Bum Jun Kim; Mi Jung Kim; Dong Seob Choi; Hye Jung Lee
Original assignee: Proteometech Inc.; Pamacotec Inc.
Priority date: 2013-06-04
Filing date: 2014-06-03
Publication date: 2014-12-11
Also published as: CN105229468A; EP3004879A1; JP2016520200A; KR101540608B1; KR20140143120A; EP3004879A4

Abstract

The present invention relates to a strip for rapid testing having a variable control line, a diagnostic kit including the strip for rapid testing, and a method for qualitative or quantitative analysis of an analyte in a sample by using the same. Since the strip for rapid testing of the present invention has a variable control line, it is possible to overcome a difficulty in quantitative analysis of the analyte due to hook effect and also to conduct highly reliable quantitative analysis as well as qualitative analysis with the naked eye in a simple, rapid manner. Furthermore, there is an effect that the dynamic range of the analyte can be improved by complementary effects of the test line and the variable control line.

Description

A STRIP FOR RAPID TESTING HAVING A VARIABLE CONTROL LINE AND A DIAGNOSIS KIT USING THE SAME

The present invention relates to a strip for rapid testing having a variable control line, a diagnostic kit including the strip for rapid testing, and a method for qualitative or quantitative analysis of an analyte in a sample by using the same.

The immunochromatographic assay, known as a rapid testing method, is a method for qualitative and quantitative analysis of a small amount of analytes by antigen-antibody reaction over a short period of time, and has been applied in various fields such as the medical field, agriculture, livestock raising, food, military, the environment and the like, as well as in the diagnosis or examination of various diseases. Typically, the immunochromatographic assay is carried out with an assay strip containing a reagent that reacts with the target analyte to produce a change, or with an assay device which includes a plastic case and the assay strip installed in the case. FIG. 1 is a cross-sectional view of the conventional assay strip used for immunochromatographic assays. As shown in FIG. 1, the conventional assay strip is composed of a sample pad for receiving a liquid sample; a conjugate pad containing a conjugate which is prepared by conjugating a label (e.g., gold particle) generating a signal detectable by the naked eye or a sensor to a ligand such as an antigen or an antibody; a detection pad, on which a test line immobilized with a binder (antibody or antigen) specifically binding to an analyte in the sample and/or the conjugate and a control line for assuring migration of the sample are formed; and an absorbent pad for finally receiving the liquid sample. These functional pads are connected with being partially overlapped each other in the order as they are described above, and consecutively arranged on a solid support. If the assay strip installed in a plastic case is used, a sample receiving hole for introducing a sample to the sample pad is formed on the upper portion of the case, and a result observation window for observing the test result is formed on the position where the binder of the detection pad is immobilized. In the immunochromatographic assay using the assay strip, when a liquid sample is injected into the sample pad, the liquid sample flows along the conjugate pad and the detection pad by a capillary action, and is finally absorbed by the absorbent pad. For example, one antibody capable of binding with a target antigen is immobilized in a line (test line) on a membrane pad, and the other antibody is conjugated with a label, such as one of gold particles, and then the sample is allowed to migrate. While the sample migrates across the conjugate pad, a part thereof produces a complex of antigen-gold particle antibody, and the antigen-gold particle antibody is captured at the immobilized test line to form ‘antibody immobilized at test line-antigen-gold particle antibody’. At this time, the test line is colored red due to gold particles. As such, the conjugate contained in the conjugate pad also migrates along with the liquid sample. If a target analyte exists in the sample, the conjugate binds to the binder which is immobilized on the detection pad via the analyte (generally, called "sandwich reaction"), or the conjugate or the analyte competitively bind to the binder (generally, called "competitive reaction"). Based on this concept, the assay strip is used to detect the existence of the analyte in the sample with the naked eye or a sensor.

The rapid testing method previously described, namely, the immunochromatographic assay using a rapid kit, is generally in the form of a strip which is prepared by immobilizing individual particular substances in several horizontal lines on a long membrane and then cutting it lengthwise. Thus, there is an advantage that its mass production is possible in a simple manner. Further, compared to ELISA or the like, it does not require an additional washing step or a secondary reaction and the reaction time is as short as 10 minutes. Therefore, the immunochromatographic assay is excellent in terms of convenience. However, there are disadvantages that its detection limit is relatively low, and accurate quantitative analysis is difficult because of naked eye-based detection. For this reason, the conventional immunochromatographic assay is usually used for qualitative analysis, and there are many limits in its use for quantitative analysis.

The present inventors have made intensive efforts to improve the reliability of the immunochromatographic strip in quantitative analysis as well as in qualitative analysis, and as a result, they have found that quantitative analysis is possible by adding a variable control line containing a substance identical to an analyte or an analog thereof and then comparing signal intensities between the test line and the variable control line. Accordingly, the present invention provides a strip for rapid testing having the variable control line, which can be used for highly reliable quantitative analysis of the analyte in the sample as well as for qualitative analysis thereof with the naked eye.

In one aspect, the present invention provides a strip for rapid testing with a conjugate pad and a detection pad, wherein the conjugate pad has a first predetermined amount of a first ligand reacting with an analyte and a signal detection label binding to the first ligand, the first ligand and the label are linked to each other to form a conjugate before migration of a sample or upon migration thereof, and the conjugate can migrate to a detection pad upon migration of the sample,

wherein the detection pad has a test line and a variable control line that are separated from each other,

wherein the test line is immobilized with a second ligand that reacts with the analyte in the sample, and

wherein the variable control line is immobilized with a second predetermined amount of a third ligand which is identical to the analyte or an analog thereof reacting with the first ligand, thereby being able to react with a bare conjugate that does not bind to the analyte in the sample.

In a preferred embodiment, the strip for rapid testing is characterized in that a portion of the first predetermined amount of the first ligand reacts with the analyte in the sample, to thereby form a complex in which the analyte binds to the conjugate having the first ligand and the label, and the amount of the complex formed increases with increasing concentration of the analyte in the sample, whereas the amount of the bare conjugate decreases.

In another preferred embodiment, the strip for rapid testing is characterized in that the complex having the first ligand, the label and the analyte reacts with the second ligand to be captured at the test line, the bare conjugate which does not bind to the analyte in the sample and has the first ligand and the label reacts with the third ligand to be captured at the variable control line, and the presence or absence of the analyte or the amount thereof in the sample, or both thereof can be determined by measuring signals from the labels of the test line and the variable control line.

In still another preferred embodiment, the strip for rapid testing is characterized in that signal intensity of the variable control line decreases with increasing concentration of the analyte in the sample.

In yet another preferred embodiment, the strip for rapid testing is characterized in that signal intensity from the variable control line is maintained at a constant level until the concentration of the analyte in the sample reaches M value, but the signal intensity gradually decreases when the concentration exceeds the M value, and finally converges to 0, wherein the M value is the maximum concentration of the analyte in the sample in the case where all the third ligands immobilized in the second predetermined amount are saturated by reacting with the bare conjugates.

In still yet another preferred embodiment, the strip for rapid testing is characterized in that the detection pad further has a constant control line for assuring migration of the sample, the constant control line being immobilized with a reporter molecule.

In a further preferred embodiment, the strip for rapid testing is characterized by comprising:

a sample pad, into which a sample containing an analyte to be analyzed is introduced;

a conjugate pad, of which one end is connected to the sample pad;

a detection pad, of which one end is connected to the other end of the conjugate pad;

an absorbent pad, of which one end is connected to the other end of the detection pad, providing a driving power for transport of the sample from the sample pad; and

a solid support placed on the lower side of the strip for rapid testing.

In another further preferred embodiment, the strip for rapid testing is characterized in that the support is made of a material selected from the group consisting of nitrocellulose, nylon, PVDF, glass and plastic.

In still another further preferred embodiment, the strip for rapid testing is characterized in that the first ligand, the second ligand and the third ligand are a protein, an antigen, an antibody, DNA, RNA, PNA or an aptamer.

In yet another further preferred embodiment, the strip for rapid testing is characterized in that the label is a colloidal gold, a latex particle, a colored polystyrene microparticle, an enzyme, a fluorescent pigment, a conductive polymer, a luminescent substance, or a magnetic particle.

In still yet another further preferred embodiment, the strip for rapid testing is an immunochromatographic strip.

In another aspect, the present invention provides a diagnostic kit comprising the strip for rapid testing as described above which is additionally installed in a case, wherein the kit has a guide and a strip support in a lower case, a sample receiving hole in an upper case, and a result observation window is placed at the position corresponding to the test line and the variable control line.

In a preferred embodiment, the diagnostic kit is characterized in that the kit is provided with standard signal data containing a standard color chart of the test line and the variable control line for the samples containing the analyte at various concentrations.

In another preferred embodiment, the diagnostic kit is characterized in that qualitative or semi-quantitative analysis can be carried out by examining the presence or absence and intensity of signals from the label of the test line and the variable control line with the naked eye.

In still another aspect, the present invention provides a method for qualitative or quantitative analysis of an analyte in a sample using the strip for rapid testing as described above, comprising the steps of:

introducing the sample into the conjugate pad or the pad positioned therebefore and permitting the sample to migrate (Step 1);

examining the presence or absence and intensity of signals from the labels of the test line and the variable control line (Step 2); and

comparing the signal intensities of the test line and the variable control line with standard signal data to determine the amount of the analyte,

wherein the standard signal data are obtained by subjecting each of the samples containing the analyte at various known concentrations to Steps 1 and 2 (Step 3).

In a preferred embodiment, the method is characterized in that in Step 2, the presence or absence and the intensity of signals from the labels of the test line and the variable control line are examined using a densitometer.

In still another aspect, the present invention provides a method for qualitative or quantitative analysis of one or more analytes in a sample, comprising reacting the sample with a ligand absorbed on a solid support, which is characterized in that two different reaction mechanisms for one analyte are applied thereto.

In a preferred embodiment, the method is characterized in that the two different reaction mechanisms are a sandwich reaction and a competitive reaction.

Since the strip for rapid testing of the present invention has a variable control line, it is possible to overcome a difficulty in quantitative analysis of the analyte due to the hook effect and also to conduct highly reliable quantitative analysis as well as qualitative analysis with the naked eye in a simple, rapid manner. Furthermore, there is an effect that the dynamic range of the analyte can be improved by complementary effects of the test line and the variable control line.

FIG. 1 is a schematic diagram showing a cross-sectional view of an assay strip used in a typical immunochromatographic assay;

FIG. 2 is a schematic diagram showing an assay strip used in an immunochromatographic assay of the present invention;

FIG. 3 is a schematic diagram showing patterns of a test line, a variable control line and a constant control line in the strip for rapid testing of the present invention, before and after introduction of a sample;

FIG. 4 is a schematic diagram showing a strip for rapid testing according to one embodiment of the present invention; and

FIG. 5 is a photograph showing the result of analyzing analyte concentrations in various samples according to one embodiment of the present invention, in which human immunoglobulin G (IgG) was used as an analyte.

In one aspect to achieve the above object, the present invention provides a strip for rapid testing having a conjugate pad and a detection pad, in which the conjugate pad has a first predetermined amount of a first ligand reacting with an analyte and a signal detection label binding to the first ligand, the first ligand and the label are linked to each other to form a conjugate before migration of a sample or upon migration thereof, the conjugate can migrate to a detection pad upon migration of the sample, the detection pad has a test line and a variable control line that are separated from each other, the test line is immobilized with a second ligand that reacts with the analyte in the sample, and the variable control line is immobilized with a second predetermined amount of a third ligand which is identical to the analyte or an analog thereof reacting with the first ligand, thereby being able to react with a bare conjugate that does not bind to the analyte in the sample.

The present invention is characterized in that the variable control line immobilized with a substance identical to the analyte or an analog thereof is added to the conventional assay strip having the detection pad on which only the test line and the control line for assuring migration of the sample are formed, thereby performing highly reliable quantitative analysis as well as qualitative analysis.

Furthermore, the present invention is characterized in that it is possible to perform highly reliable semi-quantitative analysis with the naked eye without a specialized analyzer.

In a preferred embodiment, the present invention is characterized in that the strip for rapid testing is an immunochromatographic strip.

As used herein, the term "immunochromatography" is an analytical method combining the principle of immune reaction based on an antigen-antibody reaction with the principle of chromatography based on movement of a sample and a reagent along a medium by a mobile phase. Briefly, an antibody or an antigen to be analyzed is dispensed on a porous membrane in advance and immobilized thereto, and then blood is allowed to migrate from one end of the membrane toward the immobilized antibody or antigen to observe its reaction with an antigen or antibody in blood. An immune reaction generally refers to an antigen-antibody reaction. However, in the present invention, it also broadly includes a reaction between a receptor and a ligand which specifically bind to each other as well as a antigen-antibody reaction, but is not limited thereto. In addition, the immune reaction includes all reactions which occur by specific recognition to each other, such as a reaction between an enzyme and a substrate.

Immunochromatographic assay may occur while a sample containing an analyte moves along a mobile phase through a medium by capillary action. Therefore, as the medium for the immunochromatographic assay, a strip can be manufactured and used. Specific components of the strip for rapid testing and functions thereof will be described below.

A label can be used in order to easily detect the antigen-antibody reaction with the naked eye or a sensor. A ligand which is able to specifically bind to the analyte can be linked to the label, in order to make the label bind to the target analyte.

As used herein, the term "conjugate" means a conjugate which is formed by linking the label and the ligand with each other, and the conjugate is linked with the label for signal detection and includes the first ligand which reacts with the analyte in the sample. In order to distinguish the first ligand from other ligands described below, the first ligand means a ligand which binds with the label to form the conjugate.

As used herein, the first ligand means a substance that is able to react with the analyte and specifically bind to the analyte.

The label and the first ligand can be linked physically or chemically. That is, the label and the first ligand can be linked via passive adsorption or via a covalent bond by modifying the label to have reactive groups, but are not limited thereto. The linkage between the label and the first ligand can be carried out by a method known to those skilled in the art.

However, the label and the first ligand may exist on the conjugate pad in the form of a conjugate before migration of the sample in the strip for rapid testing, or may exist on the conjugate pad individually without linkage between them and then they bind to each other to form the conjugate during migration of the sample. In either case, the label and the first ligand move to the detection pad in the form of conjugate during migration of the sample, because they are not fixed on the conjugate pad.

As used herein, the term "label" means a substance generating signals which can be detected with the naked eye or sensor. The label may be colloidal gold (gold particles), latex particles, colored polystyrene microparticles, enzymes, fluorescent pigments, conductive polymers, luminescent substances, magnetic particles or the like, but is not limited thereto. In addition, the signal may be luminescence generated autonomously by intrinsic properties of the label, or fluorescence generated by external stimulation.

As used herein, the term "ligand" means substances that specifically bind to each other. For example, an antibody specifically binding to an antigen or a ligand specifically binding to a particular receptor functions with each other as a ligand. Non-limiting examples of the ligand may include a protein, an antigen, an antibody, DNA, RNA, PNA or an aptamer. In addition, the ligand in the present invention may be any substance without limitation, as long as it exhibits the characteristics as defined above. The ligand used throughout the present specification means substances that specifically bind to each other, as defined above, unless it is confined to a ligand which specifically binds to a particular receptor.

In one preferred embodiment of the present invention, the label and the first ligand are connected to each other in advance and exist on the conjugate pad in the form of conjugate. In this case, the label and the first ligand are individually prepared in a solution of known concentration, and then they are mixed and allowed to react for a predetermined time so as to prepare a conjugate solution. This conjugate solution is dispensed on the conjugate pad to prepare the conjugate pad containing the conjugate. At this time, the concentration of each solution and the mixing ratio can be determined, considering the binding ratio of the label and the ligand. The binding of the label to the ligand may be binding of one label to one ligand, binding of one label to a plurality of ligands, or binding of a plurality of labels to one ligand. The specific binding ratio is not important, but it may maintain at a predetermined ratio in one preferred embodiment. The binding ratio may vary depending on the type of the label and the ligand, and may be determined, considering their relative size and the amount of binding site. For example, if several micron-sized latex beads are used as the label and an antibody is used as the ligand, a plurality of antibodies can bind to one latex bead. Preferably, in order to bind an equal amount of antibodies to each of the latex surface, the concentrations of the antibody and latex solutions and the mixing ratio thereof can be controlled so that the binding of the antibodies to the surface of latex is saturated, but is not limited thereto.

As described above, a conjugate solution of known concentration can be obtained by mixing and reacting the label and the first ligand, of which concentrations are known. The conjugate solution is in the form of dispersion, in which the conjugate molecules with a predetermined concentration are uniformly dispersed in a solution.

The strip for rapid testing is an immunochromatographic strip having a variable control line, which can comprise a conjugate pad containing a first predetermined amount of the first ligand reacting with an analyte and a signal detection label binding to the first ligand; and a detection pad separately having a test line and a variable control line.

For quantitative analysis as well as qualitative analysis of the analyte in the sample, the third ligand is immobilized on the detection pad to form the "variable control line". In the variable control line of the strip for rapid testing of the present invention, a substance identical to an analyte or an analog thereof reacting with the first ligand may be immobilized as the third ligand which is able to react with a bare conjugate in the mobile phase that flows in the detection pad. The bare conjugate which does not bind to the analyte in the sample contains the first ligand, and thus, the first ligand specifically binds to the substance identical to the analyte or the analog thereof, namely, third ligand.

Furthermore, a predetermined amount of the third ligand may be immobilized on the variable control line. That is, the second predetermined amount means a specific amount of the third ligand immobilized on the variable control line. By immobilizing the second predetermined amount of the third ligand, signal intensity in the variable control line and the detectable concentration range of the analyte in the sample can be controlled, which makes highly reliable quantitative analysis possible.

In the present invention, to distinguish the third ligand from other ligands described below, the third ligand refers to a ligand that is immobilized on the detection pad to form the variable control line.

Furthermore, in order to detect the analyte in the sample, a second ligand which specifically binds thereto and selectively captures is immobilized on the detection pad, to thereby form a "test line". In the test line of the strip for rapid testing of the present invention, the second ligand reacting with the analyte in the sample is immobilized so as to react with the analyte-conjugate complex, namely, the analyte in the mobile phase that flows in the detection pad. The second ligand may be a substance identical to the first ligand, or an analog thereof or a third substance specifically binding to the analyte. While the analyte in the sample migrates along the mobile phase, it binds to the conjugate in the conjugate pad to form a complex. Because this complex includes the analyte and the first ligand specifically bind to the analyte as described above, the substance identical to the first ligand or the analog thereof, or the second ligand specifically binding with the analyte contribute to the capture of the complex in the test line via binding to the analyte.

In the present invention, to distinguish the second ligand from other ligands described below, the second ligand refers to a ligand that is immobilized on the detection pad to form the test line.

As described above, the immunochromatography utilizes the principle of chromatography based on migration of a mobile phase containing the analyte along a medium. Therefore, in an immunoassay using the strip for rapid testing according to the present invention, the mobile phase is needed for migration of the sample containing the analyte along the strip. Therefore, the immunoassay kit of the present invention may further include a buffer solution. The buffer solution functions as the mobile phase for moving the sample along the strip for rapid testing, and also functions as a solvent for dissolving the conjugate. If necessary, it functions as a diluent for diluting the sample. For example, in the case of whole blood analysis, a component for lysis of the blood cells such as red blood cell or the like can be further included. As the buffer solution, typical buffer solutions such as 10 mM to 1 M of phosphate buffered solution (PBS), a nonionic or amphoteric surfactant, or a mixture thereof may be used without limitation, and the buffer solution may be properly selected according to the type of the desired reaction such as antigen-antibody reaction or the like.

The conjugate pad is a pad having the first predetermined amount of the first ligand reacting with the analyte as described above and the signal detection label binding to the first ligand, in which the first ligand and the label may exist on the conjugate pad in the form of a conjugate before migration of the sample, or may exist on the conjugate pad individually without linkage between them, and then they bind to each other to form the conjugate during migration of the sample.

In the present invention, only a predetermined amount of the first ligand may exist in the conjugate pad. That is, the first predetermined amount means a specific amount of the first ligand that exists in the conjugate pad. Because the first ligand exists in the first predetermined amount in the conjugate pad, the amount of the complex formed by binding of the first ligand with the target increases with increasing concentration of the target in the sample, whereas the amount of the bare conjugate that does not bind to the target decreases, indicating an inverse proportion.

Preferably, in the conjugate pad, the first ligand and the label are linked to each other in advance, and they exist in the form of conjugate. At this time, the conjugate pad may be prepared by applying the conjugate solution of known concentration to a pad, as described above.

Furthermore, the conjugate pad may further include a second conjugate formed by conjugation of a fourth ligand with a label, to be used as an internal control. The fourth ligand means a ligand that is able to react with a reporter molecule specifically or non-specifically. The second conjugate is captured by the reporter molecule of the constant control line, thereby assuring migration of the sample. Detailed descriptions of the constant control line and the reporter molecule will be described in below.

The detection pad is a medium for migration of the mobile phase and the sample, and the mobile phase and the sample may migrate by a capillary action of a porous membrane constituting the detection pad. Furthermore, the detection pad has the test line and the variable control line that are separated from each other, and further has the constant control line for assuring migration of the sample. One end of the detection pad may be connected to the conjugate pad, and the other end thereof may be connected to the absorbent pad which provides a driving power for transfer of the sample. The conjugate pad and the absorbent pad are partially overlaid onto the detection pad. The detection pad can be composed of a porous membrane, and the porous membrane can be a nitrocellulose membrane, a glass fiber membrane, a polyethersulfone (PES) membrane, a cellulose membrane, a nylon membrane, or a combination thereof, but is not limited thereto. Preferably, the detection pad can be a nitrocellulose membrane having a pore size of 5 to 15 μm.

The principle of qualitative or quantitative analysis using the strip for rapid testing of the present invention will be described in more detail.

In the conventional assay strip, the test line is formed on the membrane pad, and the migrating analyte-conjugate complex is captured at the test line to generate signals (sandwich reaction). At this time, signal intensity increases with increasing concentration of the analyte. However, if the concentration of the analyte exceeds a specific concentration, signal intensity decreases due to hook effect. This phenomenon is a great obstacle to the quantitative analysis of the analyte using the conventional assay strip.

However, the strip for rapid testing of the present invention is characterized in that a specific competitive reaction where the first predetermined amount of the first ligand competitively reacts with a finite amount of the analyte in the sample in the conjugate pad as well as the sandwich reaction are applied to one strip, and therefore, it is possible to perform a more rapid, convenient quantitative analysis, compared to the conventional immunochromatographic analysis.

In particular, when a liquid sample is introduced into the sample pad of the strip for rapid testing of the present invention, migration of the mobile phase and the sample occurs. In the conjugate pad, the first predetermined amount of the first ligand competitively reacts with a finite amount of the analyte in the sample. A portion of the first predetermined amount of the first ligand reacts with the analyte in the sample to form a complex, in which the analyte binds to the conjugate containing the first ligand and the label, whereas the rest of the first ligand unreacted with the analyte migrates as the bare conjugate along the mobile phase. Herein, because the first ligand is maintained in the first predetermined amount, the amount of the complex formed increases with increasing concentration of the analyte in the sample, whereas the amount of the bare conjugate decreases.

The complex containing the first ligand, the label and the analyte reacts with the second ligand to be captured at the test line during migration, whereas the bare conjugate being unreacted with the analyte in the sample and containing the first ligand and the label reacts with the third ligand to be captured at the variable control line during migration. The complex and the bare conjugate are captured at the test line and the variable control line, respectively, because the second ligand is immobilized on the test line and the third ligand is immobilized on the variable control line.

Both the complex and the bare conjugate contain labels, and thus the presence or absence of the analyte, the amount thereof in the sample, or both of them can be determined by measuring signals from the labels of the test line and the variable control line, at which the complex and the bare conjugate are captured.

As mentioned above, the amount of the complex increases with increasing concentration of the analyte in the sample, whereas the amount of the bare conjugate decreases with increasing concentration of the analyte in the sample. Therefore, signal intensity from the variable control line at which the bare conjugates are captured decreases with increasing concentration of the analyte in the sample.

More specifically, signal intensity from the variable control line is maintained at a constant level until the concentration of the analyte in the sample reaches M value, but the signal intensity gradually decreases when the concentration exceeds the M value. Finally, the signal intensity converges to 0. Here, the M value is the maximum concentration of the analyte in the sample in the case where all the third ligands immobilized in the second predetermined amount are saturated by reacting with the bare conjugates. In one Example of the present invention, when the concentration of the analyte in the sample is ranging from 0 to 90 ng/ml, signal intensity from the variable control line maintains at the strongest level, and therefore, it can be seen that all the third ligands are reacted with the bare conjugates. Thus, it is confirmed that the M value in the present Example is about 90 ng/ml (Example 2). However, the M value is changed depending on the second predetermined amount, and the types of the third ligand and the analyte, and therefore, it can be properly selected by those skilled in the art according to the purpose of analysis.

As described above, signal intensity from the test line increases with increasing concentration of the analyte. However, the concentration of the analyte exceeds a specific level, signal intensity decreases due to the hook effect. The signal intensity patterns of the test line and the variable control line according to increasing concentration of the analyte of the present invention are shown in FIG. 3.

Consequently, it is possible to perform qualitative or quantitative analysis of the analyte by measuring signals from the labels of the test line and the variable control line. More specifically, signals from the test line and the variable control line are compared to the standard signal data of the analyte of known concentration which have been prepared in advance, thereby analyzing the concentration of the analyte. The concentration can be semi-quantitatively analyzed by detecting the signal intensity with the naked eye or can be more precisely quantitatively analyzed using a reader such as a densitometer.

In the present invention, the detection pad further has the constant control line for assuring migration of the sample, and the report molecule can be immobilized on the constant control line.

As used herein, the term "constant control line" means a portion that generates constant signals irrespective of concentration of the sample or the analyte in the sample. The constant control line can be formed in the similar way as in the formation of the test line and the variable control line. However, the constant control line can be formed by immobilizing a ligand thereon, wherein said ligand does not bind to the target material (analyte) and specifically or non-specifically binds to and captures the fourth ligand of the second conjugate migrating together with the sample along the detection pad by the mobile phase. Alternatively, the constant control line can be formed by immobilizing a ligand thereon, wherein said ligand specifically or non-specifically binds to and captures a label or a label-binding substance migrating together with the sample along the detection pad by the mobile phase. Consequently, as the ligand, the substance that is able to generate constant signals irrespective of the concentration and the presence or absence of the analyte in the sample is immobilized to form the constant control line. The ligand to be used in the constant control line is referred as the term "reporter molecule", and example of the reporter molecule can include anti-rabbit IgG, anti-chicken IgY, streptavidin, bovine serum albumin or the like.

Two or more of the constant control line can be formed by varying the concentration of the reporter molecule to be immobilized thereon. As the concentration of the reporter molecule immobilized on the constant control line increases, the constant control line is able to constantly generate strong signals.

If a particular concentration of the reporter molecule is immobilized on the constant control line, signal intensity generated therefrom maintains at a constant level, and thus, the concentration of the analyte at the signal intensity of the test line or the variable control line corresponding to the signal intensity of the reporter molecule can be determined. Several constant control lines can be formed by varying the concentration of the reporter molecule, thereby forming several constant control lines having different signal intensities. These signal intensities are compared to that of the test line or the variable control line for quantitative analysis. That is, the constant control line can be utilized as internal standard signal data.

For example, suppose two constant control lines are formed and they generate a signal intensity of 1 and 3. Based on the analyte of known concentration, it can be previously determined that when the test line has a signal intensity of 1, the concentration of the analyte is 10 ng/ml, and when the test line has signal intensity of 3, the concentration of the analyte is 30 ng/ml. If the test line shows signal intensity of 2, resulting from analysis of the analyte of unknown concentration, it can be easily seen that the signal intensity is a signal intensity corresponding to an intermediate value between two constant control lines, compared to the signal intensity of the constant control line.

Size and position of the test line, the variable control line, and the constant control line can be properly selected according to antigen-antibody reaction to be used, but are not limited thereto. Successful migration of the sample can be assured by the presence or absence of signal from the constant control line, and quantitative analysis of the analyte can be carried out by comparing signal intensities of the test line and the variable control line with the standard signal data previously determined. The standard signal data will be described below.

In the present invention, in the case of quantitative analysis through signal intensities of the test line and the variable control line, the dynamic range of the analyte in the sample can be from 1 ng/ml to 1 mg/ml. In one Example of the present invention, the result of analyzing the analyte in the sample showed that the dynamic range of the analyte in the sample was broad from 5 to 100,000 ng/ml (Example 2). That is, the conventional assay strip shows a phenomenon that signal intensity of the test line decreases due to hook effect if the concentration of the analyte in the sample exceeds a specific concentration, which was described above. However, the strip for rapid testing of the present invention is characterized in that it further includes the variable control line which gradually decreases with increasing concentration of the analyte in the sample, and the dynamic range showing a limitation in the conventional strip with only the test line is extended to the range of hook effect through combination of the test line and the variable control line, because the variable control line is not affected by hook effect. However, the dynamic range can be changed depending on the type of analyte, the amount of ligand immobilized on the detection pad, a signal detector or the like, and thus the dynamic range of the present invention is not limited to the above range.

The strip for rapid testing of the present invention will be described in more detail. The strip for rapid testing of the present invention can include the sample pad, into which the sample containing the target analyte is introduced; the conjugate pad, of which one end is connected to the sample pad; the detection pad, of which one end is connected to the other end of the conjugate pad; the absorbent pad, of which one end is connected to the other end of the detection pad, providing a driving power for transport of the sample from the sample pad; and a solid support placed on the lower side of the strip for rapid testing. Constitution of the strip for rapid testing of the present invention is shown in FIG. 2.

The solid support can be made of a material selected from the group consisting of nitrocellulose, nylon, PVDF, glass and plastic. The strip is attached onto the solid support to increase durability of the strip, and it can be easily handled and stored. Further, an additional external case can be also easily installed. The plastic material to be used as the solid support can be a polypropylene film, a polyester film, a polycarbonate film, an acrylic film or the like, but is not limited thereto.

In another aspect, the present invention provides a diagnostic kit including the strip for rapid testing of the present invention installed in the case, in which a guide and a strip support are included in a lower case, and a sample receiving hole is formed in an upper case, and a result observation window is placed at the position corresponding to the test line and the variable control line.

The strip for rapid testing of the present invention can be further installed in a case. A plurality of guides and/or strip supports for placing the strip for rapid testing at the proper position and for fixing or compressing it can be included in the lower case. Optionally, the guides and strip supports can be also included in the upper case at the position corresponding to the position of the guides and strip supports in the lower case. That is, the guides and/or strip supports can be formed in the lower case, or in both the upper case and the lower case, if necessary. Further, the sample receiving hole and the result observation window for detecting signals from the labels at the position corresponding to the test line, the variable control line and the constant control line can be included in the upper case. The sample receiving hole can be formed in the form of a hole or slit at one end of the detection pad, that is, at the end opposite to the absorbent pad with respect to the test line and at the point sufficiently separated from the test line so that the sample can migrate along the membrane. The result observation window can be formed at the point of the detection pad where the test line and the variable control line are placed, and/or if necessary, it can be formed to include the constant control line so that its size is sufficiently distinguishable with the naked eye or sensor, if the constant control line is further formed. It can be formed without limitations in its size and shape, as long as the test line, the variable control line and the constant control line can be distinguishable.

The upper case and the lower case can be manufactured by using a typical plastic material, for example, polycarbonate, acrylonitrile butadiene styrene (ABS) or the like, but are not limited thereto. The upper case and the lower case can be manufactured individually, and then assembled by typical connecting means such as connecting grooves and protrusions. Alternatively, they may be manufactured as an integrated form.

Furthermore, the diagnostic kit can be provided with standard signal data containing a standard color chart of the test line and the variable control line for the samples containing the analyte at various concentrations.

As used herein, the standard signal data means data of summarizing signal intensities of the test line and the variable control line obtained by using the strip for rapid testing of the present invention with respect to the samples containing the analyte at various known concentrations. These standard signal data can be summarized in different ways, which are representatively summarized as colors of the test line and the variable control line corresponding to the concentration of each analyte. Similarly, it can be exemplified by litmus paper for measuring pH, which is provided with a standard color chart of summarizing colors of the paper corresponding to each pH. In particular, when the standard signal data containing the standard color chart is used, signal intensity of the analyte can be easily compared with the standard signal data only with the naked eye, and therefore, a simple and rapid semi-quantitative analysis is possible. In this regard, the diagnostic kit can be used to examine the presence or absence and intensity of signals from the label of the test line and the variable control line with the naked eye, which makes it possible to perform qualitative or semi-quantitative analysis. In other word, after applying the sample to the kit, the presence or absence of signals and intensity thereof from the test line and the variable control line are compared with the standard signal data provided with the standard color chart with the naked eye, thereby analyzing the presence or absence of the analyte in the sample and the concentration thereof.

In still another aspect, the present invention provides a method for qualitative or quantitative analysis of the analyte in the sample using the strip for rapid testing, comprising: introducing the sample to the conjugate pad or the pad positioned therebefore and permitting the sample to migrate (Step 1); examining the presence or absence of signals and intensity thereof from the labels of the test line and the variable control line (Step 2); and comparing the signal intensities of the test line and the variable control line with standard signal data to determine the amount of the analyte (Step 3), in which the standard signal data are obtained by subjecting each of the samples containing the analyte at various known concentrations to Steps 1 and 2.

In still yet another aspect, the preset invention provides a method for qualitative or quantitative analysis of one or more analytes in a sample, comprising reacting the sample with a ligand absorbed on a solid support, which is characterized in that two different reaction mechanisms for one analyte are applied thereto. The two different reaction mechanisms suitable for the method of the present invention can be a a sandwich reaction and a competitive reaction.

The detailed principle of the method for qualitative and/or quantitative analysis of the analyte in the sample using the strip for rapid testing of the present invention is the same as described above. Also, the standard signal data are the same as described above.

The specific analysis method can be carried out in the following order. First, a liquid sample containing a target analyte can be introduced into the conjugate pad or the pad positioned therebefore. That is, the liquid sample can be introduced into the strip by introducing the sample into the conjugate pad, but it can be preferably introduced into the pad positioned before the conjugate pad, for example, the sample pad. In addition, a buffer solution such as PBS is added to the sample, they are mixed homogeneously, and then the mixture can be introduced into the strip in the same manner.

When the sample is loaded (introduced) onto the strip for rapid testing of the present invention, the sample begin to migrate, and then successful migration of the sample can be assured by the constant control line. When successful migration of the sample is assured, the presence or absence of signals and intensity thereof from the labels of the test line and the variable control line are examined. Signals observed from the test line indicate that the analyte is contained in the sample (qualitative analysis). Furthermore, the concentration of the analyte in the sample can be determined by comparing the signal intensities of the test line and the variable control line with standard signal data (quantitative analysis).

In one specific embodiment of the present invention, analysis of human immunoglobulin G (human IgG; analyte) was carried out using the strip for rapid testing further having the variable control line. It was confirmed that highly reliable quantitative analysis is possible with the naked eye, even though hook effect occurs at the concentration of the analyte in the sample ranging from 5 to 100,000 ng/ml (Table 1 and FIG. 5).

The sample for immunoassay of the present invention can include all biological samples such as whole blood, blood cells, serum, plasma, bone marrow, sweat, urine, tears, saliva, skin, mucosa, hair or the like, which are isolated from mammals, preferably, human. Preferably, the sample is blood. The blood can be serum or plasma prepared by removing blood cells. If the whole blood is used, a component for lysis of the blood cells can be added to the buffer solution. These are for illustrative purposes only, and the sample for the immunoassay of the present invention is not particularly limited.

The immunoassay of the present invention is useful for diagnosis of the diseases, in which the whole blood is mainly used as samples, such as malaria antigen (Ag), HIV, C hepatitis, B hepatitis, syphilis, ulcer causing bacteria, cancer markers (AFP, PSA, CEA), tuberculosis, SARS, dengue fever, leprosy, or the like.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the invention is not intended to be limited by these Examples.

Example 1: Manufacture of a strip for rapid testing

A. Manufacture of detection pad having test line, variable control line, and constant control line

Three analysis lines were made on a nitrocellulose membrane. The nitrocellulose membrane was laminated on a plastic card using a laminator. Thereafter, goat anti-human immunoglobulin (anti-human IgG from goat, Arista, USA) as a second ligand of a test line, human immunoglobulin (human IgG) as a third ligand of a variable control line, and bovine serum albumin (BSA) as a reporter molecule of a constant control line were dispensed thereto using an automatic dispenser, respectively, and then dried at 25~30°C for 2 days (48 hours).

B. Manufacture of conjugate pad

A pad was fully soaked in a Tris buffer solution (10 mM, pH 8.5) containing 0.5% PVA (polyvinylalcohol), and then completely dried in a dryer for pre-treatment of the conjugate pad.

A first conjugate solution was prepared by conjugating colloidal gold particles having a diameter of about 40 nm with goat anti-human immunoglobulin (anti-human IgG from goat), and a second conjugate solution was prepared by conjugating colloidal gold particles having a diameter of about 40 nm with streptavidin. The first conjugate solution and the second conjugate solution were applied to the pre-treated conjugate pad, and completely dried in a dryer. Subsequently, the pad was prepared by cutting into a proper size.

C. Manufacture of sample pad

A sample pad was fully soaked in a 0.08 M borate buffer solution containing 1% Triton X-100, 0.5% NaN3, and 0.1% BSA, and completely dried in a dryer. Subsequently, the pad was prepared by cutting into a proper size.

D. Manufacture of absorbent pad

An absorbent pad was completely dried in a dryer, and used as it is without treatment. Subsequently, the pad was prepared by cutting into a proper size.

E. Manufacture of a strip for rapid testing

The detection pad, the conjugate pad, the sample pad and the absorbent pad prepared by the above procedures were assembled as in the structure of FIG. 4.

That is, the sample pad with a sticker was attached to overlap with one end of the conjugate pad, one end of the detection pad was attached to overlap with the other end of the conjugate pad, and the other end of the detection pad was attached to overlap with one end of the absorbent pad with a sticker indicating the top. This assembly was cut into a size of about 2 ± 1.0 mm using a cutter, and finally, a strip as in FIG. 4 was manufactured.

In FIG. 4, the meaning of each reference numeral is the same as follows.

1: Sample pad; 16 ± 4 × 4 ± 2 mm

2: Conjugate pad of anti-human immunoglobulin and gold particle; 6 ± 1.0 × 4 ± 2 mm

3: Nitrocellulose detection pad; 25 ± 5 × 4 ± 2 mm

4: Plastic solid support

5: Absorbent pad; 18 ± 4 × 4 ± 2 mm

6: Anti-human immunoglobulin-immobilized test line

7: Human immunoglobulin-immobilized variable control line

8: Bovine serum albumin-immobilized constant control line

Example 2: Immunoassay using a strip for rapid testing

100 μl of each of the sample solutions containing the analyte human immunoglobulin (human IgG) and phosphate buffer (PBS) was applied to a 96-well plate. The sample solutions were prepared by using human immunoglobulin at a concentration of 0 ng/ml, 5.6 ng/ml, 11.2 ng/ml, 22.5 ng/ml, 45 ng/ml, 90 ng/ml, 187 ng/ml, 375 ng/ml, 750 ng/ml, 1.5 μg/ml, 3.12 μg/ml, 6.25 μg/ml, 12.5 μg/ml, 25 μg/ml, 50 μg/ml, or 100 μg/ml.

The strip for rapid testing manufactured in Example 1 was dipped in the 96-well plate containing the sample solutions, and allowed to migrate for 10 minutes.

FIG. 5 is a photograph showing the development of chromatographic strip according to the concentration of human immunoglobulin as an analyte.

Further, signal intensities from the test line, the variable control line, and the constant control line were examined with the naked eye, and shown in the following Table 1. In this regard, each of the signal intensities was determined by color intensity, in which the highest color intensity was arbitrarily regarded as 5, and the lowest color intensity was regarded as 0.

Table 1

Conc. of target (ng/ml)	Constant control line	Variable control line	Test line
0	3	5	0
5.6	3	5	0.5
11.2	3	5	1
22.5	3	5	2
45	3	5	2
90	3	5	2
187	3	4	3
375	3	4	4
750	3	4	4
1500	3	3	3
3120	3	3	3
6250	3	2	2
12500	3	1	2
25000	3	0.5	2
50000	3	0	2
100000	3	0	1

As shown in Table 1, as the concentration of the analyte (IgG) in the sample increased, the signal intensity of the test line gradually increased, and the strongest intensity was observed at about 375 ng/ml to 750 ng/ml. After that, the signal intensity gradually decreased, which is attributed to hook effect as mentioned above. Therefore, the analyte cannot be quantitatively analyzed by using only the signal intensity of the test line.

For example, if the signal intensity of the test line is 2, the concentration of the analyte (IgG) in the sample was observed around 40 ng/ml and 10 μg/ml. Although the test line showed the same signal intensities, the analyte concentration may differ.

However, the signal intensity of the variable control line gradually decreased, as the concentration of the analyte (IgG) in the sample increased. In detail, it can be seen that the signal intensity almost maintained at the highest level of 5 at the analyte concentration of 0 to 90 ng/ml. As such, if the analyte concentration is low, no hook effect occurs in the test line, and thus the analyte concentration can be determined by the signal intensity of the test line. If the analyte concentration increases, the signal intensity of the test line becomes irregular due to hook effect. However, the signal intensity of the variable control line continuously decreases, and therefore, the analyte concentration can be determined through the variable control line.

For example, if the signal intensity of the test line is 2, the analyte may have two concentrations of about 40 ng/ml and 10 μg/ml, as described above. However, the variable control line exhibits the signal intensity of 5 or 2 with respect to the two concentrations, indicating that quantitative analysis of the concentration is possible, unlike the conventional method of using only the test line.

Further, it was found that the strip for rapid testing of the present invention has a wide dynamic range of the analyte from 5 ng/ml to 100,000 ng/ml.

Claims

A strip for rapid testing having a conjugate pad and a detection pad,

wherein the conjugate pad has a first predetermined amount of a first ligand reacting with an analyte and a signal detection label binding to the first ligand, the first ligand and the label are linked to each other to form a conjugate before migration of a sample or upon migration thereof, and the conjugate can migrate to a detection pad upon migration of the sample,

wherein the detection pad has a test line and a variable control line that are separated from each other,

wherein the test line is immobilized with a second ligand that reacts with the analyte in the sample, and

wherein the variable control line is immobilized with a second predetermined amount of a third ligand which is identical to the analyte or an analog thereof reacting with the first ligand, thereby being able to react with a bare conjugate that does not bind to the analyte in the sample.
The strip for rapid testing according to claim 1, wherein a portion of the first predetermined amount of the first ligand reacts with the analyte in the sample, to thereby form a complex in which the analyte binds to the conjugate having the first ligand and the label, and the amount of the complex formed increases with increasing concentration of the analyte in the sample, whereas the amount of the bare conjugate decreases.
The strip for rapid testing according to claim 2, wherein the complex having the first ligand, the label and the analyte reacts with the second ligand to be captured at the test line, the bare conjugate which does not bind to the analyte in the sample and has the first ligand and the label reacts with the third ligand to be captured at the variable control line, and the presence or absence of the analyte or the amount thereof in the sample, or both thereof can be determined by measuring signals from the labels of the test line and the variable control line.
The strip for rapid testing according to claim 3, wherein signal intensity of the variable control line decreases with increasing concentration of the analyte in the sample.
The strip for rapid testing according to claim 4, wherein signal intensity from the variable control line is maintained at a constant level until the concentration of the analyte in the sample reaches M value, but the signal intensity gradually decreases when the concentration exceeds the M value, and finally converges to 0,

wherein the M value is the maximum concentration of the analyte in the sample in the case where all the third ligands immobilized in the second predetermined amount are saturated by reacting with the bare conjugates.
The strip for rapid testing according to claim 1, wherein the detection pad further has a constant control line for assuring migration of the sample, the constant control line being immobilized with a reporter molecule.
The strip for rapid testing according to claim 1, comprising

a sample pad, into which a sample containing an analyte to be analyzed is introduced;

a conjugate pad, of which one end is connected to the sample pad;

a detection pad, of which one end is connected to the other end of the conjugate pad;

an absorbent pad, of which one end is connected to the other end of the detection pad, providing a driving power for transport of the sample from the sample pad; and

a solid support placed on the lower side of the strip for rapid testing.
The strip for rapid testing according to claim 7, wherein the support is made of a material selected from the group consisting of nitrocellulose, nylon, PVDF, glass and plastic.
The strip for rapid testing according to claim 1, wherein the first ligand, the second ligand and the third ligand are a protein, an antigen, an antibody, DNA, RNA, PNA or an aptamer.
The strip for rapid testing according to claim 1, wherein the label is a colloidal gold, a latex particle, a colored polystyrene microparticle, an enzyme, a fluorescent pigment, a conductive polymer, a luminescent substance, or a magnetic particle.
The strip for rapid testing according to claim 1, which is an immunochromatographic strip.
A diagnostic kit comprising the strip for rapid testing of any one of claims 1 to 11 which is additionally installed in a case,

wherein the kit has a guide and a strip support in a lower case, a sample receiving hole in an upper case, and a result observation window placed at the position corresponding to the test line and the variable control line.
The diagnostic kit according to claim 12, wherein the kit is provided with standard signal data containing a standard color chart of the test line and the variable control line for the samples containing the analyte at various concentrations.
The diagnostic kit according to claim 13, wherein the presence or absence and intensity of signals from the label of the test line and the variable control line can be examined by qualitative or semi-quantitative analysis with the naked eye.
A method for qualitative or quantitative analysis of an analyte in a sample using the strip for rapid testing of any one of claims 1 to 11, comprising the steps of:

introducing the sample into the conjugate pad or the pad positioned therebefore and permitting the sample to migrate (Step 1);

examining the presence or absence and intensity of signals from the labels of the test line and the variable control line (Step 2); and

comparing the signal intensities of the test line and the variable control line with standard signal data to determine the amount of the analyte,

wherein the standard signal data are obtained by subjecting each of the samples containing the analyte at various known concentrations to Steps 1 and 2 (Step 3).
The method according to claim 15, wherein in Step 2, the presence or absence and the intensity of signals from the labels of the test line and the variable control line are examined using a densitometer.
A method for qualitative or quantitative analysis of one or more analytes in a sample, comprising reacting the sample with a ligand absorbed on a solid support, which is characterized in that two different reaction mechanisms for one analyte are applied thereto.
The method according to claim 17, wherein the two different reaction mechanisms are a sandwich reaction and a competitive reaction.