KR20140143120A - Assay strip having variable control line, and diagnosis kit using the same - Google Patents

Abstract

The present invention relates to an analysis strip equipped with a variable control line, a diagnosis kit equipped with the analysis strip, and a method for qualitatively or quantitatively analyzing a target material in a specimen using the same. The analysis strip of the present invention is equipped with a variable control line so that the difficulty of quantitatively measuring a target material due to a hook phenomenon is overcome, and not only a qualitative analysis but also a reliable quantitative analysis can be rapidly and easily performed with the naked eye. Furthermore, by complementing an inspection line and the variable control line, the measured scope of the concentration of an analyzed material is improved.

Description

[0001] The present invention relates to an assay strip having a variable contrast line and a diagnostic kit comprising the strip,

The present invention relates to an assay strip having a variable contrast line, a diagnostic kit having the assay strip, and a method for qualitatively or quantitatively analyzing a target substance in a sample using the assay strip.

Various diagnostic and analytical methods using analytical strips are easy to test and, if necessary, multiple ligands can be immobilized on one strip to obtain many analytical results in a single experiment. Rapid testing is a method for qualitatively and quantitatively analyzing a trace amount of analyte in a short period of time. It is used for diagnosing or examining various diseases, medical, agricultural, livestock, food, military, environment It is used in various fields.

The most widely used immunochromatographic assay among the rapid test methods is an assay strip comprising a reaction substance capable of reacting with a target substance to be detected by using an antigen-antibody reaction or an analyte strip containing the reaction substance, A device type analyzer attached to the case is generally used.

Figure 1 is a cross-sectional view of an assay strip used for conventional immunochromatographic analysis. As shown in Figure 1, a typical analytical strip comprises a sample pad for receiving a liquid sample; A conjugate pad containing a conjugate in which a label (for example, gold particles) that generates a signal that can be detected with the naked eye or a sensor is bonded to a ligand such as an antigen, an antibody, or the like; A detection line to which a target substance in the sample and / or a binding agent (antibody or antigen) that specifically binds to the conjugate is immobilized, and a detection line on which a check line for confirming the development of the sample is formed; And a hygroscopic pad for ultimately housing a liquid sample. These functional pads are connected in a partially overlapping manner in the order listed above, and are continuously arranged on the solid support.

When the analytical strip is used in a plastic case, a sample inlet for dropping the sample is provided at the position of the sample pad on the upper part of the case, and a result confirmation window for confirming the result of the inspection is provided at the position where the binding agent of the detection pad is fixed .

As described above, in the immunochromatographic analysis using the analytical strip, when the liquid sample is dropped onto the sample pad, the liquid sample moves through the conjugate pad and the detection pad by capillary action, and finally, the liquid sample is accommodated in the moisture absorption pad. For example, an antibody capable of binding to an antigen to be measured is fixed to a membrane pad in a line form (test line), another antibody is bound to a labeling substance such as gold particles, and a specimen is developed . The developed specimen develops by forming a complex of antigen-gold particle antibody while passing through the conjugate pad, and the antigen-gold particle antibody is captured on the fixed test line and is labeled with 'test-fixed antibody- Antibody " is formed. As a result, the inspection line line becomes red due to the gold particles.

Thus, when the conjugate contained in the conjugate pad moves with the liquid sample and the substance to be analyzed is present in the sample, the conjugate binds to the binding agent immobilized on the detection pad through the target substance (typically, (sandwich) reaction "). Alternatively, the conjugate and the target substance may be competitively bound to the binding agent (commonly referred to as a "competition reaction"). The presence or absence of the target substance in the sample can be detected visually or by using a sensor by using the analysis strip through the above principle.

In the immunochromatographic assay using the rapid test, that is, the rapid kit described above, mainly, a plurality of specific substances are drawn horizontally and horizontally in a line form, and then cut vertically to form a strip So that the production process is simple and can be mass-produced. In comparison with ELISA, the washing process or the second reaction is not required separately, and the reaction time is about 10 minutes, which is excellent in simplicity. However, the disadvantage is that the detection limit is relatively low, and the quantification is insufficient because it is generally determined by the naked eye. For this reason, conventional immunochromatographic assays are mainly used for qualitative analysis, and there are many limitations in quantitative analysis.

As a result of intensive researches to improve the reliability of quantitative analysis as well as qualitative analysis of an analysis strip, the present inventors have found that by adding a variable trajectory containing the same substance or analogue as the target substance, the signal intensity of the test line and the variable control line are compared And it is confirmed that quantitative analysis is possible. Since the analytical strip according to the present invention has a variable contrast line, it has an advantage that it is possible to perform not only a qualitative analysis of a target material in a sample but also a reliable and quantitative analysis with the naked eye.

In one aspect, the present invention provides an assay strip having a conjugate pad and a detection pad,

And a signal detecting labeling substance that binds to the first ligand, wherein the first ligand and the labeling substance are connected to each other before or after the sample development, The conjugate can move to the detection pad when the sample is developed,

Wherein the detection pad is provided with a check line and a variable control line isolated from each other,

The second ligand in which the inspecting line reacts with the target substance in the sample is fixed,

The third ligand which is the same as the target substance or the analogue of the target substance which reacts with the first ligand is immobilized at the second constant amount so that it can react with the bare conjugate not bound to the target substance in the sample Lt; / RTI >

In one embodiment, the analytical strip of the present invention comprises

Wherein a portion of the first ligand is reacted with the target substance in the sample to form a complex in which the target substance is bound to the conjugate having the first ligand and the labeling substance,

As the concentration of the target substance in the sample increases, the number of the complexes formed increases and, conversely, the number of the bare conjugates decreases.

In another embodiment, the analytical strip of the present invention comprises

The complex comprising the first ligand, the labeling substance and the target substance reacts with the second ligand to be captured on the inspection line,

The bare conjugate having the first ligand and the labeling substance not bound to the target substance in the specimen is captured in the variable label by reacting with the third ligand,

The signal from the inspecting line and the signal from the marking substance on the variable rectifying line can be measured to measure the presence or absence or both of the target substance in the specimen.

In another embodiment, the analytical strip of the invention is characterized in that the signal intensity from the variable canister decreases as the concentration of the target material in the sample increases.

In another embodiment, the analytical strip of the present invention comprises

The intensity of the signal from the variable control line is maintained constant up to the M value of the target substance in the sample, gradually decreases when the concentration exceeds the M value, and finally the signal intensity converges to zero,

The maximum concentration of the target substance in the sample when the third ligand in which the M value is fixed to the second predetermined amount is saturated and reacts with all of the bare conjugate.

In another embodiment, the assay strip of the present invention is further characterized in that the detection pad is further provided with an immovable contrast line capable of confirming the development of the specimen, and the immovable contrast line is fixed to the reporter molecule.

In another embodiment, the analytical strip of the present invention comprises

A sample pad into which a sample to be inspected for inclusion of a target substance is introduced;

The conjugate pad having one end connected to the sample pad;

The detection pad having one end connected to the other end of the conjugate pad;

A moisture absorption pad connected at one end to the other end of the detection pad and providing a driving force for sample transport from the sample pad; And

And a solid support located below the analysis strip.

In another embodiment, the analytical strip of the present invention is characterized in that the support is formed of a material selected from the group consisting of nitrocellulose, nylon, PVDF, glass and plastic.

In another embodiment, the analytical strip of the present invention is characterized in that the first ligand, the second ligand and the third ligand are proteins, antigens, antibodies, DNA, RNA, PNAs or platemers.

In another embodiment, the analytical strip of the present invention is characterized in that the labeling substance is a colloidal gold, a latex particle, a colored polystyrene microparticle, an enzyme, a fluorescent dye, a conductive polymer, a luminescent material, or a magnetic particle do.

In another embodiment, the analytical strip of the present invention is characterized by being a strip for a rapid test, preferably an immunochromatographic strip.

In another aspect, the present invention is a diagnostic kit in which the assay strip is additionally immobilized in a case,

The lower case is provided with a guide and a strip supporting part,

The upper case includes a sample inlet; And

And a result confirmation window is provided at a position corresponding to the inspection line and the variable control line.

In one embodiment, the diagnostic kit of the present invention is further characterized in that standard signal data is included, including a check line for specimens containing target substances of various known concentrations and a color reference chart of the variable contrast line.

In another embodiment, the diagnostic kit of the present invention is characterized in that it can perform qualitative or semi-quantitative analysis for visually confirming presence or absence of signals of the labeling substance from the inspection line and the variable control line.

According to another aspect of the present invention, there is provided a method for qualitatively or quantitatively analyzing a target substance in a sample using the analytical strip,

A first step of injecting a specimen into the conjugate pad or a pad positioned before the conjugate pad to develop the specimen;

A second step of checking the signal presence and intensity of the labeling substance from the inspection line and the variable control line; And

And a third step of measuring the amount of the target material by comparing the signal strength detected from the inspection line and the variable contrast line with the standard signal data,

And the standard signal data is obtained by performing the first step and the second step, respectively, on the specimens including target substances of various known concentrations.

In one embodiment, the assay method of the present invention is characterized by confirming signal presence and intensity of the labeling substance from the inspection line of the second step and the variable control line by a densitometer.

In another aspect, the present invention provides a method for qualitative or quantitative analysis of one or more target substances in a sample, comprising reacting a sample with a ligand adsorbed on a solid phase, wherein two different reaction mechanisms for one target substance And an analysis method is provided.

In one embodiment, the assay method of the present invention is characterized in that the two different reaction mechanisms are competitive and sandwich reactions.

In one aspect, the present invention provides an assay strip having a conjugate pad and a detection pad, wherein the conjugate pad comprises a second amount of a first ligand which reacts with the target substance and a second ligand which binds to the first ligand And the first ligand and the labeling substance are connected to each other before or during the development of the specimen to form a conjugate and the conjugate can move to the detection pad when the specimen develops, Wherein the test line is immobilized with a second ligand which reacts with the target substance in the sample and the variable control line is a third ligand which is the same as the target substance or which is a similarity of the target substance reacting with the first ligand, Is capable of reacting with a bare conjugate that is not bound to the target material in the sample, Provide lip.

In the present invention, the analytical strip may be a strip for rapid analysis, preferably for immunochromatography.

The present invention is a conventional analytical strip having a detection pad on which only a check line capable of confirming the development of a test line and a specimen is formed can be obtained by adding a variable control line to which the same substance or analogue as the target substance is immobilized, And reliable quantitative analysis can be performed.

Further, the present invention is characterized in that a semi-quantitative analysis with high reliability can be performed through the naked eye without using a special analysis mechanism.

The term "immunochromatography" of the present invention is an analytical method that combines the principle of immunoreaction based on an antigen-antibody reaction and the chromatographic principle in which a sample and a reagent migrate along a medium by a mobile phase. Briefly, the antibody or antigen to be analyzed is preliminarily packed in a porous membrane and fixed, and the blood is expanded toward the antibody or antigen immobilized from one end of the membrane to observe the reaction with the antigen or antibody in the blood . In general, the term " immune response " refers to a reaction of an antigen-antibody. However, the present invention includes not only the reaction of an antigen-antibody but also the reaction between a receptor specifically binding to each other and a ligand specifically binding thereto. And reactions occurring between the enzyme and the substrate, such as the reaction between the enzyme and the substrate.

Analysis by immunochromatography is carried out by moving a specimen containing a target substance together with a mobile phase by capillary action through a medium. Thus, as a medium for such immunochromatographic development, strips can be prepared and used. The specific components of each of these analysis strips and their respective functions will be described later.

A labeling substance can be used so that the antigen-antibody reaction can be easily confirmed visually or by using a sensor. Also, a ligand capable of specifically binding to a target substance can be used by linking the labeling substance to the labeling substance so that the labeling substance can bind to the target substance to be analyzed.

The term "conjugate " of the present invention means a conjugate formed by linking the labeling substance and the ligand, wherein the conjugate is bound to a labeling substance for signal detection, Ligand. The first ligand means a ligand which binds to the labeling substance to form a conjugate in order to distinguish it from other ligands described later.

In the present invention, the first ligand refers to a substance capable of reacting with a target substance, and capable of specifically binding to a target substance.

The labeling substance and the first ligand may be physically or chemically connected. That is, the labeling substance and the first ligand may be connected by passive adsorption, and the labeling substance may be modified to have a reactive group to form a covalent bond with the first ligand. However, the present invention is not limited thereto, The linkage of the labeling substance and the first ligand can be carried out using a method known to a person skilled in the art.

However, the labeling substance and the first ligand may be present in the conjugate pad in the form of a conjugate, before the specimen is developed on the assay strip. In addition, they are not connected to each other but exist in a separated state in the conjugate pad, and they can be connected to each other to form a conjugate. In any case, the labeling substance and the first ligand can migrate to the detection pad in the state of the conjugate upon sample development, since they are not fixed to the conjugate pad.

The term "labeling substance" of the present invention means a substance which generates a signal that can be detected visually or by using a sensor. As the labeling substance, gold colloid (gold particles), latex particles, colored polystyrene microparticles, enzymes, fluorescent dyes, conductive polymers, luminescent materials or magnetic particles can be used, but the present invention is not limited thereto. Further, the signal may be generated by itself due to the inherent characteristics of the labeling substance such as luminescence, or may be generated by external stimulation such as fluorescence.

The term "ligand" of the present invention means a substance that specifically binds to each other. For example, an antibody that specifically binds to an antigen, a ligand that specifically binds to a specific receptor, etc. act as ligands with each other. The ligand may be a protein, an antigen, an antibody, a DNA, an RNA, a PNA, or an aptamer. In addition, the ligand of the present invention may be used without limitation as long as it exhibits the characteristics defined above. The term ligand as used throughout the present specification means a substance that specifically binds to each other as defined above unless specified to the contrary as a ligand that specifically binds to a specific receptor.

In a preferred embodiment of the present invention, the labeling substance and the first ligand are pre-connected and may be present in the conjugate pad as a conjugate. In this case, it is possible to prepare a conjugate solution by preparing a labeled substance and a first ligand as a solution of a known concentration, reacting it for a predetermined time, and dispensing the conjugate solution on the conjugate pad to prepare a conjugate pad provided with the conjugate. At this time, the concentrations and mixing ratios of the respective solutions can be determined in consideration of the binding ratio between the labeling substance and the ligand. The binding of the labeling substance to the ligand may be a case where one ligand binds to one labeling substance, a case where a plurality of ligands bind to one labeling substance, or a case where a plurality of labeling substances bind to one ligand. The specific binding ratio is not critical, but the preferred embodiment may be to maintain a constant ratio. The binding ratio may vary depending on the types of the labeling substance and the ligand, and may be estimated in consideration of their relative sizes and the number of binding sites. For example, when a latex bead of several microns in size is used as a labeling substance, and an antibody is used as a ligand, a plurality of antibodies can bind to one latex. Preferably, the concentration and mixing ratio of the antibody and the latex solution may be adjusted so that the antibody binds to the latex surface in order to bind a uniform number of antibodies to each latex surface. However, the present invention is not limited thereto.

As described above, a conjugate solution having a known concentration can be obtained by mixing and reacting a labeled substance and a first ligand at a known concentration, respectively. The conjugate solution is in the form of a dispersion in which conjugate molecules of a certain concentration are uniformly dispersed in the solution.

The analytical strip is an analytical strip having a variable rectilinear line,

A conjugate pad comprising a second amount of a first ligand that reacts with the target material and a signal detection label material that binds to the first ligand; And

And a detection pad provided separately from the inspection line and the variable control line.

The third ligand was immobilized on the detection pad to form a "variable control line" so that the target substance in the sample could be detected qualitatively as well as quantitatively. In the variable contrast line of the analytical strip of the present invention, an analogue of the target substance which is the same substance as the target substance or reacts with the first ligand so as to be able to react with the bare conjugate in the mobile phase deployed on the detection pad is referred to as a third ligand Can be fixed. Since the bare conjugate not bound to the target substance in the sample contains the first ligand, the first ligand specifically binds to the same substance or analogue as the target substance, i.e., the third ligand.

Furthermore, the third ligand can fix the specified amount of the ligand to the variable vessel. That is, the second predetermined amount means a specific amount in which the third ligand is fixed to the variable control line. By fixing the third ligand in the second predetermined amount as described above, it is possible to control the signal intensity in the variable control tank, to adjust the concentration range of the target substance in the sample to be detected, and further to assure reliable quantitative analysis.

In the present invention, the third ligand means a ligand which is fixed to the detection pad to form a variable control line, in order to distinguish it from other ligands described later.

Further, in order to detect the target substance in the sample, a second ligand capable of specifically binding with the ligand and capable of selectively capturing was fixed to the detection pad to form a "test line ". In the test strip of the analytical strip of the present invention, a second substance which reacts with the target substance in the sample is immobilized so as to be able to react with the target substance-conjugate complex, i.e., the target substance, in the mobile phase developed on the detection pad. The second ligand may be the same substance or analogue as the first ligand, or may be a third substance that specifically binds to the target substance. As the target material in the specimen develops with the mobile phase, it binds to the conjugate within the conjugate pad to form a complex and develops. Such a complex comprises a target material, and as described above, the first ligand can specifically bind to the target substance, so that the same substance or analogue as the first ligand; Or the second ligand, which is a separate substance that specifically binds to the target substance, can capture the complex to the test line through binding with the target substance.

In the present invention, the second ligand means a ligand which is fixed to the detection pad and forms an inspection line to distinguish it from other ligands described later.

As described above, immunochromatography utilizes the principle of chromatography in which a mobile phase containing a target substance moves along a medium. Thus, immunoassay using analytical strips requires a mobile phase to move the sample containing the target material along the strip. Accordingly, the immunoassay kit of the present invention may further comprise a buffer solution. The buffer not only acts as a mobile phase for moving the sample along the assay strip but also acts as a solvent to dissolve the conjugate and may also serve as a diluent for diluting the sample if necessary. Further, for example, when performing whole blood analysis, a component for lysis of a blood cell component such as red blood cells may be further included. As the buffer solution, a conventional buffer solution such as a phosphate buffered solution (PBS) of 10 mM to 1 M concentration, a nonionic or an amphoteric surfactant or a mixture thereof may be used without limitation, and a desired reaction such as an antigen- As shown in FIG.

Wherein the conjugate pad is a pad having a first predetermined amount of a first ligand reacting with the target substance and a signal detecting labeling substance binding to the first ligand, wherein the first ligand and the labeling substance are present before the specimen is developed They may be connected to each other in the form of a conjugate and may be present in the conjugate pad or may be present in the conjugate pad in a separated state without being connected to each other and then they may be connected to each other to form a conjugate.

In the present invention, in the case of the first ligand existing in the conjugate pad, only a predetermined specific amount can be present. That is, the second predetermined amount means a specific amount in which the first ligand exists in the conjugate pad. Since the first ligand in the conjugate pad is defined as the second predetermined amount, the number of complexes formed by binding the first ligand to the target substance increases as the concentration of the target substance in the sample increases, and conversely, And the number of the bare conjugates decreases.

Preferably, the conjugate pad may be present in the pad in a conjugate state in which the first ligand and the labeling substance are already linked. At this time, as described above, the conjugate solution of the known concentration can be applied to the pad.

Furthermore, the conjugate pad may further include a second conjugate in which a fourth ligand is bound to a labeling substance so as to be used as an internal control. The fourth ligand means a ligand capable of reacting specifically or nonspecifically with the reporter molecule. The second conjugate can be captured by the reporter molecules of the invariant cannula to confirm the development of the specimen. A more detailed description of the invariant vector and the reporter molecule will be described later.

The detection pad can be moved by a capillary phenomenon of a porous membrane in which a mobile phase and a sample form a detection pad, in which a mobile phase and a specimen are developed. Further, the detection pad may be further provided with an inspective line which is isolated from the inspection line and the variable control line, and can confirm the development of the specimen. One end of the detection pad may be connected to the conjugate pad, and the other end may be connected to a hygroscopic pad providing a driving force for sample transport. The conjugate pad and the hygroscopic pad may partially overlap the detection pad. The detection pad may be a porous membrane, and the porous membrane may include a nitrocellulose membrane, a glass fiber membrane, a polyethersulfone membrane (PES) membrane, a cellulose membrane, a nylon membrane nylon film, and a combination thereof, and preferably a nitrocellulose film having pores of 5 to 15 m may be used, but the present invention is not limited thereto.

The principles of qualitative and quantitative analysis using the analytical strip of the present invention will be described in more detail.

Conventional analytical strips form test lines on the membrane pad, and the developed target substance-conjugate complex is captured on the test line to generate a signal (sandwich reaction). At this time, the intensity of the signal increases as the concentration of the target substance increases, but when the target substance increases above a predetermined concentration, the intensity of the signal decreases as a hook phenomenon. Due to this phenomenon, conventional analytical strips have great limitations for quantitative analysis of the target material.

However, the present invention applies not only a sandwich reaction but also a special competitive reaction using a characteristic in which a second predetermined amount of the first ligands in the conjugate pad competitively binds to a finite target substance in the sample, Compared to chromatographic methods, there is an improvement in the promptness, ease of use and especially the possibility of quantitative analysis.

Specifically, when a liquid sample is introduced into the sample pad of the strip of the present invention, the development of the mobile phase and the sample starts. In the conjugate pad, a second constant amount of the first ligands competitively reacts to a finite target material in the sample. A part of the second predetermined amount of the first ligand reacts with the target substance in the sample to form a complex in which the target substance is bound to the conjugate having the first ligand and the labeling substance, The ligand is developed with the mobile phase in the state of the bare conjugate. Here, since the first ligand is constant at the second predetermined amount, the number of the complexes formed increases as the concentration of the target substance in the sample increases, and conversely, the number of the bare conjugates decreases.

The complex comprising the first ligand, the labeling substance, and the target substance reacts with the developed second ligand to be trapped on the inspecting line and is not bound to the target substance in the sample, The conjugate reacts with the developing third ligand and is captured in the variable vessel. Since the second ligand is fixed to the inspection line and the third ligand is fixed to the variable control vessel, the complex and the bare conjugate are trapped at each line.

Since both the complex and the bare conjugate have a labeling substance, signals from the labeling substance and the labeled labeling substance on the variable labeling line can be measured to determine the presence or absence or both of the target substance in the sample.

As mentioned above, the number of the complex increases with the concentration of the target substance in the sample, and conversely, the number of the bare conjugate decreases as the concentration of the target substance in the sample increases. Therefore, the signal intensity from the variable control vessel in which the bare conglomerate is captured decreases as the concentration of the target substance in the sample increases.

More specifically, the signal intensity from the variable control line is maintained constant up to the M value of the target substance in the sample, and gradually decreases when the concentration of the target substance is over the M value. Finally, the signal intensity converges to 0, 2 is the maximum concentration of the target substance in the sample when the third ligand immobilized at a constant amount reacts with all of the bare conjugates and becomes saturated. In one embodiment of the present invention, the concentration of the target substance in the sample is maintained at 0 to 90 ng / ml as the signal with the strongest signal intensity from the variable control line, so that the third ligand is all reacted to the bare conjugate State, indicating that the M value in this example is about 90 ng / ml (see Example 2). However, the M value varies depending on the second constant amount, the third ligand, and the type of the target substance, and accordingly, a person skilled in the art can appropriately select the M value according to the analysis purpose.

In the case of the inspection line, as described above, the intensity of the signal increases as the amount of the target material increases, and then decreases from a certain level due to the hook phenomenon. FIG. 3 shows signal intensity patterns of the inspection line and the variable control line according to the increase of the concentration of the target material of the present invention.

As a result, qualitative and quantitative analysis of the target material is possible through signals from the inspection line and the labeling substance on the variable control line. More specifically, it is possible to analyze the concentration of the target substance by comparing the signal at the inspection line and the variable control line with the standard signal data that has been previously completed with respect to the target substance at the known concentration. This can be analyzed semi-quantitatively by visually checking the intensity of the signal, or more rigorously quantitatively using a reader such as a densitometer.

In the present invention, the detection pad is further provided with an invariant control line for confirming the development of the specimen, and the invariant control line may have fixed reporter molecules.

As used herein, the term " constant control line "refers to a portion of a specimen or a specimen that produces a constant signal regardless of the concentration of the target material. The invariant can be formed using a method similar to the inspection line and the variable control line. However, the constant invariant may specifically or non-specifically bind to the fourth ligand of the second conjugate moving along the detection pad by the mobile phase together with the specimen, without binding to the substance (target substance) to be analyzed And the ligand can be fixed. Alternatively, the constant invariant can be formed by immobilizing a ligand capable of binding specifically or non-specifically with a labeling substance or a separate substance to which a labeling substance is bound, which is moved along with the specimen by the mobile phase along with the detection pad. As a result, it is formed by fixing a substance capable of emitting a constant signal with a ligand regardless of the concentration and presence or absence of the target substance in the sample. The ligand that can be used as the invariant vector is expressed using the term "reporter molecule ", for example, the reporter molecule includes anti-rabbit IgG, anti-chicken IgY, streptavidin, bovine serum albumin Etc. may be used.

The constant invariant may be formed in two or more different concentrations of fixed reporter molecules. The higher the concentration of reporter molecules immobilized on the invariant cannula, the more constant the constant invariant can emit a strong signal.

When the reporter molecule of a specific concentration is immobilized on the invariant control line, since the intensity of the signal generated here is always constant, the intensity of the signal on the inspection line or the variable control line corresponding to the signal intensity is fixed to a certain level Can be determined. By forming a number of repetitive control lines with different concentrations of the reporter molecules, it is possible to form several constant control lines having different signal intensities. They can be quantitatively analyzed by comparing them with the signal intensity at the inspection line or the variable control line. That is, the invariant can be used as internal standard signal data.

For example, assume that two invariant lines are formed and emit signal strengths of 1 and 3, respectively. When the target substance concentration is 10 ng / ml when the test line has a signal intensity of 1 and the signal intensity is 3 when the test substance has a signal intensity through the target substance at a known concentration, the concentration of the target substance is 30 ng / Ml can be predetermined. When analyzing the target substance of unknown concentration, it can be easily seen that the signal intensity corresponding to the value between the two invariant cannulas is compared with the signal strength of the invariant cannon if the inspecting line shows signal strength of 2 .

The size and position of the inspection line, the variable control line, and the invariant control line may be appropriately selected depending on the antigen-antibody reaction used, and the present invention is not limited thereto. It is possible to confirm whether the specimen is successfully developed from the presence or absence of the invariant rectilinear signal and quantitatively analyze the target material by comparing the intensity of the signal of the inspecting line and the intensity of the signal of the variable rectifying line with predetermined standard signal data. The standard signal data will be described later.

In the present invention, in the case of quantitative analysis through the signal intensity of the inspection line and the variable control line, the dynamic range of the target substance in the sample may be between 1 ng / ml and 1 mg / ml. In one embodiment of the present invention, the target substance in the sample was analyzed and it was confirmed that the dynamic range of the target substance in the sample was widely ranged from 5 to 100,000 ng / ml (see Example 2). That is, in the conventional analysis strip, the signal intensity at the inspection line is rather reduced due to occurrence of the hook phenomenon when the target substance in the sample exceeds a predetermined concentration. However, since the analytical strip of the present invention further includes a variable contrast line that gradually decreases as the concentration of the target substance in the sample increases, and since the variable contrast line is not affected by the hook phenomenon, the combination of the inspection line and the variable control line The concentration measurement range limited by the conventional inspection line can be extended to the range where the hook phenomenon occurs. However, since the concentration measurement range may vary depending on the type of the target substance, the amount of the ligand fixed on the detection pad, the signal detection device, and the like, the concentration measurement range of the present invention is not limited to the above range.

The analysis strip of the present invention will be described in more detail.

The assay strip may include a sample pad into which a sample to be tested for the target substance is introduced; The conjugate pad having one end connected to the sample pad; The detection pad having one end connected to the other end of the conjugate pad; A moisture absorption pad connected at one end to the other end of the detection pad and providing a driving force for sample transport from the sample pad; And a solid support located below the assay strip. A schematic diagram of the analytical strip of the present invention is shown in Fig.

The solid support may be formed of a material selected from the group consisting of nitrocellulose, nylon, PVDF, glass, and plastic. By attaching the strip on the solid support, the durability of the strip can be improved and handling and storage can be facilitated. Further, it is possible to easily mount an additional external case. As a plastic material that can be used as the solid support, a polypropylene film, a polyester film, a polycarbonate film, an acrylic film, or the like can be used, but the present invention is not limited thereto.

According to another aspect of the present invention, there is provided a kit in which the assay strip is further fixed in a case, wherein the lower case is provided with a guide and a strip supporting part, and the upper case includes a sample inlet; And a result confirmation window is provided at a position corresponding to the inspection line and the variable control line.

The analytical strip may additionally be fixed within the case. The lower case may be provided with a plurality of guides and / or strip supports for positioning and fixing or pressing the analytical strip in a suitable position. A guide and a strip supporting part may be provided in the upper case at positions corresponding to the guide and the strip supporting part provided in the lower case. That is, the guide and / or the strip supporting part may be formed in the lower case or may be formed in both the upper case and the lower case, if necessary.

The upper case may be provided with a result confirmation window for detecting a signal from the labeling substance at a position corresponding to the sample inlet, the inspection line, the variable rectifying line, and the invariant rectifying line. The sample inlet may be formed at one end of the detection pad, that is, at the opposite end of the hygroscopic pad on the basis of the inspection line, and at the same time spaced sufficiently apart from the inspection line so that the sample may be developed along the membrane. have.

The result confirmation window may have a size sufficient to be visually recognizable from the outside, including the point where the inspection line and the variable control line are located on the detection pad and / or the immovable control line, As shown in FIG. The size and shape of the inspection line, the variable rectifying line, and the constant rectifying line can be determined without limitation.

The upper and lower cases may be manufactured using a common plastic material. For example, materials such as polycarbonate, acrylonitrile butadiene styrene (ABS) may be used, but the present invention is not limited thereto. The upper and lower cases may be separately manufactured and provided with coupling grooves, coupling protrusions, and the like, and they may be coupled by conventional means, and in some cases, they may be integrally manufactured.

Further, the diagnostic kit may further include standard signal data including a check line for specimens containing target substances of various known concentrations and a color reference chart of the variable contrast line.

In the present invention, the standard signal data refers to data obtained by summarizing the signal intensities on the test lines and the variable control lines for each sample using the analyte strip of the present invention for a sample containing target substances of various known concentrations. Such standard signal data can be arranged in various forms, and can be typified by a check line corresponding to the concentration of each target substance and a color of the variable contrast line. Similarly, in the case of a litmus test strip for measuring pH, it is possible to provide a color reference table together with the color of the test strip corresponding to each pH. In particular, in the case of the standard signal data including the color reference chart, since the intensity of the signal with respect to the target material can be easily compared with the standard signal data simply by the naked eye, semi-quantitative analysis can be performed easily and quickly. In this regard, the diagnostic kit can perform qualitative or semi-quantitative analysis by visually checking the signal presence or intensity of the labeling substance from the inspection line and the variable control line. That is, the standard signal data including the color reference chart that can be provided before, and the presence or absence of the signal appearing on the inspection line and the variable reference line after the specimen is developed in the kit are visually compared, Its concentration can be analyzed.

According to another aspect of the present invention, there is provided a method for qualitatively or quantitatively analyzing a target substance in a sample using the above-described analytical strip, comprising: a first step of putting a specimen into the pad of the conjugate body or a previously located pad; A second step of checking the signal presence and intensity of the labeling substance from the inspection line and the variable control line; And a third step of measuring the amount of the target material by comparing the signal intensity detected from the inspection line and the variable contrast line with the standard signal data, wherein the standard signal data includes, for samples containing various known concentrations of the target substance, Wherein the first step and the second step, respectively, are carried out.

In yet another aspect, the present invention provides a method for qualitative or quantitative analysis of one or more target substances in a sample, comprising reacting a sample with a ligand adsorbed on a solid, comprising the steps of reacting two different reactions And a mechanism is applied. The analysis method is characterized in that the two different reaction mechanisms are a competitive reaction and a sandwich reaction.

The analytical method of the present invention can be carried out by using an analytical strip in which two ligand reaction regions are present at different positions in one strip or using the analytical strips in which the two ligand reaction regions are respectively present in the separated strip . Further, the analytical method of the present invention is not limited to the development method such as the lateral flow method or the one-time reaction method in the reaction vessel.

The specific principle of the method for qualitative and / or quantitative analysis of a target substance in a sample using the assay strip of the present invention is the same as described above. Furthermore, the standard signal data is the same as described above.

In carrying out the specific analysis method, the following procedure can be carried out.

First, a liquid sample containing the substance (target substance) to be analyzed can be put into the pad or the pad located before the conjugate pad. That is, the liquid sample can be introduced directly into the conjugate pad and introduced into the strip, but preferably introduced into the conjugate pad, for example, into the sample pad. The specimen may be uniformly mixed with a buffer such as PBS, and the same may be introduced into the strip in the same manner.

When the sample is loaded (introduced) into the assay strip as described above, the development is started, and then it can be confirmed whether the sample is developed well through the invariant control line. If the sample develops well, check the signal presence and intensity of the labeling material from the inspection line and the variable control line. If a signal is confirmed from the test line, this indicates that the target substance is contained in the sample (qualitative analysis). Further, it is possible to confirm that the target substance is included in a certain concentration in the sample by comparing the signal intensity detected from the test line and the variable control line with the standard signal data (quantitative analysis).

In a specific embodiment of the present invention, the concentration of the target substance in the sample is in the range of 5 to 100,000 ng / ml (for example, in the range of 5 to 100,000 ng / ml) by performing analysis of human IgG (target substance) using the analytical strip, It is confirmed that reliable quantitative analysis is possible with naked eyes even though the hook phenomenon occurs (see Table 1 and FIG. 5).

The sample for immunoassay of the present invention includes all biological samples such as whole blood, blood cells, serum, plasma, bone marrow fluid, sweat, urine, tears, saliva, skin, mucous membrane and hair separated from mammals, preferably human For example, blood. Blood may be serum or plasma components from which blood components have been removed, or components that can lyse blood components may be added to the buffer when whole blood is used. This is an illustrative example, and the specimen for the immunoassay of the present invention is not particularly limited.

The immunoanalytical method according to the present invention is characterized in that the immune analysis method according to the present invention is a method for immunologically analyzing the whole blood such as malaria antigen (Ag), AIDS, hepatitis C, hepatitis B, syphilis, gastric ulcer, cancer marker (AFP, PSA, CEA), tuberculosis, It can be useful for the diagnosis of diseases used as specimens.

Since the analyzing strip of the present invention has a variable contrast line, it is possible to overcome the difficulty of the quantitative measurement of the target material due to the hook phenomenon, and perform quick and convenient quantitative analysis with high reliability as well as qualitative analysis with the naked eye. Furthermore, the concentration range of the analyte can be improved by complementing the test line with the variable control line.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram showing a cross-section of an assay strip used in a conventional immunochromatographic assay.
2 is a schematic diagram showing an assay strip used in the immunochromatographic analysis of the present invention.
Fig. 3 is a schematic view showing aspects of test lines, variable control lines, and invariant control lines before and after the introduction of a specimen in the assay strip of the present invention.
4 is a schematic diagram of an analytical strip according to an embodiment of the present invention.
FIG. 5 is a photograph showing an analysis result of concentration of a target substance in various samples according to an embodiment of the present invention. Human IgG (human IgG) was used as the target material.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example 1: Preparation of analytical strips

A. Manufacture of detection pads with inspecting lines, variable toners and invariant toners

Three analytical lines were placed on a nitrocellulose membrane. The nitrocellulose membrane was laminated to a plastic card using a laminator. Thereafter, anti-human IgG (Arista, USA) was used as the second ligand of the test line, human IgG (human IgG) was used as the third ligand of the variable control line, and reporter molecules Bovine serum albumin (BSA) was used for each of these, and each of these was dispensed using an automatic dispenser, followed by drying at 25 to 30 ° C for 2 days (48 hours).

B. Fabrication of conjugate pad

The pad was fully wetted with Tris buffer (10 mM, pH 8.5) containing 0.5% PVA (polyvinylalcohol) and completely dried in a dryer to pre-treat the pad of the conjugate.

A colloidal gold particle with a diameter of about 40 nm and a conjugate solution with an anti-human IgG from goat were prepared and colloidal gold particles with a diameter of about 40 nm A second conjugate solution to which streptavidin was bound was prepared. The conjugate solution and the second conjugate solution were dispensed onto the pre-treated conjugate pad, completely dried, and then cut to an appropriate size and prepared.

C. Sample pad production

The sample pad was thoroughly wetted with 0.08 M borate buffer solution containing 1% Triton X-100, 0.5% NaN ₃ , and 0.1% BSA, completely dried in a drier, and cut into an appropriate size.

D. Fabrication of absorbance pad

The absorbent pad was dried without any treatment in a state where the moisture was completely dried in the dryer, and the absorbent pad was prepared by cutting it to an appropriate size.

E. Fabrication of analytical strips

The detection pad, the conjugate pad, the sample pad, and the absorption pad prepared through the above processes are assembled according to the structure shown in FIG.

That is, the sample pad with the sticker is attached so as to overlap with the one end of the conjugate pad, the one end of the detection pad is attached so as to overlap with the other end of the conjugate pad, Was attached so that one end of the absorbing pad was overlapped with the other. This was cut to about 21.0 mm using a cutter to produce a strip as shown in Fig.

In Fig. 4, the respective reference numerals denote the following.

1: sample pad; 16442 mm

2: conjugate pad of anti-human IgG and gold particles; 61.042 mm

3: nitrocellulose detection pad; 25542 mm

4: plastic solid support

5: hygroscopic pad; 18442 mm

6: test line immobilized with anti-human IgG

7: Variable cannulas fixed with human IgG

8: Invariant cannula fixed with bovine serum albumin

Example 2: Immunoassay using assay strips

To the 96-well plate, 100 sample solutions containing human IgG and phosphate buffer (PBS) as target substances were dispensed. The concentration of human IgG in the sample solution was adjusted to 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, 50 μg / ml, and 100 μg / ml, respectively, in the same manner as described above.

The analytical strip prepared in Example 1 was placed in a 96-well plate containing the sample solution, and developed for 10 minutes.

Fig. 5 is a photograph showing the development result of the chromatography strip according to the concentration of human IgG as a target substance.

Further, the intensities of the signals appearing on the inspection line, the variable control line and the invariant control line are visually confirmed, and are shown in Table 1 below. At this time, each signal intensity was judged by the intensity of color, the highest chromaticity was arbitrarily set at 5, and the lowest chromaticity was set at 0.

As shown in Table 1, as the concentration of the target substance (IgG) in the sample increases, the signal intensity of the test line gradually increases to show the strongest intensity between about 375 ng / ml and 750 ng / ml, . This is due to the aforementioned hook phenomenon. Therefore, the concentration of the target substance can not be quantitatively analyzed only by the signal intensity of the test line.

For example, if the signal intensity of the inspecting line is 2, it can be confirmed that the concentration of the target substance (IgG) in the specimen appears in the vicinity of about 40 ng / ml and about 10 μg / ml, Even if the signal intensity is the same, the concentration of the target substance may be different.

However, in the case of the variable control tank, the signal intensity gradually decreased as the concentration of the target substance (IgG) in the sample increased. Specifically, it can be seen that the intensity of the signal is kept at 5, which is the highest, from 0 to 90 ng / ml of the target substance. When the concentration of the target material is low, the hook phenomenon does not occur in the inspection line. Therefore, the concentration of the target substance can be analyzed through the signal intensity of the inspection line. When the concentration of the target material further increases, the intensity of the signal is irregular due to the occurrence of the hook phenomenon in the inspection line, but the intensity of the signal continuously decreases in the variable contrast line, so that the concentration of the target substance can be analyzed through the variable contrast line .

For example, when the signal intensity of the inspecting line is 2, there may exist two types of inspecting line, one having a concentration of the target substance of about 40 ng / ml and one having a concentration of about 10 μg / ml as described above, The signal intensity is 5 or 2 for each concentration. Therefore, it can be confirmed that the concentration which can not be measured by only one conventional inspecting line can be quantitatively measured.

Also, it can be seen that the dynamic range of the analyte strip to the target substance is broadly from 5 ng / ml to 100,000 ng / ml or more.

Claims (19)

In an assay strip having a conjugate pad and a detection pad,
Wherein the conjugate pad has a first amount of a first ligand reacting with a target substance and a signal detection labeling substance binding to the first ligand, wherein the first ligand and the labeling substance are connected to each other before or after the sample development, And when the sample is developed, the conjugate can move to the detection pad,
The detection pad is provided with a check line and a variable control line isolated from each other,
Wherein the inspecting line has a second ligand immobilized thereon, the second ligand reacting with the target substance in the specimen,
The variable control line may have a third ligand, which is the same as the target substance or is similar to the target substance reacting with the first ligand, is immobilized at a second predetermined amount so that it can react with a bare conjugate not bound to the target substance in the sample Analytical strips. The method of claim 1, wherein a portion of the second ligand is reacted with a target substance in the sample to form a conjugate having the target substance bound to the conjugate having the first ligand and the labeling substance,
Wherein the number of the formed complexes increases as the concentration of the target substance in the sample increases and conversely the number of the bare conjugates decreases. 3. The method of claim 2, wherein the complex comprising the first ligand, the labeling substance and the target substance is captured by the inspection line by reaction with the second ligand,
The bare conjugate having the first ligand and the labeling substance not bound to the target substance in the specimen is captured in the variable label by reacting with the third ligand,
The signal from the inspecting line and the marking substance on the variable rectifying line can be measured to measure the presence, quantity, or both of the target substance in the sample. 4. An assay strip according to claim 3, wherein the signal intensity from the variable contrast line decreases as the concentration of the target material in the sample increases. 5. The method according to claim 4, wherein the intensity of the signal from the variable control line is maintained at a constant M value until the concentration of the target substance in the sample is constant,
Wherein the M value is the maximum concentration of the target substance in the sample when the third ligand immobilized at the second predetermined amount is saturated and reacts with all of the bare conjugate. 2. The apparatus according to claim 1, wherein the detection pad further comprises an invariant check line for confirming the development of the specimen,
Wherein said immobile pigments have fixed reporter molecules. The method of claim 1,
A sample pad into which a sample to be inspected for inclusion of a target substance is introduced;
The conjugate pad having one end connected to the sample pad;
The detection pad having one end connected to the other end of the conjugate pad;
A moisture absorption pad connected at one end to the other end of the detection pad and providing a driving force for sample transport from the sample pad; And
And a solid support located below the analysis strip. 8. An assay strip according to claim 7, wherein the support is formed of a material selected from the group consisting of nitrocellulose, nylon, PVDF, glass and plastic. 2. The assay strip of claim 1, wherein said first ligand, said second ligand and said third ligand are proteins, antigens, antibodies, DNA, RNA, PNAs or platamers. 2. The assay strip of claim 1, wherein the labeling material is a colloidal gold, a latex particle, a colored polystyrene microparticle, an enzyme, a fluorescent dye, a conductive polymer, a luminescent material, or a magnetic particle. The assay strip of claim 1, wherein the assay strip is a strip for rapid assay. 2. The assay strip of claim 1, wherein the analyte strip is a strip for immunochromatography. 13. A kit in which the assay strip of any one of claims 1 to 12 is additionally fixed within a case,
The lower case is provided with a guide and a strip supporting part,
The upper case includes a sample inlet; And
And a result confirmation window is provided at a position corresponding to the inspection line and the variable control line. 14. The diagnostic kit of claim 13, further comprising standard signal data comprising a check line for samples containing various known concentrations of target material and a color reference table of variable contrast lines. 15. The diagnostic kit according to claim 14, wherein qualitative or semi-quantitative analysis capable of visually confirming presence or absence of signal and intensity of a labeling substance from the inspection line and the variable control line can be performed. A method for qualitative or quantitative analysis of a target substance in a sample using the analytical strip according to any one of claims 1 to 12,
A first step of injecting a specimen into the conjugate pad or a pad positioned before the conjugate pad to develop the specimen;
A second step of checking the signal presence and intensity of the labeling substance from the inspection line and the variable control line; And
And a third step of measuring the amount of the target material by comparing the signal strength detected from the inspection line and the variable contrast line with the standard signal data,
Wherein the standard signal data is obtained by performing the first step and the second step, respectively, on the samples containing target substances of various known concentrations. 17. The analysis method according to claim 16, wherein signal presence and intensity of the mark substance are confirmed by a densitometer from the inspection line of the second step and the variable contrast line. 17. The analysis method according to claim 16, wherein signal presence and intensity of the mark substance are confirmed by a densitometer from the inspection line of the second step and the variable contrast line. 19. The method of claim 18, wherein the two different reaction mechanisms are competitive and sandwich reactions.

Images