KR101406923B1 - Strip for quantitative immunoassay analysis having herringbone pattern signaling and quantitative immunoassay analysis method using the same - Google Patents

Abstract

The present invention relates to an inspection strip for analyzing quantitative immunochromatography and a method for analyzing the quantitative immunochromatography using the same. Spots which are formed of first captors are arranged on a measuring pad of an inspection strip as a herringbone pattern and the first captors of various concentrations are fixated. Therefore, the present invention facilitates the clear quantitative signaling than a linear captor alignment method of an existing inspection strip for a qualitative analysis. Especially, color formation particles are formed on each spot. The color of many points is formed if the concentration of an analyzing material is high. The color of few points is formed if the concentration of an analyzing material is low. Therefore, the present invention quantifies the concentration of an analyzing material with the unaided eye by counting the number of points directly.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a quantitative immunoassay analysis method using quantitative immunochromatographic assay strips and a quantitative immunoassay analysis method using the same,

The present invention relates to a calibration strip for quantitative immunochromatographic analysis having a captor section in the form of a vertebrate and a quantitative immunochromatographic analysis method using the same.

Lateral flow immunoassay, a representative class of immunochromatography, is a disease detection method that detects an analyte generally through a detector-acceptor response based on a sandwich immunoassay on a porous membrane.

In general, the detectors are labeled with a coloring substance, which is dropped onto the porous membrane together with the analyte, and the capturing agent is present in the form of a linear linear regression in a limited region of the porous membrane.

When the sample with the analyte and the detector mixed moves the trapped porous membrane, the immunocomplex with the analyte and the detector is aligned with the trapping section of the porous membrane and the optical signal by the coloring substance labeled on the detector . In this case, the optical signal appears only in a single linear form, so that only the presence or absence of the analyte can be effectively examined visually.

In case of using separate optical measuring equipment, it is possible to analyze the severity of the disease by analyzing the intensity of the optical signal shown in the capturing compartment, and the concentration of the analyte is quantitatively analyzed. In this case, a complex light source and a light receiving unit are required, Detectors' labeling materials for optical signals also require expensive materials, limiting their application to on-site diagnostic equipment, which is the original purpose of the lateral flow immunoassay.

Therefore, the conventional lateral flow immunoassay technique is used for qualitative analysis rather than quantitative analysis, and when performing quantitative analysis, an additional optical signal analyzer is used instead of visual detection for signal analysis. In this regard, there is an increasing need to develop a lateral flow immunoassay for quantitative analysis that does not require additional optical signal analysis equipment.

Korean Patent Laid-Open Publication No. 2012-0132249 discloses that among the test strips, a plurality of captors capable of binding each target antigen with the test portion of the assay pad are formed in front of the control portion and immobilized on the test portion, Discloses a quantitative flowable lateral flow assay strip capable of quantitatively measuring the concentration of an antigen by counting. However, the patent does not disclose a lateral flow immunoassay kit incorporating a captivator in the form of a vertebrate.

In particular, in the case of the above-mentioned patent, the measurement of the actual antigen concentration takes a method of measuring the intensity of color development of a coloring point composed of a zig-zag form, which is an incomplete method which is incompatible with the quantification method based on the target color- The invention is designed and actualized to quantify the concentration of the antigen through the factor of the coloring point actually through the structure of the captivator in the form of a vertebrate.

An object of the present invention is to provide a test strip for quantitative immunochromatography, which has a captor section in the form of a protruding pattern capable of overcoming the limitations of the conventional immunoassay method and quantitatively analyzing the concentration of the analyte with high sensitivity, And a method for quantitative immunochromatography using the same.

In order to attain the above object, the present invention provides a test pad comprising a sample pad, a conjugate pad, a measurement pad, and an absorbent pad, wherein the measurement pad comprises a test part including a first capturing element and a control part including a second capturing element, And a black spot for quantitative immunochromatographic analysis, characterized in that the spot of the first captor is arranged in a dichromatic pattern.

The protrusive pattern may include 3 to 7 protrusions each having 3 to 9 points.

The first captor in the form of the vermilion pattern can fix a captor with a low concentration of the front protrusive pattern, a medium concentration of the middle protrusive pattern, and a high concentration of the rear protrusive pattern in the direction of the analyte.

The material of the black strip may be a hydrophobic porous film selected from the group consisting of nitrocellulose, nylon, and glass fiber to which the liquid material can migrate.

The test strips may be useful for quantitatively diagnosing diseases selected from the group consisting of osteoarthritis, infectious diseases, endocrine metabolic diseases, autoimmune diseases and tumors.

The method may further include injecting an analytical sample into the sample pad of the assay strip; Moving the analyte to a conjugate pad to form a conjugate with a detector labeled with a chromogenic particle present in the conjugate pad; And analyte-detector conjugate moves to a measurement pad and combines with a first captor arranged in a habitat pattern to develop a colorimetric immunochromatography assay.

The first captor arranged in the order of the vermilion pattern can fix the catcher in the direction of the direction of the analyte at a low concentration, a medium concentration at the middle level, and a high concentration at the backside level.

In the above analysis method, the analytical sample may be blood, joint fluid, lymph fluid, spinal fluid or urine of a patient having or suspected of having a disease selected from the group consisting of osteoarthritis, infectious disease, endocrine metabolic disease, autoimmune disease and tumor. But is not limited thereto.

In the calibration strip for quantitative immunochromatographic analysis according to the present invention, since the spot composed of the first captor is arranged in the form of protruding pattern on the measurement pad and the first capturer of various concentrations is fixed, In particular, it is possible to distinguish the coloring particles at the spot by the visual color, and when the concentration of the analyte is high, many points are developed and the concentration is increased. The concentration of the analyte can be determined visually by directly counting the number of points by using the fact that a small number of points are colored at low temperatures.

FIG. 1 is a perspective view illustrating a calibration strip for quantitative immunochromatography analysis capable of quantitative analysis according to an embodiment of the present invention,
FIG. 2 is a plan view showing a calibration strip for quantitative immunochromatography analysis capable of quantitative analysis according to an embodiment of the present invention,
FIG. 3 is an exemplary view showing a resultant analysis and a validity criterion of a calibration strip for quantitative immunochromatography analysis according to an embodiment of the present invention,
The left side of FIG. 4 is a schematic view of the immobilized form of the first captor, and the right side shows the results of the immunochromatography performing experiment according to the immobilized form of the first captor,
FIG. 5 shows experimental results of immunochromatography performed according to the first captor concentration using a test strip for quantitative immunochromatography analysis according to the present invention,
FIG. 6 is a graph showing the results of immunochromatography performed according to COMP concentration using an assay strip for quantitative immunochromatography analysis according to the present invention.

Hereinafter, the present invention will be described in more detail.

The present invention was devised to overcome the limitations of the existing qualitative analysis-based immunochromatography method. The present invention provides a method of sorting a capturing array in a test section of a measurement pad in a herringbone form in a conventional linear test line, The concentration of the analyte can be quantitatively analyzed with high sensitivity by adjusting the concentration of the captor.

Particularly, when the concentration of the captor fixed to the test part is the same, the color of the anterior captor differs only depending on the concentration of the analyte, and the color of the captor of the posterior part hardly occurs. Therefore, it is possible to control the concentration of the trapped substance to be aligned so as to prevent the binding of the majority of the analyte in the anterior captor and to bind a large amount of the analyte to the captor of the rear part. ) In order to indicate a valid signal.

1, the structure of a calibration strip for quantitative immunochromatographic analysis implemented in the present invention is composed of a sample pad, a conjugate pad, a measurement pad, and an absorbent pad, which are general forms of a lateral flow assay strip.

2, after analyte (1) to be measured is injected into a sample pad, the analyte is moved to a conjugate pad in which a detector (3) labeled with coloring particles (2) exists, To form a complex. As the coloring particles, gold nanoparticles, colored fluorescent beads, and the like are used. At this time, bovine serum albumin (BSA), skim milk, and a blocker (④) such as a polymer are used together to prevent nonspecific binding to the coloring particles. The analyte forming the complex moves to the assay pad and develops color in the inspecting unit (5) including the first captor that specifically binds to the analyte. At this time, the control unit (6) including the second captor for comparison, Performs a specific combination with the detector. In particular, it is possible to arrange the spot of the first capturing member in the form of a dichromatic pattern on the measurement pad, thereby realizing a calibration strip capable of accurate quantitative analysis rather than the qualitative analysis used in the past.

As the material of the black strip for analyzing the quantitative immunochromatography method implemented in the present invention, a hydrophobic porous membrane such as nitrocellulose, nylon, glass fiber and the like through which the liquid material can move can be used. When a liquid substance containing the analyte is dropped on the sample pad, the liquid moves through the medium from the compartment to the conjugate pad due to the effect of pure capillary action without external force. The analyte forms a complex with the detectors labeled with the chromogenic particles on the conjugate pad, and the analyte-detector complex continues to migrate due to the capillary phenomenon and binds to the first capturer aligned in the measuring pad . This is done by sandwich bonding to create a first capture analyte-detector complex. The analyte that became the first captor-analyte-detector complex was no longer moved through the medium by the first captor fixed to the assay pad, but was left on the assay pad and developed by the color- Lt; / RTI >

At this time, the first captor fixed to the black strip was fixed at various concentrations, and the front retina pattern was low in concentration, the middle retina pattern was in the middle concentration, and the rear retina pattern was fixed in a high concentration of the first capturing element do. This results in the majority of the analyte in the liquid material binding when the concentration of the front first captor is high, resulting in fewer analytes to bind at the rear first captor. When the combined analyte becomes smaller, the binding with the first captor at the back is reduced, so that the color development due to the binding is difficult to be visually observed. In addition, the first captor, which is fixed at the back of the black strip, should be combined with a small amount of analyte to obtain a visual color that can be observed with the naked eye. Therefore, a higher concentration first captor is fixed Combine with large quantities of analytes. When analyzing using various concentrations of the first captor fixed on the black strip, it is possible to count the coloring points according to the concentration of the analyte, so that more sophisticated results can be obtained.

In one embodiment, the total number of five spotted spots is 5 (the total number of coloring spots is 25). The concentration of the first trapped spot is fixed from the direction of the analyte to the first spot For example, a medium concentration of 0.8 mg / mL is used for a low concentration, for example, 0.6 mg / mL for a medium concentration, a medium concentration is 0.8 mg / mL for a medium concentration, For example, the concentration of 1 mg / mL can be determined by visual inspection.

On the other hand, an analyte-detector combination or an analyte-unidentified detector that has not been combined with the capturing partner continues to move through the medium to meet with the second capturer to form a second capturing-detector combination, Is observed. The second capturer can grasp the reaction due to errors in the experimental procedure, such as the Hook's effect, as a control with the first captor, and can see if the combination has moved the pads of the black strip without problems. In the case of the spots that are developed in the measurement pad by the combination, if the concentration of the analyte is high, the detector-analyte complex binds to the first captor at a lot of spot, When the concentration is low, a small spot is developed and the number of points to be developed depending on the concentration tendency is changed. This provides a new method of lateral flow assay that allows the amount of analyte to be visually confirmed without a separate external device.

Therefore, in the measurement pad of the assay strip for quantitative immunochromatography according to the present invention, the spot composed of the first captor is arranged in the form of a stripe pattern, Enabling more precise quantization (see FIG. 3).

The quantitative analysis on the conventional linear capturing array calibration strip depends on the difference in sensitivity such as the color development and the light intensity of the developed test unit using an external device, which requires the introduction of additional measurement equipment. In contrast, in the case of the present invention, in which the captors are arranged in a cluster of dots in the shape of a triangle, the coloring particles are colored at each spot, and when the concentration of the analyte is high, many points are developed. The concentration of the analyte can be quantified by the naked eye by directly counting the number of points.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

Example 1 Production of black strips

In order to measure COMP (Cartilage Oligomeric Matrix Protein) used as a diagnostic marker of osteoarthritis as an analyte, the test strip shown in Fig. 1 was prepared as follows.

The black strips were cut using a nitrocellulose (Model: HF09004XSS) manufactured by Millipore, cut into a length of 60 mm and a width of 5 mm. The test section was immobilized with 1.0 mg / mL of 16F12 antibody, which specifically binds to COMP (Cartilage Oligomeric Matrix Protein: cartilage damage marker protein), at 1.5 cm from the end of the strip. spot) in total. At this time, each antibody spot spot aligned with the protruding pattern was formed using a pen tip, and the tip was carried on the nitrocellulose test strip by using the tip after being carried on the antibody solution. The control was prepared by aligning 3 points (1 mg / mL anti-mouse IgG) at 1 cm to the right of the end of the black strip. Gold nanoparticles (AuNP, 30 nm) used as chromogenic particles were labeled with 1 mg / mL 12C4, which serves as an analyte detector. An absorbent pad capable of absorbing liquid was attached to the end of the black strip. Kimberly-Clark's science wiper product was used as the absorption pad. Due to the absorption of the liquid in the absorbent pad, the solution containing the reactant reacted in the test part and the control part, and after reaching the end of the black strip, it could continue to flow without stopping so that the materials could continue to pass without clogging.

<Example 2> Evaluation of black strip

1. Immunochromatographic performance test according to the type of immobilization of the first captor

Using the assay strip prepared in Example 1, 19 μg / mL COMP and AuNP-Ab were linearly reacted in a 1: 1 ratio at room temperature for 5 minutes in a sample pad, 25 μL was added to the conjugate pad, (40 μL) of loading buffer was used as a mobile phase and immunochromatography was performed to confirm the spot coloration.

On the left side of FIG. 4 is a schematic view of the immobilized form, and on the right is an experimental result of immunochromatography according to the immobilized form. In FIG. 4A, more than 10 spots were observed. In B and C, the former point developed color. In addition, D and E showed color at all points, but this appeared to be inadequate for quantitative analysis because all points were developed at low concentrations of COMP. From these experimental results, it was found that the capturing arrangement method of the zebra pattern is the most effective form for quantitative analysis.

2. Immunochromatographic performance test according to the concentration of immobilized first captor

Experiments were performed to determine whether a valid signal was produced according to the concentration of the first captor arranged in the form of protrusions on the black strip. That is, the 16F12 antibody that specifically binds to COMP in the test strip of the test strip was immobilized in a variety of concentrations ranging from 0.6 to 1.0 mg / mL in the form of a rhombus. The assay used was 19 μg / mL COMP. The subsequent process was the same as the previous experiment.

As a result, in the case of (1) and (2) of FIG. In the case of (3), the concentration of immobilized captor was low and all the captors were able to confirm the color. However, even at a low concentration of COMP, the color was confirmed in all fixed captors and no valid signal was obtained. (4), the coloring of the C portion was not confirmed because the concentration of the fixed portion A capturing agent was high and the concentration of the C portion was low. In (5), the concentration of fixed A-captor was low and the fixed concentration of C-captor was high.

Therefore, it was found that the method of immobilizing the captor in a fixed concentration from a low concentration to a high concentration is most suitable.

3. Immunochromatographic performance test according to the concentration of analyte

The number of color spots according to the COMP concentration at various concentrations was measured. In the test section, the 16F12 antibody, which specifically binds to COMP, was administered at a concentration gradient of 0.6 to 1 mg / mL (0.6, 0.8, 0.8, 1, 1 mg / 25 points (spot) were arranged. The assay used was 19 μg / mL COMP. The subsequent process was the same as the previous experiment.

As a result, as shown in FIG. 6, it was found that as the concentration of COMP increases, the number of points to be developed increases proportionally.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It is to be understood that various modifications and changes may be made without departing from the scope of the appended claims.

Claims (8)

Wherein the measurement pad is composed of a sample pad, a conjugate pad, a measurement pad, and an absorption pad, the measurement pad being composed of a check part including a first capturing element and a control part including a second capturing element, Characterized in that it is arranged in a pattern shape. [3] The assay strip for quantitative immunochromatography analysis according to claim 1, wherein the vermilion pattern comprises 3 to 7 patterns of a single vermilion pattern consisting of 3 to 9 points. [3] The method according to claim 1, wherein the first captor of the vermilion pattern has a capturing element fixed at a low concentration in the front protruding pattern, a medium concentration in the middle protruding pattern, and a high concentration in the rear protruding pattern, Black strip for chromatographic analysis. The assay strip of claim 1, wherein the material of the assay strip is a hydrophobic porous membrane selected from the group consisting of nitrocellulose, nylon, and glass fibers to which the liquid material can migrate. [3] The assay strip of claim 1, wherein the assay strip diagnoses a disease selected from the group consisting of osteoarthritis, infectious diseases, endocrine metabolic diseases, autoimmune diseases and tumors. 6. A method for testing a test strip, comprising: injecting an analytical sample into a sample pad of a test strip according to any one of claims 1 to 5;
Moving the analyte to a conjugate pad to form a conjugate with a detector labeled with a chromogenic particle present in the conjugate pad; And
A step of moving to the analytical sample-detector complex measurement pad and combining with a first captor arranged in a pattern of protrusions to develop color
Lt; RTI ID = 0.0 &gt; immunochromatography &lt; / RTI &gt; assay. [3] The method according to claim 1, wherein the first captor aligned with the vermilion pattern has a capturing element fixed at a low concentration in the front protruding pattern, a medium concentration in the middle protruding pattern, and a high concentration in the rear protruding pattern, Immunochromatographic assay method. [Claim 7] The method according to claim 6, wherein the analytical sample is blood or urine of a patient having or suspected of having a disease selected from the group consisting of osteoarthritis, infectious disease, endocrine metabolic disease, autoimmune disease and tumor. Graphical analysis method.

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