WO2019050321A1 - Chromatographic strip comprising multiple test lines, diagnostic kit comprising same, and qualitative, semi-quantitative or quantitative analysis method comprising multiple competitive reaction measurement steps - Google Patents

Abstract

The present invention relates to: a chromatographic strip comprising a plurality of test lines for a competitive assay; a diagnostic kit comprising the chromatographic strip; and a method for qualitatively or quantitatively analyzing a target material within a specimen by using the same. By using the chromatographic strip of the present invention, qualitative analysis and highly reliable quantitative analysis can be rapidly and conveniently carried out with the naked eyes or a sensor even for a target material, which has one ligand binding position or is small. Furthermore, the present invention has an effect of improving the concentration measurement range of an analyte by the mutual supplementation of test lines and control lines.

Description

A qualitative, semi-quantitative, quantitative analysis method comprising a chromatographic strip having a plurality of inspection lines, a diagnostic kit comprising the same, and a plurality of competitive reaction measurement steps

The present invention relates to a chromatography strip having a plurality of inspection lines, a diagnostic kit having the chromatography strip, and a method for qualitatively and / or quantitatively analyzing a target substance in a sample using a plurality of competitive reactions. The device for chromatographic analysis of the present invention can be used for qualitative, semi-quantitative and quantitative analysis in various fields such as diagnosis and prediction of disease, food safety inspection, environmental analysis and compound analysis. Do.

The chromatographic analysis, known as the rapid test method, utilizes the binding reaction between a target substance to be analyzed and a ligand that specifically binds to the target substance, and quantitatively and quantitatively analyzes a trace amount of the analyte in a short period of time It is used in various fields such as medicine, agriculture, animal husbandry, food, military, environment as well as diagnosis or inspection of various diseases. In such a chromatographic assay, an assay strip containing a reactant capable of reacting with the target substance to be detected to change or a device-type analyzer equipped with the analytical strip in a plastic case is generally used. Figure 1 is a cross-sectional view of an analytical strip used in conventional chromatographic analysis. As shown in Figure 1, a typical analytical strip comprises a sample pad for containing a liquid sample; A conjugate pad containing a conjugate in which a labeling substance (for example, a fluorescent substance or gold particle) generating a signal that can be sensed by the naked eye or a sensor is bonded to a ligand such as a nucleic acid, an antigen, or an antibody; A detection line on which a target substance in a specimen and / or a binding agent (nucleic acid, antibody or antigen) specifically binding to the conjugate is immobilized, and a detection line on which a check line for confirming the development of the specimen 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 chromatographic analysis using the analytical strip, when a 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 is housed in the moisture absorption pad. For example, an antibody capable of binding to an antigen to be measured is linearly fixed to a detection pad (test line, first binding antibody), and another antibody (second binding antibody) is labeled with a labeling substance such as gold particles And the specimen is developed. The developed specimen develops by forming a complex of antigen-gold particle second binding antibody while passing through the conjugate pad, and the antigen-gold particle second binding antibody is captured by the first binding antibody of the fixed test line, The first binding antibody-antigen-gold particle second binding antibody conjugate immobilized on the test line is formed. As a result, the inspection line line becomes red due to the gold particles. As such, the conjugate contained in the conjugate pad moves with the liquid sample, and when 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 ").

However, when analyzed by the sandwich assay, the epitope of the antibody or ligand should be at least two so that the target substance binds to the first binding antibody and the second binding antibody-labeled substance and the sandwich reaction can occur. (Peptide protein, drug, allergen, etc.), the binding affinity is weakened due to sterical hinderence, etc., the specificity, sensitivity and specificity of the antibody, ) And cross-reactivity may arise.

Therefore, in order to overcome this problem, the target substance is immobilized on the detection line of the detection pad to compete the target substance in the sample with the immobilized target substance in the test line against the binding position of the antibody-labeled substance in the conjugate pad, (Often referred to as a " competition reaction ") in which the signal of the test line weakens as the temperature increases. The presence or absence of the target substance in the sample can be detected visually or using a sensor by using the analysis strip through the above principle.

Rapid kits using this competitive reaction, however, have advantages of easy mass-production, easy usage, quick results, expensive and large professional analytical equipments and low cost of analysis but relatively low detection limit And the fact that it is generally judged by the naked eye, it is considered to be a disadvantage that the quantitative property is relatively insufficient. For this reason, conventional chromatographic methods are mainly used for qualitative analysis, and there are many limitations in quantitative analysis.

The present inventors have made extensive efforts to quantitatively analyze not only the qualitative analysis but also the quantitative analysis of the target substances which are difficult to analyze using the strip for chromatography using the sandwich reaction in the strip for chromatography. As a result, , It is necessary to make the chromatogram strips with the inspection lines so that a plurality of competition reactions occur differently from the case of the conventional chromatography strips having one inspection line including one inspection line including one inspection line, , It is confirmed that quantitative analysis of the target substance can be performed in a wide measurement concentration range.

Accordingly, an object of the present invention is to provide a chromatography strip capable of quantitative analysis using a plurality of competitive reactions and having a high detection limit.

It is another object of the present invention to provide a diagnostic kit comprising the strip for chromatography.

It is still another object of the present invention to provide a method for qualitative analysis and quantitative analysis of a target substance in a sample using the chromatography strip or the diagnostic kit.

In accordance with one aspect of the present invention, there is provided a chromatographic strip comprising a conjugate pad and a detection pad, wherein the conjugate pad comprises a first ligand which reacts with the target substance and a second ligand which binds to the first ligand Wherein the first ligand and the labeling substance are connected to each other before or after the specimen development to form a first conjugate and the conjugate can move to the detection pad when the specimen develops, Wherein the two or more test lines are each capable of reacting with a bare conjugate that is immobilized to the target substance in the sample, wherein the same substance or analogue thereof as the target substance is immobilized, Provide a strip for printing.

The present invention further provides a conjugate pad further comprising a second ligand which does not react with the target substance and a signal detection labeling substance which binds to the second ligand, wherein the second ligand and the labeling substance are present before or after the sample development, And the second bonding member can move to the detection pad. The detection pad further includes a reference line for confirming the development of the specimen, and the reference line is a target substance or a first Wherein the material to be immobilized to the conjugate is immobilized, and the substance immobilized on the control line is capable of reacting with the second conjugate that does not bind to the target substance.

The present invention relates to a conventional chromatography strip having a detection pad on which the same substance as the target substance or its analogue is immobilized, and a detection pad on which only a check line for confirming the development of the specimen is formed, It is also possible to perform reliable quantitative analysis as well as qualitative analysis by comparing the signals of the reference lines which can show a certain signal.

In addition, the present invention is characterized in that a target substance, which is relatively small in size and has only one ligand binding site, is difficult to apply the sandwich assay method to a qualitative analysis as well as a reliable quantitative analysis by a competitive analysis method.

Furthermore, the present invention is characterized in that a reliable semi-quantitative analysis can be performed through visual or general sensors without using a special analysis instrument.

The term " chromatography " of the present invention refers to the principle of immune reaction based on an antigen-antibody reaction, the principle of binding of a ligand and a receptor, the principle of binding of a nucleic acid to a complementary nucleic acid, This is an analytical method that combines chromatographic principles. Briefly, a target substance or an analogue to be analyzed is preliminarily dispensed and fixed on a porous membrane, and a solution containing the sample is developed from the one end of the membrane toward the target substance or analogue immobilized thereon, . In general, the term " immune response " refers to a reaction of an antigen-antibody. However, the present invention includes not only the reaction of antigen-antibody but also the reaction between a receptor and a ligand specifically binding to each other. And reactions that occur by specifically recognizing each other.

Chromatographic analysis is accomplished by moving the specimen containing the target material along with the mobile phase by capillary action through the medium. Thus, as a medium for such chromatographic development, strips can be prepared and used. The specific components of each of these chromatography strips and their respective functions will be described later.

A labeling substance can be used to easily confirm various specific binding reactions including the antigen-antibody by visual observation or 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 or the target substance to be analyzed.

The term " conjugate " of the present invention means a binding body in which the labeling substance and the ligand are connected to each other, and the binding body includes a first binding body in which a labeling substance for signal detection and a first ligand are connected to each other .

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 be covalently bonded to 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 prior to development of the sample in the strip for chromatography, or they may exist in the conjugate pad in a separated state without being connected to each other. They may be connected to each other to form a conjugate, or they may be present in a solution form and added to the conjugate pad before use. 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. Examples of the labeling material include, but are not limited to, gold colloid (gold particles), latex particles, colored polystyrene fine particles, enzymes, fluorescent dyes, conductive polymers, luminescent materials or magnetic particles. 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. Alternatively, the prepared solution may be added to the conjugate pad by a user before use. 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 chromatography strip is a chromatography strip having two or more check lines fixed by using a predetermined amount of the same or similar material as the target substance. The chromatography strip includes a first ligand which reacts with a target substance in an inspection line or a sample, A conjugate pad having a signal detection labeling substance binding to the ligand; And a detection pad having two or more inspection lines and a reference line separated from each other.

In order to qualitatively detect and quantitatively analyze the target substance in the sample, two or more test lines are fixed on the detection pad to form a " multiple competition assay test line " Respectively. In the control line of the strip for chromatography of the present invention, the label substance in the mobile phase developed on the detection pad-a substance or a target substance which reacts with the first ligand of the first ligand-bearing conjugate but does not react with the target substance, A substance which does not react with the ligand and reacts with the second ligand of the labeling substance-second ligand conjugate may be further fixed to the detection pad.

In the present invention, the first ligand means a ligand which reacts with a target substance or an analogue of two or more test lines fixed to a target substance or a detection pad in the sample.

In the present invention, the second ligand refers to a ligand which reacts only with a substance fixed to the reference line.

The second ligand adjusts the signal intensity using a predetermined amount and adjusts the concentration range of the target substance in the detectable sample by comparing the signal intensity with the test line that changes according to the concentration of the target substance in the sample And can further assist in reliable quantitative analysis.

Two or more check lines of the strip for chromatography of the present invention may be immobilized with a certain amount of target substance or target substance analogue reacting with the first ligand of the conjugate pad and two or more check lines may be fixed with different amounts or the same amount have. Since the first ligand immobilized on the conjugate pad can specifically bind to the target substance, the first ligand labeling substance conjugate is developed with the mobile phase when the target substance is not present in the sample, and the target substance or analogue Binds to the target substance of the immobilized test line, forms a complex of the target substance-first ligand-labeled substance, and exhibits a strong signal. When the target substance in the sample is present, it forms a complex of the target substance with the target substance - the first ligand - labeled substance in the sample, and develops into the mobile phase with the detection pad. The amount of the complex of the target substance - first ligand - labeled substance formed at this time , The amount of the test substance to form a complex with the target substance or the analogue thereof is reduced, thereby reducing the signal. That is, the signal of the inspecting line decreases proportionally depending on the amount of the target substance in the specimen.

The second ligand, which is further immobilized on the conjugate pad, does not react with the target substance or the first ligand and binds to only the substance of the control line immobilized on the detection pad, and displays a signal.

Quantitative analysis can be performed over a wide range of target substances by comparing the ratio of each signal of two or more inspecting lines to the signal of the control line depending on the concentration of the target substance in the specimen.

As described above, chromatography employs the principle of chromatography in which a mobile phase containing a target substance moves along a medium. Therefore, analysis using chromatographic strips requires a mobile phase to move the sample containing the target material along the strip. Accordingly, the assay kit of the present invention may further comprise a buffer. The buffer solution acts not only as a mobile phase for moving a sample along a strip for chromatography but also as a solvent for dissolving the conjugate, and may also serve as a diluent for diluting a 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. The buffer solution may be 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. The composition and the use ratio of the buffer solution may be appropriately selected depending on the kind of the buffer solution.

The conjugate pad is a pad having a first predetermined amount of a first ligand reacting with a target substance as described above and a signal detecting labeling substance binding to the first ligand, wherein the first ligand and the labeling substance are 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, Can be added to the whole conjugate pad

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 first 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 first constant 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 having a second ligand and a labeling substance bound thereto so as to be used as an internal control. The second ligand means a ligand capable of reacting specifically or nonspecifically with the substance of the control line. The second junction body may be bonded to the substance of the reference line to confirm the development of the specimen. A more detailed description of the control line 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 specimen form a detection pad as a medium in which a mobile phase and a specimen are developed. Further, the detection pad is provided so that the inspection line and the reference line are isolated from each other. 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. The porous membrane may be a nitrocellulose membrane, a glass fiber membrane, a polyethersulfone (PES) membrane, a cellulose membrane, a nylon membrane nylon membranes, and combinations thereof. A nitrocellulose membrane having pores of preferably 5 to 15 mu m may be used, but is not limited thereto.

The principle of qualitative and quantitative analysis using the strip for chromatography of the present invention will be described in more detail.

Conventionally, a strip for chromatography using a competitive reaction forms a test line by fixing the same substance or its analogue as a target substance to a detection pad, and when a target substance is not present in a mobile phase containing the developed test substance, a first ligand- It is combined with the target material of the inspecting line to generate a signal. When the target substance is present in the sample, the first ligand specifically reacts with the target substance to form a complex of the target substance-first ligand-labeled substance, develops along the mobile phase, and forms a complex with the target substance of the test line. (The competitive reaction), depending on the amount of target material-first ligand-labeled material included in the sample. At this time, the intensity of the signal decreases as the concentration of the target substance in the sample increases. In this case, when a small amount of target substance exists in the sample due to a strong specific binding force when the target substance of the first ligand is reacted with the target substance of the test line, if a single test line is used, even if the binding of the target substance and the first ligand occurs, - It does not affect the formation of the complex of the first ligand-labeling substance. When the color of the test line is visually observed or when it is confirmed by the sensor, there is almost no change. Therefore, when the target substance exists at a certain high concentration in the sample, And the range of quantitative analysis is small, so there is a great limitation to quantitative analysis of the target substance.

However, in the present invention, a test strip in which two or more competitive reactions in which a target substance or an analogue is immobilized is different than a single test line in a chromatographic strip using a general competitive reaction, To improve the sensitivity and to allow quantitative analysis over a wide concentration range. As shown in Fig. 2, when three test lines are used, if the test line number is given in the order of close to the pads of the conjugate, the signal of the test line 1 (T1) (the test line in which the mobile phase including the sample first comes first) becomes strongest, The quantitative graph of the competition reaction, the quantitative graph of the test line 2, and the quantitative graph of the test line 3 are plotted for each of the test samples 1, 2 (T2) and 3 (T3) Can be obtained. Therefore, depending on the concentration range of the target substance in the sample, the appropriate concentration range is selected by using the graph of the inspection line 3 at the low concentration, the graph of the inspection line 2 at the intermediate concentration, and the graph of the inspection line 1 at the high concentration, And it is possible to broaden the quantitative measurement range from a low concentration to a high concentration. The concentration of the target substance is quantitatively analyzed using the competitive reaction of the same substance or its analogue as the target substance immobilized on the test substance in the test substance and the test substance in the test substance with respect to the first predetermined amount of the first ligand in the test substance conjugate pad So that it is applied to one strip, thereby improving the promptness, ease of use and especially the quantitative analysis possibility as compared with the conventional chromatography method.

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 first predetermined amount of the first ligands specifically reacts with the target substance in the sample. A part of the first 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 first predetermined amount, the number of the complexes formed increases with the concentration of the target substance in the sample, and conversely, the number of the bare conjugates decreases relatively.

The bare conjugate having the first ligand and the labeling substance not bound to the target substance in the specimen is caught by a plurality of inspection lines to which the intermediate target substance or the analogue is developed, appear. Also, the complex comprising the first ligand, the labeling substance, and the target substance is not captured on the developed inspecting line but developed with the mobile phase. The bare conjugate is captured on the test line to indicate a signal, and the complex comprising the first ligand, the labeling substance and the target substance is not captured and does not display a signal.

Since both the complex and the bare conjugate have the labeling substance, signals from the labeling substance of the inspection line by the bare conjugate captured on the inspection line among them are measured to determine presence or absence or both of the target substances in the sample .

As mentioned above, the number of the bare conjugates decreases as the concentration of the target substance in the sample increases, and on the contrary, the number of the complexes increases as the concentration of the target substance in the sample increases. Therefore, the signal intensity from the test line in which the bare conjugate is captured decreases as the concentration of the target substance in the sample increases. Since a plurality of inspection lines are used, if three inspection lines and one reference line are used as shown in FIG. 4, if the inspection line is numbered in the order of proximity to the bonding pad, the inspection line 1 (the inspection line ) Due to the binding of the target substance to the bare conjugate and the test line, the signal becomes strongest and the response of the signal decreases depending on the amount of the target substance in the sample. Next, in the case of the test line 2, the amount of the bare conjugate developed with the mobile phase decreases in response to the test line 1, and the response of the test line decreases according to the amount of the target substance in the test sample, In the case of the test line 3, the amount of the bare conjugate developed with the mobile phase reacts with the test lines 1 and 2 to further decrease, and the response of the test line signal decreases according to the amount of the target substance in the test sample, A quantitative graph for the competitive reaction according to the concentration of the target substance in the sample for each test line 1, a quantitative graph for the test line 2, and a quantitative graph for the test line 3 can be obtained as shown in FIG. Therefore, in the graph of the inspection line 3 in the low-concentration measurement range T3, the line of the inspection line 2 in the intermediate-concentration measurement range T2, and the high-concentration measurement range T1 in the low concentration test range T3 By using the graph of test line 1, it is possible to select the appropriate quantitative graph according to the concentration range in one experiment, so that it can be used for quantification, so that sensitivity of the target substance is improved and quantitative measurement test from low to high concentration (T1 to T3, You can expand the dynamic range.

More specifically, as shown in FIG. 7, the signal intensity from the inspecting line 1 is maintained constant up to a low M1 concentration of the target substance in the sample, the signal gradually decreases with increasing concentration, and finally, The signal intensity converges to zero and the signal intensity from the inspecting line 2 is kept constant up to the low M2 value of the target substance in the sample, and the signal gradually decreases with increasing concentration, 0, and the signal intensity from the inspection line 3 is kept constant up to the M3 value of the low concentration of the target substance in the sample. Then, as the concentration increases, the signal gradually decreases and finally the signal intensity converges to zero , It is possible to measure quantitatively by the range T1 of the quantitative measurement by the inspection line 1, the measurement range T2 of the quantitative measurement by the inspection line 2, and the inspection line 3 It appears above T3, which will move to the low concentration range, depending on the measuring range of the number of scan lines to T1 ~ TN corresponding to the N in the scan line one. In the case of the test line, as described above, the intensity of the signal decreases as the amount of the target material increases. FIG. 4 and FIG. 6 show intensity patterns of several test lines according to the increase of the concentration of the target substance of the present invention.

As a result, qualitative and quantitative analysis of the target substance is possible through a signal from the labeled substance bound to the first ligand captured in two or more check lines in which the competitive reaction occurs. More specifically, the signal of the inspecting line can be compared with the standard signal data that has already been completed with respect to the target substance of known concentration, and the concentration of the target substance can be analyzed. This can be analyzed semi-quantitatively by visually confirming the intensity of the signal or can be quantitatively analyzed more precisely using a reader such as a densitometer.

In the present invention, the detection pad may further include a control line for confirming the development of the specimen.

As used herein, the term " control line " refers to a portion of a specimen or a specimen that gives a constant signal regardless of the concentration of the target material. The invariant check line may be formed using a method similar to the inspection line. However, the control line can be specifically or non-specifically bound to the second ligand of the second conjugate moving along the detection pad together with the sample together with the substance to be analyzed (target substance) And then fixing the material. Alternatively, the control line may be formed by immobilizing a substance capable of binding specifically or non-specifically with a labeling substance or a separate substance to which the 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 irrespective of the concentration and presence or absence of the target substance in the specimen. Materials that can be used in the above-mentioned control line include anti-Rabbit IgG, anti-chicken, anti-IgY, streptavidin, bovine serum albumin and the like.

Since the intensity of the generated signal is always constant when fixing the substance of a specific concentration to the reference line, the signal intensity at the N test lines can be more easily compared with the signal intensity of the reference line, When the concentration of the substance is certain, it is possible to determine how much the intensity of the inspection line occurs. The signal intensity of the reference line can be used for quantitative analysis compared with the signal intensity of the inspection line. That is, the ratio of the signal intensity of the inspection line to the inspection line can be used as internal standard signal data.

For example, as shown in FIG. 7, three test lines are formed. Test lines 1, 2, and 3 for the reference line are respectively 10 times, 5 times, and 3 times The concentration of 10 ng / ml was 5 times and 3 times. When 1 × signal was detected, it was confirmed that the concentration was 30 ng / ml. After analyzing the unknown substance, the inspecting lines 1, 2 and 3 emitted 7, 4 and 2 times of signal intensity respectively The concentration of the target substance is between 10 and 30 ng / ml. Compared with the signal intensity of the test lines, it is easy to see the concentration not only by simple POCT medical devices such as a reader but also by the naked eye.

The position, number and position of the test line using the plurality of competitive reactions can be appropriately selected according to the antigen-antibody reaction used, and the like, but are not limited thereto. It is possible to confirm whether the specimen is successfully developed from the presence or absence of the control line signal and to quantitatively analyze the target substance by comparing the intensity of the signal of each inspection line and the intensity of the signal of the control line with predetermined standard signal data. The standard signal data will be described later.

In the present invention, in the case of quantitative analysis using different signal intensities and signal intensity of the test line appearing at the same concentration on a plurality of fixed test lines, the dynamic range of the target substance in the test sample is 0.1 ng / 1 mg / ml. Since the strip for chromatography of the present invention includes a plurality of inspection lines, a plurality of inspection lines exhibiting different intensities at the same concentration are used so that a low concentration and a high concentration Since a plurality of inspection lines are used in combination with each other in a single strip, it is possible to carry out a quantitative measurement with a limit of conventional inspection lines alone, and to widen the measurement range of the concentration and improve the sensitivity.

The configuration of the strip for chromatography of the present invention will be described in more detail.

The chromatographic strip includes a sample pad into which a sample to be tested for the presence or absence of a target substance is introduced; A conjugate pad having one end connected to the sample pad; A 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 strip for chromatography. A schematic diagram of the strip for chromatography 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 strip for chromatography is additionally fixed in a case, wherein the lower case is provided with a guide and a strip supporting part, and the upper case is provided with a sample inlet; And a result confirmation window is provided at a position corresponding to the plurality of inspection lines and the reference lines.

The strip for chromatography may additionally be fixed within the case. Inside the lower case, a plurality of guides and / or strip supports may be provided for positioning and fixing or pressing the chromatographic 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 have a result confirmation window for detecting a signal from a labeling substance at a position corresponding to a sample inlet, an inspection line, and a reference line. The sample inlet is formed at the one end of the detection pad, that is, at the opposite end of the hygroscopic pad with respect to the inspection line, and at the same time sufficiently spaced from the inspection line so that the sample can be developed along the membrane, . The result confirmation window may include a plurality of inspection lines and a reference line on the detection pad and may be formed to be large enough to be visually recognizable from the outside or through a sensor. The size and shape of the inspection lines and the reference lines can be determined without limitation as long as they can be confirmed.

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 plurality of inspection lines for the samples containing the target substances at various known concentrations and color reference tables for the reference lines.

In the present invention, the standard signal data refers to data obtained by summarizing signal intensities of a plurality of test lines and reference lines for each sample using the chromatography strip of the present invention for a sample containing target substances of various known concentrations it means. These standard signal data can be arranged in various forms, and can be grouped into a plurality of inspection lines and a color of a reference line corresponding to the concentration of each target substance. 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 signal presence and intensity of the labeling substance from the plurality of inspection lines and the control line. That is, standard signal data including a color reference table that can be provided before, and the presence or absence of a signal appearing on the plurality of inspection lines and the reference line after visual inspection of the specimen in the kit, And 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 strip for chromatography, 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 signal presence and intensity of the labeling substance from a plurality of inspection lines and a reference line; And a third step of measuring the amount of the target material by comparing the signal intensity detected from the plurality of inspection lines and the reference line with the standard signal data, wherein the standard signal data includes , Respectively, and the first step and the second step, respectively.

The specific principle of the method for qualitative and / or quantitative analysis of a target substance in a sample using the strip for chromatography 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 chromatography strip as described above, the development starts and after that, it can be confirmed whether the specimen is well developed through the reference line. If the sample develops well, check the signal presence and intensity of the labeling material from the multiple test lines and the control line. If a change in signal from multiple inspectors is confirmed, this indicates that the target substance is contained in the specimen (qualitative analysis). Furthermore, it is possible to confirm that the target substance is contained in a certain concentration in the specimen by comparing the signal intensity detected from the plurality of test lines and the reference 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 1 to < RTI ID = 0.0 > A wide range of reliable quantitative analysis was possible with a fluorescent reader in the range of 5,000 μg / ml (FIG. 6).

The specimen for the analysis of the present invention includes not only a general aqueous solution but also all kinds of living bodies such as whole blood, blood cells, serum, plasma, bone marrow fluid, sweat, urine, tears, saliva, skin, mucous membrane, Sample, and may be, 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 analysis of the present invention is not particularly limited.

The analytical method according to the present invention can be useful for semi-quantitative or quantitative diagnosis of various drugs such as protein, vitamin D, hIgG, hIgE, allergen, antibiotic, etc., which are small in size,

The strip for chromatography of the present invention includes a plurality of inspection lines using competitive measurement reactions, thereby overcoming the difficulties of quantitative measurement of target materials and performing quick and convenient quantitative analysis as well as qualitative analysis with a fluorescent reader . Furthermore, since a plurality of inspecting lines are used, the sensitivity and the concentration measuring range of the analyte are improved.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram showing a cross-section of an analytical strip used for conventional chromatographic analysis.

2 is a schematic diagram illustrating an assay strip used in the chromatographic analysis of the present invention.

3 is a schematic diagram of a strip for chromatography according to an overview of the present invention.

4 is a schematic view showing the appearance of a plurality of inspection lines and reference lines before and after the introduction of a sample in the strip for chromatography of the present invention.

Figure 5 is a schematic diagram for a dip strip method in accordance with one embodiment of the present invention.

FIG. 6 is a graph illustrating the results of analyzing the concentrations of target substances in various samples according to an embodiment of the present invention. Rabbit immunoglobulin (rabbit IgG) was used as the target material.

7 is a quantitative graph derived by a plurality of inspection lines according to the outline of the present invention.

FIG. 8 is a quantitative graph showing the intensity of each signal according to the concentration of a target substance of a plurality of test lines represented by the experimental results of the present invention, and shows a quantitative measurement range.

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. The embodiment utilized a dip strip method, one of the chromatographic methods.

Example 1: Preparation of a dip strip for chromatography

A. Fabrication of detection pads with inspection lines

Three test lines were dispensed onto a nitrocellulose membrane. The nitrocellulose membrane was laminated to a plastic card using a laminator. Then, rabbit IgG (rabbit immunoglobulin G, Arista, USA) was injected to the test specimens 1, 2 and 3 at a predetermined interval using an automatic dispenser as a target substance or an analogue of the test line, And dried for 2 days (48 hours).

B. 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.

C. Fabrication of Strips for Chromatography

The prepared detection pad and the absorbent pad are assembled into the rescue unit shown in FIG. 5 through the above processes.

That is, a detection pad was attached to a plastic support with a sticker, and one end of the detection pad and one end of the absorption pad were attached so as to overlap with each other. This was cut to about 2 1.0 mm using a cutter to produce a strip as shown in Fig.

In Fig. 5, the respective reference numerals denote the following.

1: a hygroscopic pad; 18 ± 4 Х 4 ± 2 mm

2: nitrocellulose detection pad; 25 ± 5 Х 4 ± 2 mm

3: Rabbit immunoglobulin fixed test lines 1, 2, 3

4: plastic support

Example 2 Analysis Using a Dip Strip for Chromatography

100 μl of a sample solution containing rabbit IgG and phosphate buffer (PBS) as target substances was dispensed into a 96-well plate. The sample solutions were prepared so that the concentrations of rabbit immunoglobulin were 0, 5, 10, 50, 100, 500, 1000, and 5000 ug / ml, respectively. To the sample solution, add a labeling substance (fluorescence) -first ligand (anti-rabbit immunoglobulin) solution in a first predetermined amount.

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

FIG. 6 is a diagram showing the development result of a strip for chromatography according to the concentration of rabbit immunoglobulin as a target substance. FIG.

It was confirmed that the intensity of the signal varies depending on the concentration of the target substance and that the quantitative curve according to different concentrations can be obtained for the test line 1. 2. 3.

In addition, it can be seen that the dynamic range of the chromatographic strip for the target substance ranges from 5 ug / ml to 5,000 ug / ml or more.

Claims (19)

A chromatographic strip comprising a conjugate pad and a detection pad, Wherein the conjugate pad has a first ligand that reacts with the target substance and a signal detection label substance that binds to the first ligand or forms a first conjugate when the sample is developed or when the sample develops, Can be moved to the detection pad, Wherein the detection pad is provided with two or more inspection lines isolated from each other, Wherein the two or more test lines are each capable of reacting with a bare conjugate that is immobilized to the target material in the sample, such that the same material or analogue thereof as the target material is immobilized. The method according to claim 1, Wherein the first ligand is present in a predetermined amount in the conjugate pad and a portion 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 first conjugate having the first ligand and the labeling substance, Wherein the number of the formed complexes increases as the concentration of the target material in the sample increases and conversely the number of the bare conjugates decreases. 3. The method of claim 2, The complex comprising the first ligand, the labeling substance and the target substance is not captured on the inspection line, The bare conjugate having the first ligand and the labeling substance without being bound to the target substance in the sample reacts with the same substance or its analogue as the target substance fixed to the test line in a predetermined amount, Wherein a signal from the labeling substance of the inspecting line is measured to measure the presence or absence or both of the target substance in the sample. The method of claim 3, Wherein the signal intensity from all test lines fixed to the detection pad is reduced as the concentration of the target material in the sample increases. 5. The method of claim 4, The intensity of the signal from the inspection line is maintained constant up to the M value of the target substance in the specimen, and gradually decreases when the concentration exceeds the M value. Finally, the signal intensity converges to zero, Wherein the M value is the maximum concentration of the target substance in the sample when the same substance or the same substance as the target substance fixed to the inspection line at a predetermined amount is saturated and reacted with the whole of the bare conjugate. 6. The method of claim 5, In case of using a plurality of test lines, the intensity of the signal is maintained constant up to the concentration Mn value as the number of test lines increases to N (N > = 2) The dragon strip. The method according to claim 1, Wherein the conjugate pad has a second ligand that does not react with the target substance and a signal detection labeling substance that binds to the second ligand, and the second ligand and the labeling substance are connected to each other before or after the sample development, The second conjugate can move to the detection pad when the sample is developed, The detection pad is further provided with a check line for confirming the development of the specimen, Wherein the control line is capable of reacting with a target substance or a substance that does not react with the first conjugate, and wherein the substance for the control line is capable of reacting with a second conjugate that does not bind to the target substance. The chromatography strip according to 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 Wherein the chromatographic strip comprises a solid support located below the chromatographic strip. 9. The method of claim 8, Wherein the support is formed of a material selected from the group consisting of nitrocellulose, nylon, PVDF, glass and plastic. The method according to claim 1, Wherein the ligand is a protein, an antigen, an antibody, a DNA, an RNA, a PNA or an extramammary. The method according to 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 method according to claim 1, Characterized in that the target substance has only one site capable of binding to the ligand. The method according to claim 1, Wherein the dynamic range of the target substance in the sample is between 1 ng / ml and 1 mg / ml. A kit comprising the chromatographic strip of any one of claims 1 to 13 further fixed within the 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 or the reference line. 15. The method of claim 14, A diagnostic kit further comprising standard signal data including a color reference chart of test lines and shots for specimens containing different concentrations of target material. 16. The method of claim 15, Wherein a qualitative or semi-quantitative analysis for visually confirming the presence or absence of the signal of the labeling substance from the inspection line and the control line can be performed. 16. The method of claim 15, And the signal presence and intensity of the mark substance from the test line and the control line can be measured and quantitatively analyzed using a medical device including a reader. A method for qualitative or quantitative analysis of a target substance in a sample using the strip for chromatography according to any one of claims 1 to 13, 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 confirming the signal presence and intensity of the labeling substance from the inspection line and the reference line; And And a third step of measuring the amount of the target substance by comparing the signal intensity confirmed from the inspection line and the reference line with the standard signal data, Wherein the standard signal data is obtained by performing the first and second steps, respectively, on the samples containing target substances of various known concentrations. 19. The method of claim 18, Wherein the signal presence and intensity of the labeling substance are confirmed by a densitometer from the inspection line and the control line of the second step.

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