KR20160120675A - Rapid Quantitative Diagnostic Kit - Google Patents

Abstract

The present invention relates to a strip or a kit for immunochromatographic analysis which quantifies analysis antigen by measuring the length of a coloring section by a complex consisting of a conjugate of the analysis antigen and a first antibody-marker in a detection region, wherein the detection region included in a signal detection pad of the trip or the kit for immunochromatographic analysis is a section of the predetermined length.

Description

[0002] Rapid Quantitative Diagnostic Kit [

The present invention relates to a strip or kit for immunochromatographic analysis, and more particularly to a strip or kit for immunochromatographic analysis, wherein the detection region contained in the signal detection pad of the strip is a constant length region and the analyte and the first antibody- And measuring the length of the chromogenic region due to the complex composed of the gates to quantify the analyte antigen.

Rapid diagnostic test (DRT) is referred to as various names such as immunochromatographic analysis or rapid kit analysis, and its main components include support, sample pad, binding pad (conjugate pad), signal detection pad and absorption pad Which allows the user to easily detect analytes from biological or chemical samples within 2-30 minutes with 1-100 microliter samples without special techniques or equipment. Immunochromatography is a combination of immunochemistry and chromatographic methods. It is a unique immunological response of an antibody to an antigen, a coloring property and fluidity of a gold particle (colloidal gold), a porous membrane ) Of the capillary phenomenon.

The rapid diagnostic test is a method that can qualitatively and quantitatively inspect the analyte in a short time using the property that the biological substance or the chemical substance attaches to each other in a specific manner. Specifically, the immunochromatography method has advantages such as sample dilution, washing, and color development through reaction of an enzyme conjugate with a substrate, which can be found in a conventional multistage immunoassay, into a single step for rapid inspections. It also has the advantages of being easy and economical to judge the test result without using the specific equipment, and quickness of reading the test result. It is a method mainly to judge that the test result is a positive result when two markers appear in the control area and the sample area. A strip used for such immunochromatography and a diagram showing positive results using it are shown in Fig.

Therefore, the rapid diagnostic test is one of the most advanced analytical methods developed in recent years in terms of simplicity and promptness, and is useful for diagnosing various disease causing substances such as antigens or antibodies of infectious pathogens, cancer factors, and cardiac markers have. Using such immunochromatographic strips or immunochromatographic kits containing the same, samples of human or animal whole blood, plasma, serum, tears, saliva, urine, runny nose, body fluids, And the like. They may also be used to confirm the presence of pregnancy, ovulation, cancer markers, heart disease markers, etc., and may be used to confirm the administration of various drugs.

The diagnostic principle of the conventional rapid diagnosis kit is shown in Fig. In the conventional rapid diagnostic kit, the first antibody reacted with the antigen in the sample moves along the kit and goes to the control line through the detection line . In this process, the antigen in the sample reacted with the first antibody binds to the second antibody at the detection line and develops color. The first unreacted antibody moves to the control line and binds to the third antibody specifically binding to the first antibody . By comparing the color of the detection line and the color of the reference line, it is possible to know whether or not an antigen exists in the sample. Therefore, the rapid diagnostic kits are suitable for qualitative analysis and have some limitations in quantitative detection.

In addition, the rapid diagnostic kit has the advantage of being able to visually confirm the test result in a short time, but does not provide the result of how much the antigen is present, so that the judgment of the disease based on the amount of the antigen present in the past It is not possible to use it. In addition, if the quantity of the antigen is known, it should be confirmed by using expensive equipment which can confirm the quantity of the antigen. Therefore, in order to overcome the limitations of the conventional rapid diagnosis kit, there is a need for a technique capable of quantifying the antigen in the sample using a rapid diagnosis kit without any additional equipment.

The object of the present invention is to provide a strip or kit for immunochromatographic analysis for quantitatively analyzing antigen by measuring the length of a chromogenic segment due to a complex composed of an analyte antigen and a conjugate of a first antibody-markers in a detection region do.

Yet another object of the present invention is to provide a method for quantifying an antigen contained in a sample using a strip or kit for immunochromatographic analysis for quantifying the analytic antigen.

The object of the present invention is to provide a strip or kit for immunochromatographic analysis for quantitatively analyzing antigen by measuring the length of a chromogenic segment due to a complex composed of an analyte antigen and a conjugate of a first antibody-markers in a detection region do.

Yet another object of the present invention is to provide a method for quantifying an antigen contained in a sample using a strip or kit for immunochromatographic analysis for quantifying the analytic antigen.

The rapid diagnostic kit according to the present invention can be used for quantitative measurement of specific antigens in a sample which can not be realized by the conventional rapid diagnostic kit while maintaining the portion of quick and easy inspection which is an advantage of the conventional rapid diagnostic kit, It has the advantage to be implemented.

1 is a schematic diagram of a conventional immunochromatographic assay strip.
FIG. 2 is a view showing a process of qualitative analysis of antigens contained in a sample using a conventionally used strip for immunochromatographic analysis. FIG.
3 is a diagram illustrating a method for determining the amount of a second antibody to be applied to a detection section in accordance with an embodiment of the present invention.
Figure 4 is a diagram of a strip for immunochromatographic analysis according to one embodiment of the invention.
5 is a diagram showing an example of a strip for immunochromatography analysis according to an embodiment of the present invention.
FIG. 6 is a view illustrating a process of analyzing an antigen contained in a sample using a strip for immunochromatography according to an embodiment of the present invention. FIG.
FIG. 7 is a graph showing the results of quantitative analysis of SP-10 protein using gold colloid or HRP-labeled immunochromatographic strip prepared according to the present invention.

The present invention is characterized in that the detection region included in the signal detection pad of the strip for immunochromatographic analysis is a section having a constant length and the length of the color development region due to the complex composed of the analyte antigen and the first antibody- To a strip or kit for immunochromatographic analysis.

The present invention also relates to a method for quantifying an antigen contained in a sample using a strip or kit for immunochromatographic analysis for quantifying the analytic antigen of the present invention.

The strip for immunochromatographic analysis according to the present invention can be used for quantitative measurement of a specific antigen in a sample which can not be realized by a conventional rapid diagnostic kit while maintaining the function of detecting a rapid and easy analytical sample, It can be implemented by itself. That is, the strip for immunochromatographic analysis according to the present invention can be used as a strip for immunochromatographic analysis for quantitative analysis of an antigen in a sample.

A strip for immunochromatographic analysis according to an example of the present invention is shown in FIG. 4, and a strip for immunochromatographic analysis will be described in detail below with reference to the exemplary diagram of FIG. The strip for immunochromatographic analysis according to the present invention comprises a) a test area including a first signal detection pad, a sample pad, a binding pad and an absorption pad and b) a second signal detection pad, a sample pad, A first signal detection pad provided on one side of the first signal detection pad, the first signal detection pad including a detection section for detecting an analytic antigen contained in the liquid sample, A sample pad for containing a sample, a binding pad including a conjugate of a first antibody and a label specifically binding to the antigen, and a binding pad provided on the other side of the first signal detection pad, And an absorbing pad for absorbing the sample, wherein the detecting section of the first signal detecting pad recognizes an epitope that is different from the epitope of the first antibody as an antibody to the analyzing antigen, and the first antibody- A test region in which a second antibody binding to an antigen to which a gate is bound is treated; And b) a binding pad comprising a conjugate of a first antibody and a label specifically binding to an analyte antigen, a third antibody that specifically recognizes the first antibody is treated so that the liquid sample migrates to the absorbent pad A sample pad provided at one side of the second signal detection pad and accommodating a liquid sample, and a sample pad provided at the other side of the second signal detection pad, Wherein the detection section has a length of 20 mm or more and 80 mm or less on the basis of the direction of the absorbent pad on the sample pad, and wherein the detection section includes the analyte antigen and the first antibody- And measuring the length of the chromogenic region due to the complex composed of the conjugate of the sieve.

The strip for immunochromatographic analysis according to an example of the present invention may further include a support under the signal detection pad. The support may be made of a plastic material, and may be manufactured by attaching a strip formed on the support, thereby improving durability and facilitating handling and storage.

The sample pad is a part for injecting the liquid sample to be analyzed, and absorbs the sample and moves it to the bonding pad by the capillary phenomenon. In the immunochromatographic strip, a liquid sample may be added to a single or a plurality of injection sections, and then a buffer may be added or a mixture of a liquid sample and a buffer may be introduced. The sample pad is located above the bonding pad and flows from the upper sample pad to the lower bonding pad through the porous material portion that does not include the conjugate when the sample is supplied. In the binding pad, a conjugate in which a substance to be detected in a sample and an antibody to a substance to be detected are bound with a labeling substance forms a complex (aggregate). For example, when the substance to be detected is SP-10 protein in the sperm, a monoclonal antibody-immobilized gold colloid particle conjugate to SP-10 binds to SP-10 to form a complex (aggregate). Thereafter, the sample is developed into first and second signal detection pads arranged in contact with the lower portion of the bonding pad.

The binding pad is the site where the first antibody-labeled conjugate is located. The sample transferred to the binding pad is selectively bound by the first antibody-labeled conjugate and the first antibody-labeled conjugate, for example the gold-conjugated first antibody, forms a complex with the antigen. The bonding pad used in the present invention is made of a porous material on a pad capable of expanding the sample and maintaining the conjugate, and has a sample pad and a bonding pad in a line on a part thereof. In addition, a porous material portion that does not include a conjugate exists between the sample pad and the bonding pad, and the sample supplied to the sample pad preferably extends to the bonding pad by expanding the porous material. The material of the bonding pad may be glass fiber, polyethylene fiber, or the like. The bonding pad is a site containing a conjugate and is formed in a line on the porous material on the downstream side of the sample pad.

The absorbent pad may be a pad capable of promoting the movement of the liquid sample, and may be made of, for example, pulp, cellulose, cotton fiber, or the like. And serves as a reservoir for the sample flowing through the nitrocellulose membrane. In the immunochromatographic strip of the present invention, it is preferable to provide an absorbent pad at one end of the lower part of the signal detection pad or the control line. For example, filter paper can be used. Suitably Wattmann, or 740-E is used.

The first or second signal detection pad may include at least one detection period, and may be formed of a porous membrane pad, and may be made of nitrocellulose, cellulose, polyethylene, polyethersulfone, nylon or the like.

A second antibody recognizing an epitope that is different from the epitope of the first antibody as an antibody to the analyte antigen is treated in the detection period of the first signal detection pad and the control line of the second signal detection pad is provided with a specific Lt; RTI ID = 0.0 > 3 < / RTI >

The first antibody is an antibody that specifically recognizes an antigen in a sample to be analyzed and binds to a label, for example, gold particles to form a conjugate and is located on the binding pad. When the analyte in the sample is present, the analyte binds to the first antibody-label conjugate to form a complex, and reacts with the second antibody located in the detection region to produce a color reaction. The amount of antigen in the sample is proportional to the degree of color development. When the antigen in the sample is not present, the first antibody-labeled conjugate can not bind to the second antibody located in the detection region, so that it can not develop color in the detection region.

The label binds to the first antibody to form a conjugate and is located in the binding pad. The analyte and the conjugate supplied through the sample pad form a complex, and the second antibody and the analyte antigen , A coloring shape is generated.

The label may be selected from a variety of reagents capable of exhibiting color development in an antigen-antibody reaction, such as gold colloid or horse radish peroxidase, without limitation.

The particle size of the label is preferably a label having an appropriate particle size according to the label to be used. For example, when gold colloid particles are used as markers, the particle size thereof is preferably from 20 to 60 nm, particularly preferably from 45 to 55 nm. The above-mentioned gold colloid particles can be produced by a generally known method, for example, by dropwise adding a heated aqueous solution of tetrachloro gold (III) acid to an aqueous solution of trisodium citrate.

A method for producing a gold colloid-labeled antibody having the first antibody bound to a gold colloid is not particularly limited, including a known method. For example, a gold colloid solution adjusted to pH 9.0 with 0.2 M potassium carbonate solution is added to 2 mM boric acid buffer 9.0), added with a solution of the monoclonal antibody dissolved therein, reacted at room temperature for 30 minutes, added with 10% BSA solution, reacted again for 15 minutes and centrifuged. The gold colloid labeled antibody carrier may be prepared by coating the prepared gold colloid labeled antibody on a conjugate pad made of, for example, glass wool, followed by drying.

The second antibody is an antibody that binds to an epitope that is different from the epitope of the first antibody, and provides information to quantify the antigen in the sample by binding to the antigen bound to the first antibody-labeled conjugate.

The third antibody has the property of being able to bind to the first antibody and always binds with the first antibody-labeled conjugate at the time of sample analysis to provide information that the assay kit is operating normally.

The first antibody and the second antibody according to the present invention are preferably monoclonal antibodies. By using a monoclonal antibody, the specificity of the reaction can be increased. For example, if the substance to be detected is SP-10, which is known as a representative indicator for determining the concentration of sperm in anatomical, genetic and biochemical analyzes, the second antibody immobilized on the label and detection region is SP-10 Any antibody that can be detected can be used, but an anti-SP-10 monoclonal antibody is preferred.

In addition to the whole molecule of the antibody, a functional fragment of an antibody having an antigen-antibody reaction activity is also included. Examples of the functional fragment of the antibody include those obtained using an immunological process for an animal, as well as those obtained using a gene recombination technique, and chimeric antibodies. Examples of the functional fragment of the antibody include F (ab ') 2, Fab and the like. These functional fragments can be produced by treating the antibody with a protease (for example, pepsin or papain). In the present invention, it is particularly preferable to use F (ab ') 2 as an antibody immobilized on a label. By using F (ab ') 2, which is a functional fragmentation antibody, on the label, for example, the size of the conjugate to which the antibody binds to the label can be made small, and the spreadability in the binding pad and the signal detection pad is excellent . In addition, depending on the substance to be detected, the specificity of the reaction can be enhanced by using the functional fragmentation antibody.

Methods for placing the second antibody and the third antibody on the first and second signal detection pads include direct or indirect immobilization by physical or chemical means. Direct immobilization is preferred in view of ease of sensitivity control. As a method of directly immobilizing, physical adsorption may be used or a covalent bond may be used. In general, when the material of the signal detection pad is a nitrocellulose membrane or a mixed nitrocellulose ester membrane, physical adsorption can be performed. In the case of covalent bonding, the material of the signal detection pad is activated by activation with cyanogen bromide, glutaraldehyde, carbodiimide or the like. The first signal detection pad and the second antibody, or the second signal detection pad and the third antibody may be adsorbed or bound by, for example, a microsyringe, a pen with a regulating pump, ink jet printing, or the like.

The amount of the second antibody to be applied to the first signal detection pad is determined by the quantitative section of the antigen. Prepare a sample composed of various concentrations of the antigen in advance, apply the second antibody at a specific concentration, The amount of the second antibody to be applied to the signal detection pad can be determined by adjusting the concentration of the second antibody. If the color of the prepared antigen is darkened without any difference according to the concentration of the antigen, the amount of the second antibody is decreased. On the contrary, if the coloration becomes soft and the concentration range is difficult to confirm, the amount of the second antibody is increased.

For example, when the analyte antigen is SP-10, which is a representative protein in sperm, the correlation between the number of spermatids and the amount of SP-10 is determined. Then, SP-10 at various concentrations is sampled . The concentration of SP-10 corresponding to the number of spermatozoa (15 million, WHO) which is a normal / abnormal standard is applied by applying the anti-SP-10 antibody to be used as the second antibody at a specific concentration and applying various SP- Or the amount of the second antibody is adjusted after confirming that the color is properly formed.

A method by which the concentration of the second antibody can be determined is shown in Fig. In FIG. 3, if the amount of the second antibody is reduced as shown in Case 1, and the amount of the second antibody is increased as shown in Case 2, the resultant coloring range is adjusted as shown in FIG. It is preferable that the normal / abnormal reference point is located in the middle of the color development range.

The amount of the third antibody recognizes the first antibody-labeled conjugate regardless of the amount of the antigen, and determines the conditions under which the color of the second signal detecting pad may appear.

The detection interval is a position where the second antibody is coated and fixed long without margins. When the conjugate complex of the analyte antigen and the first antibody-labeled antibody formed on the first binding pad passes the detection interval by the capillary phenomenon, The antibody binds by the double antibody sandwich immunoassay principle, and red color is developed by the gold. When the amount of the antigen in the sample is small, the coloring length is short because the amount trapped by the second antibody is small, whereas when the amount of the antigen is large, the amount of the antibody captured by the second antibody is large and the coloring length becomes long.

The detection section is a section having a length of 20 mm or more and 80 mm or less in the direction of the absorption pad direction, that is, the direction in which the sample moves, in the sample pad.

However, in the conventional immunochromatographic assay strip, the detection line is present and has a length of about 2 to 3 mm in the direction of the absorption pad in the sample pad, and the complex composed of the analyte antigen and the first antibody- It was confirmed that the analyte in the sample was present due to color development due to the presence of the analyte and the analyte was not present in the sample because the complex composed of the analyte antigen and the conjugate of the first antibody- And provides a qualitative analysis result that detects only the presence or absence of the analyte in the sample. The detection results using such a conventional diagnostic kit are shown in Fig. In many cases of diagnosis, it is difficult to judge normal / abnormal with the presence or absence of antigen. When the amount of antigen does not reach a certain level, it is often abnormal. Conventional immunochromatographic strips have limitations in application because the amount of antigen can not be quantified in this case. For example, in the case of the sperm count diagnostic strip, it is possible to distinguish between a state in which the number of spermatozoa is equal to or higher than the normal sperm count (15,000,000 / ml, WHO) Is also possible.

Due to the conjugation of the analyte and the first antibody-labeling agent within the detection interval, a coloring interval is formed within the detection interval. First, the normal / abnormal level of the antigen to be analyzed is checked through WHO data, etc., and then the amount of the second antibody is determined to determine the length of the coloring region according to the concentration of the analyte, The method comprising: applying a liquid sample containing the analytic antigen to a sample pad of the immunochromatographic strip; measuring a length of a color development interval of the immunochromatographic strip; The concentration of the analyte in the sample can be determined through the length of the color development interval.

In the example of the present invention, in the immunochromatographic strip, since the color development period according to the concentration of the antigen was confirmed in the process of determining the amount of the second antibody, the scale indicating the concentration or amount of the analysis antigen according to the length of color development in the detection period And a concentration of the analyte can be determined by applying a liquid sample containing the analyte to the sample pad of the immunochromatographic strip and reading the scale of the coloring interval. An example of an immunochromatographic strip showing the scale of the color development period is shown in Fig. The normal / abnormal judgment of the quantitative diagnostic kit can be judged based on whether or not color is developed beyond the normal reference line. Quantitative items can be applied or extended in mg / ml, number of cells / ml, and the like. When the analyte antigen is the protein SP-10 in the sperm, the amount of SP-10 quantified through the color development period can be converted into the number of sperm after confirming the correlation between the number of sperm and SP-10.

In the present invention, the control line of the second signal detection pad is a site where the third antibody is located, and the first antibody-labeled conjugate continues to flow toward the absorbent pad regardless of whether the antigen is bound or not, and the third antibody Combined, color appears on the contrast line.

The analyte antigen according to the present invention refers to a substance to be detected, and examples of the substance to be detected include viruses and physiologically active substances such as proteins that can be generally measured using an antigen-antibody reaction.

Examples of the virus include influenza viruses such as influenza A virus and influenza B virus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus. Examples of the virus include, for example, Representative proteins SP-10, human hemoglobin, human albumin, hepatitis B virus antibody, hepatitis C virus antibody, and human immunodeficiency virus antibody. Among them, SP-10, a representative protein in sperm, is preferably used as the substance to be detected.

Examples of the sample containing the analyte in the present invention include substances derived mainly from living organisms such as body fluids and extracts obtained by extracting the substances to be detected from them. Specific examples of substances derived from living organisms (biologicals) include nasal fluid, runny nasal aspirate, sputum or swab samples collected from blood, urine, stool, nostrils, nasal cavity, pharynx, nasopharynx, , Semen, and the like. Among them, semen is preferable as a sample only when the substance to be detected is SP-10, which is a protein in sperm, and it is preferable that the semen which has been liquefied by allowing to stand for 20 minutes after ejaculation is dissolved by using a lysis solution and used as a sample desirable. The above-described living body (biological) derived material or its extract can be used as a sample as it is, or can be diluted with a diluent as appropriate to form a sample. In addition, appropriately filtered or dissolved may be used as a sample.

According to still another aspect of the present invention, there is provided an immunochromatographic kit comprising a lower housing for housing an immunochromatographic strip and an upper housing complementary to the lower housing, Wherein the strip is one of the immunochromatographic strips according to all the aspects of the present invention enumerated above.

The method for producing the test strip for immunochromatography is not particularly limited, but a conjugate pad is prepared by applying a conjugate solution in a line form to a part of a porous material (a portion where a conjugate part is located) on a binding pad and drying, A test strip for immunochromatographic analysis can be prepared by contacting the binding pad and the signal detection pad. According to one example of the present invention, the method for producing a test strip for immunochromatography of the present invention comprises a step of immersing a part of a porous material on a pad as a binding pad, with a conjugate containing a conjugate of the first antibody and the labeling substance to the analyte antigen Applying a solution and drying to prepare a bonding pad; Producing a first signal detecting pad including at least one detection section in which the second antibody with respect to the analyte antigen is located; Producing a second signal detection pad including a reference line on which the third antibody is located; A step of laminating a sample pad, a bonding pad, and a first signal detection pad to manufacture an inspection region; And a step of laminating a sample pad, a binding pad and a second signal detecting pad to produce a check area.

In another example of the present invention, a method for quantifying an antigen contained in a sample using a strip or kit for immunochromatographic analysis for quantitating the analytic antigen. Specifically, the quantification method may include the following steps.

a) applying a liquid sample comprising the analyte antigen to a sample pad of the immunochromatographic strip;

b) measuring the length of the color development interval of the immunochromatographic strip; And

c) determining the concentration of the analyte in the sample according to the measured length of the color development interval.

In this method, the normal range of analytic antigens follows the criteria suggested by WHO and sets the concentration interval of analytic antigens containing the normal range. The amount of the second antibody is determined to determine the length of the color development interval according to the concentration of the assay antigen. The length of the color development interval is determined by the length of the color development depending on the concentration determined in the process of determining the amount of the second antibody To determine the concentration of the analyte antigen in the sample.

A quantitative analysis method of an analyte according to an example of the present invention is shown in Fig.

Example

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

One. Example  1: Quantitative diagnosis using gold colloid-labeled antibody

Immunochromatographic strips were prepared according to the following method for quantitative diagnosis using gold colloid labeled antibodies.

First, a chicken immune antibody library for SP-10 was prepared using phage display method.

Specifically, mRNA was extracted from the spleen and bone marrow of a chicken immunized with SP-10 and confirmed to have antibodies to SP-10, and cDNA was prepared by RT-PCR, and then the antibody gene was specifically amplified The primers were used to amplify the antibody gene by PCR. The amplified antibody gene was cloned into a phagemid vector, and the phage was amplified by E. coli transformation to prepare a phage display library for SP-10 having a complexity of 10 ⁹ or more.

Biopanning of SP-10 was performed using the phage display library. Biopanning was performed by screening for positive clones binding to SP-10 by immobilizing SP-10 in magnetic beads and reacting the phage display library. Biopanning was carried out up to the third step. The number of washing was three times in the first step and five times in the second and third step. Clones obtained by biopanning were identified by direct ELISA for SP-10, and clones with high binding strength were selected.

The whole antibody (full IgG) was prepared by cloning the variable region of the selected antibody with the constant region and cloned into an animal cell expression vector. The plasmid containing clone gene was transiently expressed in animal cells (HEK293F) to produce antibodies and purified through affinity column. The responses of purified whole antibodies to SP-10 were analyzed by sandwich ELISA and antibodies 1C and 9E-11F binding to different sites of SP-10 were selected.

Then, 1C of the prepared monoclonal antibody was diluted to 0.1 mg / ml with 2 mM boric acid buffer (pH 7.4), 0.025 ml of the antibody diluted in 1 ml of the gold colloid solution was added, and the reaction was carried out at room temperature for 30 minutes Colloid was labeled. Furthermore, monoclonal antibodies 9E-11F was diluted to a concentration of 0.01 mg / ml using 0.05 M carbonate buffer, and 60 μl of diluted 9E-11F was added to a nitrocellulose membrane (3 mm × 85 mm) For a period of time.

after, Human recombinant SP-10 was diluted stepwise from 3 mg / ml with PBS, and 100 μl of 1C labeled with gold colloid and 100 μl of human recombinant SP-10 solution prepared by concentration were mixed. Thereafter, the end of the nitrocellulose membrane to which 9E-11F was fixed was immersed in a mixture solution of 1C and SP-10 labeled with gold colloid and developed for 30 minutes to confirm the color development length depending on the concentration. The result is shown in FIG. As can be seen from Fig. 7, it was confirmed that the color-developing range of the gold colloid widened as the concentration of SP-10 increased.

2. Example  2: Horse radish Peroxide ( HRP ) Quantitative diagnosis using antibodies labeled

Immunochromatographic strips were prepared by the following method for quantitative diagnosis using horse radish peroxide labeled antibody.

First, monoclonal antibodies 9E-11F and 1C binding to different sites of SP-10 prepared in the same manner as in Example 1 were prepared . Then, 1 ml of 1C 0.1 mg / ml solution was labeled with peroxidase labeling kit-NH ₂ (Dojindo, Japan) according to the manufacturer's instructions. Further, monoclonal antibody 9E-11F was diluted to a concentration of 0.01 mg / ml with 0.05 M carbonate buffer, and 60 μl of diluted 9E-11F was added to a nitrocellulose membrane (3 mm × 85 mm) at a length of 50 mm And allowed to stand at 37 DEG C for 1 hour to fix. Human recombinant SP-10 was diluted stepwise from 3 mg / ml with PBS, and 100 μl of 1C labeled with HRP and 100 μl of human recombinant SP-10 solution prepared by concentration were mixed. After that, the end of the nitrocellulose membrane fixed with 9E-11F was immersed in a mixture solution of HRP labeled 1C and SP-10 and developed for 30 minutes. The nitrocellulose membrane was dried and then reacted with the TMB solution for 5 minutes, The color development length was confirmed and the results are shown in Fig. As can be seen from Fig. 7, it was confirmed that the color range of the horseradish peroxidase was broadened as the concentration of SP-10 was increased.

Claims (13)

a) a first signal detection pad including a detection section for detecting an analytic antigen included in a liquid sample, a sample pad provided at one side of the first signal detection pad for receiving the liquid sample, A binding pad including a conjugate of a first antibody and a labeling substance binding to each other, and an absorption pad disposed on the other side of the first signal detection pad for absorbing a liquid sample whose signal detection reaction has been completed,
Wherein the detection section of the first signal detection pad recognizes an epitope that is different from the epitope of the first antibody as an antibody to the analyte antigen and that the second antibody that binds to the antigen bound to the first antibody- domain; And
b) a binding pad comprising a conjugate of a first antibody and a label specifically binding to an analyte antigen, and a third antibody specifically recognized by the first antibody are treated to determine whether the liquid sample has migrated to the absorbent pad A sample pad provided on one side of the second signal detection pad and containing a liquid sample, and a sample pad provided on the other side of the second signal detection pad, And an absorbent pad for absorbing the absorbent pad,
The detection interval has a length of 20 mm or more on the basis of the absorption pad direction on the sample pad, and the length of the chromogenic section due to the complex composed of the analyte antigen and the first antibody-labeled conjugate in the detection interval is measured, Wherein the strip is for immunochromatographic analysis.
2. The method according to claim 1, wherein the concentration of the analyte contained in the liquid sample is proportional to the length of the coloring interval, thereby measuring the unknown antigen concentration in the sample. By measuring the length of the coloring interval according to the concentration of the analyte antigen, Lt; RTI ID = 0.0 > immunochromatographic < / RTI >
The strip for immunochromatography according to claim 1, wherein the concentration of the second antibody to be treated in the detection section is adjusted to adjust the degree of color development and the length of the color development section.
4. The method according to claim 3, wherein the specific concentration range of the analyte antigen is determined, the amount of the second antibody is varied to determine the length of the color development interval according to the concentration of the analyte antigen,
Wherein the specific concentration range of the analyte antigen is set so that the color development interval is included within the detection interval.
2. The strip for immunochromatographic assay according to claim 1, wherein the label is a reagent which shows color development using an antigen-antibody reaction.
The strip for immunochromatographic analysis according to claim 1, wherein the label is gold colloid or horseradish peroxidase.
The strip for immunochromatographic analysis according to claim 1, wherein the length of the chromogenic section is read by visual, optical, electrochemical, or electrical conductivity.
2. The strip for immunochromatographic analysis according to claim 1, wherein a scale for indicating the amount of analytic antigen is displayed in the detection section of the first or second signal detection pad.
The strip for immunochromatographic analysis according to claim 1, wherein the strip for immunochromatographic analysis is a strip for immunochromatography analysis, which is selected from the group consisting of hepatitis C, hepatitis A, hepatitis B, influenza, avian flu, rotavirus, AIDS, syphilis, chlamydia, malaria, Antibody, Albumin, Pregnancy, Ovulation, Diabetes Marker, Enzyme, Cancer Marker, Heart Disease Indicator, C Reaction Protein, Metabolite, Antibody, Dandruff, Dandruff, Leprosy, The detection of proteins in lipids, carbohydrates, proteins, drug dosages, lysine, anthrax, brucella, botulinum, Vexinia, Salmonella, cholera, Staphylococcal enterotoxin B, Tularemia, Salmonella, Vibrio campylobacterium, enterohemorrhagic Escherichia coli, For immunochromatographic analysis.
1. An immunochromatographic kit comprising a lower housing for storing a strip for immunochromatographic analysis and an upper housing complementary to the lower housing, wherein the immunochromatographic strip contained in the kit comprises an immunochromatographic strip according to any one of claims 1 to 9, ≪ / RTI > wherein the immunochromatographic strip is an immunochromatographic strip according to any one of claims 1 -
Providing an immunochromatographic strip according to any one of claims 1 to 9
Applying a liquid sample containing the analytic antigen to a sample pad of the immunochromatographic strip,
The length of the color development period of the immunochromatographic strip was measured
And determining the concentration of the analyte in the sample according to the measured length of the color development interval.
The method according to claim 11, wherein the detection interval of the immunochromatographic strip is determined by determining a specific concentration range of the analyte antigen, changing the amount of the second antibody to determine the length of the coloration interval according to the concentration of the analyte antigen, Wherein the specific concentration range of the analyte antigen is set so that the color development section is included in the detection section.
13. The quantitative analysis method according to claim 12, wherein the concentration or amount of analytic antigen is determined according to the length of the color development period indicated in the detection section.

Images