KR101270512B1 - Lateral flow assay strip for quantitative analysis - Google Patents

Abstract

The present invention relates to a lateral flow assay strip capable of quantitative analysis, and more particularly, it is possible to quantitatively measure antigens in a sample without a separate analytical device and additional measurement process, so that the presence or absence of a disease can be determined immediately in the field. To a lateral flow assay strip that may be.
To this end, the present invention is configured such that the sample pad, the conjugate pad, the measurement pad, and the absorbent pad are arranged laterally on the base member and contact between adjacent pads.
The inspection unit of the measurement pad is formed by immobilizing a plurality of capturers, each of which is capable of binding to the target antigen, in front of the control unit, and quantitative concentration measurement of the antigen is possible by counting the colored traps among the capture units of the inspection unit. A lateral flow assay strip capable of quantitative analysis is provided.

Description

Lateral flow assay strip for quantitative analysis

The present invention relates to a lateral flow assay strip capable of quantitative analysis, and more particularly, it is possible to quantitatively measure antigens in a sample without a separate analytical device and additional measurement process, so that the presence or absence of a disease can be determined immediately in the field. To a lateral flow assay strip that may be.

A lateral flow analysis device based on immunochromatography is a diagnostic method that can be used to determine whether a pregnancy or a disease has occurred on a membrane using an antigen-antibody reaction. It is measured through gold nanoparticles or colored polystyrene beads in porous thin films in which phenomena can occur.

The lateral flow analysis kit using the immunochromatography method according to the prior art checks the presence or absence of antigen contained in a sample such as blood or urine through an inspection portion of a solid-state measurement pad, that is, a trapper portion capable of binding to an antigen. Through this, it was confirmed whether pregnant or infected with the disease. In this case, only a single line of the trapping part that can bind to the antigen can be checked for the presence or absence of the antigen. Quantitative analysis had an impossible problem.

Although a device or method for measuring the concentration of an antigen using a fluorescence coloration or a scanner has been disclosed and published by using a fluorescence coloration or a scanner using a different signal size of color development depending on the concentration of the antigen in the sample, As the methods require separate measurement equipment and additional diagnostic procedures for quantitative determination of antigen concentration, the diagnostic process is complicated and the convenience is reduced. Also, expert knowledge is required, which results in subjective analysis of the diagnosis result of the analyst. Due to this, there is always a problem that the error is lowered reliability.

The present invention has been invented to solve the above problems, by forming an inspection unit of the measurement pad as a plurality of traps, by measuring the antigen concentration through the count of the number of trapped color, separate analysis equipment and The aim is to provide a lateral flow assay strip capable of quantitative analysis that can readily determine the presence or absence of disease by measuring antigen concentrations easily without additional measurement.

In order to achieve the above object, the present invention is that the sample pad, the conjugate pad, the measurement pad, the absorbent pad on the base member is configured to be laterally arranged and to contact between adjacent pads,

The inspection unit of the measurement pad is formed by immobilizing a plurality of capturers, each of which is capable of binding to the target antigen, in front of the control unit, and quantitative concentration measurement of the antigen is possible by counting the colored traps among the capture units of the inspection unit. A lateral flow assay strip capable of quantitative analysis is provided.

Preferably, each trap of the inspection unit is characterized in that formed in a spot (spot) or line (line) shape.

Specifically, the inspection unit is characterized in that the spot-shaped trap is formed by aligning in the form of a plurality of lines arranged in the spaced apart along the longitudinal direction of the measuring pad, in particular the spot-type trap arranged in the rear spot-type capture Spaced apart on both sides of the ruler to increase the contact of the first composite to the capturer.

Alternatively, the inspection unit is characterized in that the linear trap is formed by being arranged side by side along the longitudinal direction of the measuring pad.

By using the lateral flow assay strip according to the present invention, it is possible to quickly and easily diagnose the infection of various diseases, as well as to overcome the measurement limitations of the conventional lateral flow assay device, which was able to determine whether or not the pregnancy was infected. By directly measuring the concentration of antigen in the sample without additional optical analysis equipment and additional experimental or diagnostic procedures, it is possible to immediately determine the severity of the disease in the field.

1 is a perspective view showing a lateral flow assay strip capable of quantitative analysis according to an embodiment of the present invention
Figure 2 is a plan view showing a lateral flow assay strip capable of quantitative analysis according to an embodiment of the present invention
Figure 3 is a plan view showing a lateral flow assay strip capable of quantitative analysis according to another embodiment of the present invention
4 is an exemplary view showing a result analysis and validity criterion of a lateral flow assay strip according to an embodiment of the present invention.
5 is a diagram showing signal color development points according to COMP concentration in an experimental example using a lateral flow assay strip according to the present invention;

The present invention relates to a lateral flow assay strip capable of quantitative analysis of a target antigen in a lateral flow assay using immunochromatography. The concentration of the antigen contained in the sample can be measured easily and simply through the count of spots or lines, thereby immediately determining the severity of the disease in the field.

That is, the present invention provides a lateral flow assay strip capable of quantitative analysis of a target antigen by counting signal development points that increase with the concentration of the antigen by an alignment method of capture antibodies constituting the test unit.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

In general, the lateral flow assay strip is composed of about 4 to 6 pads according to the sample to be measured (whole blood, plasma, urine, etc.) and the measurement method, and the present invention is described as an example by applying the most common lateral flow assay strip. Do it.

As shown in FIG. 1, the assay strip according to an embodiment of the present invention has a sample pad 1, a conjugate pad 2, a measurement pad 3, an absorbent pad 4 on a base member 5. Are arranged in a sequential order in a lateral direction, and have a structure in contact between adjacent pads (1, 2, 3, 4), preferably a structure in which some sections are overlapped (or stacked) between adjacent pads. Have

In immunochromatographic analysis, gold nanoparticles or colored polystyrene beads are used as the chromogenic particles (8) for signaling through the colorization of the test trap (6). And gold nanoparticles that are simple to combine and easy to visually identify. The test sample uses a target analyte, that is, a liquid biological sample (whole blood, plasma, urine, synovial fluid, etc.) containing a target antigen and the like.

The sample pad 1 is made of a pad capable of diffusion flow, i.e., a material having a porosity sufficient to contain and contain a sample to be analyzed, and at one end of the base member 5 made of a thin film of plastic. (Left end in this embodiment).

The sample pad 1 may be made of a porous absorbent material, which may include fibrous paper, microporous membranes of cellulose materials such as paper, cellulose derivatives such as cellulose, cellulose acetate, nitrocellulose, glass fibers, Naturally occurring cotton, fabrics such as nylon or porous gels, and the like, but are not limited thereto.

The conjugate pad 2 is configured separately from the sample pad 1, and the left end portion is disposed in contact with the right end portion of the sample pad 1. The conjugate pad 2 also has a number of pores to receive the blood diffused from the sample pad 1.

In addition, the conjugate pad 2 is included in a form in which the conjugates 8 to 10 are coupled to each other so as to diffuse together with the movement of the sample.

The conjugate comprises a detector 9 (detection antibody) and a chromophoric particle 8 which are specifically attached to a target antigen, wherein the detector 9 and the chromophoric particle 8 are oriented in the conjugate. Are bound together in conjugated form.

Accordingly, the first complex is formed by binding the antigen and the conjugate present in the sample in the conjugate pad 2 to each other by the detector 9.

As shown in FIG. 2, a blocking agent 10 is attached to the colored particles 8 in a form filled in the space between the detector 9 and the colored particles 8.

The blocker 10 prevents unwanted non-target substances other than the target antigen from adhering to the chromophoric particles. As the blocker, BSA (bovine serum albumin), skim milk, polymer, or the like may be used. Can be.

This causes the chromogenic particles 8 to accumulate in proportion to the amount of the target antigen in the sample in the measurement pad 3 so that a chromogenic signal is generated, and the detector 9 contained in the conjugate specifically binds to the target antigen. This is chosen to be possible.

The measurement pads 3 are arranged such that their left ends are in contact with the right ends of the conjugate pads 2 so as to receive a blood sample diffused and moved from the conjugate pads 2.

The measurement pad 3 is a pad made of a porous thin film capable of capillary action. The capture pad 6 (capture antibody) that is specifically attached to and bound to a target antigen in a sample is immobilized (flow of sample). And a control part immobilized with a catcher 7 (an antibody that specifically binds to the detector) that is specifically bound to the detector 9 of the conjugate. Formed.

The detector 9 of the conjugate pad 2 moves with the flow of the sample, whereas the capturers 6, 7 (the catcher of the test and control portions) of the measurement pad 3 move with the flow of the sample. It is fixed to an inspection part or a control part without doing so.

In this embodiment, the control part is formed at the right rear end of the measurement pad 3, and the inspection part is formed over a part of the measurement pad 3 at the left side of the control part.

In detail, the inspection part of the measurement pad 3 is formed by arranging a plurality of capturers 6 fixed in a spot shape in a predetermined section in front of the control part of the measurement pad 3. By counting the colored traps in the trap 6 of the inspection unit, it is possible to measure the quantitative concentration of the antigen.

In other words, the catcher 6 (or the first catcher) of the inspection part fixed to the measuring pad 3 is located at each spot arranged over a predetermined interval in front of the control part of the measuring pad 3. The antigen in the sample transferred from the conjugate pad 2 is selectively bound to the first trap 6 of the test part while being moved laterally along the measurement pad 3 by capillary flow. .

That is, since the sample contains the first complex formed by binding the antigen contained in the conjugate pad with the conjugated particles 8 through the antigen-antibody reaction in the conjugate pad 2, the conjugate pad 2 The formed first complex may be combined with the first capturer 6 in the inspection unit to form a second complex by a sandwich assay principle, and thus the first complex is fixed as the second complex is fixed to the inspection unit. The first capturer 6 bound to the target antigen is allowed to develop through the chromogenic particles 8.

As a result, the assay strip of the present invention colors and signals the first capturer 6 of the test unit by using the colored particles 8 of the conjugate attached to the first capturer 6, thus depending on the antigen concentration. Counting is possible through visual identification of colored spots, i.e. signal spots.

Preferably, the inspection unit arranges the spot-shaped first traps 6 spaced apart in the longitudinal direction of the measuring pad 3 in the form of lines in a plurality of rows to facilitate immediate concentration measurement in the field.

That is, the inspection unit forms a single capture line by arranging a plurality of first traps 6 in a line along the width direction of the measurement pad 3, and the capture line includes a plurality of rows to measure the measurement pad 3. It is formed in a shape arranged spaced along the longitudinal direction of the.

The first complex flowing out of the conjugate pad 2 travels along the measurement pad 3 and first meets the first capture line of the test section to develop the first capturer 6 and then depending on the concentration of antigen in the sample. Develop a heat capture line.

For example, if the concentration of antigen in the sample is relatively low, the first complex in the sample will bind to the first capture line 6 of the first capture line, and thus cannot bind to the second capture line. Therefore, only the first capture line will be developed. When the concentration of the antigen is relatively high, the first capture line of the test part is developed, and the remaining first complex is sequentially developed in the next row of capture lines.

It is easier to measure the concentration of the antigen in the sample through the count of such colored capture lines, ie signal developing lines or color spots.

More preferably, the capture lines are alternately arranged up and down along the width direction of the measuring pad 3 and are arranged in a zigzag fashion as shown in FIG. 2.

The first composite moved to the measuring pad 3 is resisted by the first trap 6 fixed to the measuring pad 3 when moved by the capillary flow force, and thus flows to the measuring pad 3. The first composite is divided into left and right sides of the capturer 6 by receiving the resistance of the capturer 6 arranged in the relatively front row, and accordingly, contactability with the capturer 6 arranged in the next row is maintained. It can be reduced, and the possibility of equal contact with all the trappers 6 of the inspection section is reduced, making it difficult to obtain an accurate color signal according to the concentration of the antigen.

Therefore, by configuring the inspection unit in a zigzag form in which each capture line arranged along the longitudinal direction of the measurement pad 3 is alternately arranged up and down along the width direction of the measurement pad 3, the side of the measurement pad 3 is sided. The resistance received by the first composite flowing in the direction can be reduced and contact with all the trappers 6 of the inspection unit, so as to increase the contact of the first composite to the first trap (6).

In other words, in the present embodiment, the spot-type traps 6 arranged in the rear row at the inspection portion of the measurement pad 3 are arranged to be spaced apart to the left and right sides of the spot-type traps 6 arranged in the front row (of the measurement pads). It is arranged to the left and right sides on the basis of the width direction) to improve the contact between the first trap (6) and the first composite.

On the other hand, as another embodiment of the present invention, as shown in Figure 3, the test unit may be made of a line-shaped capturer (6-1) capable of binding to the target antigen.

The inspection unit is formed by arranging the linear traps 6-1 side by side in the longitudinal direction of the measuring pad 3 from the front end of the measuring pad 3 to the rear end side, whereby the first composite is connected to the measuring pad 3. As it flows, the linear trapping agent 6-1 is colored in proportion to the concentration of the antigen, and the quantitative concentration measurement is made possible through the count of the signaled coloring line.

As such, the measurement pad 3 measures the quantitative concentration of the antigen by counting the colored traps or capture lines of the test unit, that is, signal spots and lines that increase in proportion to the concentration of the antigen. Measurement is possible.

In other words, the first composite is combined with the first capturing member 6, 6-1 at the front of the inspection section (in this embodiment, the left side of the inspection section of the measurement pad), so that the capillary flow of the measurement pad 3 The concentration in the sample gradually decreases as the force moves, so that the first traps 6 and 6-1 arranged at the rear of the inspection section (in the present embodiment, the right side of the inspection pad) are combined with the first complex. The probability of doing so decreases.

Therefore, the higher the concentration of the antigen in the sample moving along the measurement pad 3, the higher the probability of binding between the first capturer 6,6-1 and the target antigen arranged behind the inspection unit, and the lower the concentration of the antigen. The probability of binding between the first capturer (6, 6-1) and the target antigen arranged behind the inspection unit is lowered, and the number of signal emission points generated in the inspection unit of the measurement pad 3 is dependent on the antigen concentration in the sample. In other words, they appear proportionally.

On the other hand, in the conjugate pad 2, the conjugate that is not bound to the antigen in the sample passes through the inspection unit along with the blood flow.

The conjugate penetrating into the conjugate pad 2 is present in a large amount relative to the amount of the target antigen which is supposed to be contained in the sample. It attaches to the capture | capturing agent (referred to as 7 or a 2nd capture | capturing agent) which is an antibody which carries out specific binding with the detector 9 of a conjugate, while passing through the control part of 3).

That is, as described above, the measuring pad 3 is immobilized on the control part with a second capturer 7 which is not attached to the target antigen but is specifically attached to the detector 9 of the conjugate. The trapper 7 is immobilized on the control and does not flow with the flow of the sample.

The control unit is formed by arranging a plurality of second capturers (reference numeral 7 of FIG. 2) fixed in a spot form or arranged in a line or a second capture collector fixed in a line form (FIG. 3). 7-1), located in the downstream direction of the inspection section, the joined body passing through the inspection section is attached to the second traps 7 and 7-1 and fixed to the control section.

The second capturer (7,7-1) of this control indicates whether the sample and the conjugate formed the first complex and migrated to the control by capillary flow, regardless of the presence or absence of the target antigen in the sample, thereby measuring The pad 3 checks the occurrence of the capillary phenomenon and serves to determine the validity of the immunoassay and analysis results of the inspection unit.

Specifically, as shown in Fig. 4, the measurement is invalid if the second trapper 7 of the control is not colored by the colored particles 8 of the conjugate, i.e. if no signal color spot occurs in the control. When the second capturer (7) is combined with the conjugate in the sample (the conjugate that is not combined with the antigen, but simply mixed in the sample), the color is developed. This results in a positive or negative result of the immunoassay.

For example, if no target antigen is present in the sample, the test part will not develop and the control part will develop. That is, if only the control part is developed, there is an immunological negative indication that the target antigen is not present in the sample.

In addition, for example, when the antigen concentration in the sample is low, the signal development point appears only in front of the test unit, and when the antigen concentration is high, the signal development point appears in the back of the test unit.

On the other hand, the sample passing through the control part of the measuring pad 3 is moved to the absorbent pad 4 which is disposed on the other end of the base member 5 (right end in this example) and is in contact with the measuring pad 3. Is absorbed.

The absorbent pad 4 is configured in the form of a plurality of pores in order to sufficiently absorb all the samples from the measuring pad.

As described above, in the assay strip according to the present invention, when the sample containing the target antigen is dropped on the sample pad 1, the conjugate pad 2 in which the chromogenic particles 8 and the detector 9 are coupled by diffusion flow. The antigen in the sample selectively bound to the detector 9 through the antigen-antibody reaction in the conjugate pad 2 is moved by the capillary flow force to the measurement pad 3 together with the sample flow. The first capturers 6 and 6-1 fixed to the inspection unit are combined.

The assay strip of the present invention performs an immunochromatography-based lateral flow assay analysis by a so-called "sandwich assay" as described above, and the excess conjugate passing through the test part is the second trap (7) of the control part. And 7-1), which is used as a criterion for determining the validity of the measurement results.

Hereinafter, as an immunoassay of the lateral flow assay strip according to the present invention, the following examples are performed to confirm that the number of signal chrominance points increases according to the antigen concentration in the sample, but the present invention is not limited thereto. .

In particular, a measurement in which a lateral flow assay strip capable of measuring Cartilage oligomeric matrix protein (COMP), which is a diagnostic marker of osteoarthritis, is prepared, but the measurement is performed in which the concentration of antigen in the sample is substantially performed for experiments to confirm the measurability of antigen concentration in the sample. A simple assay strip consisting of only pads was prepared and used, and the measurement results according to the concentration of COMP and the capture process of antigen in the sample moved by capillary flow force in the measurement pad will be described in detail.

For reference, COMP is a diagnostic marker for osteoarthritis, a disease marker in which general humans maintain a concentration of about 0.67 μg / ml in blood, and the concentration of COMP in blood increases greatly when osteoarthritis or joint tissue is damaged.

Example

In order to confirm that the number of actual signal development points increases proportionally with the concentration of COMP, high-density COMP samples taken directly from actual patients were diluted twice with 100 mM PBS buffer, and the total six concentrations of samples were obtained. Was prepared, and a buffer solution without COMP was added as a control.

As a measurement pad, a nitrocellulose membrane was used, and it was cut into 6 cm in length x 0.5 cm in width. The control section immobilized 1 mg / ml concentration of anti-mouse IgG antibody in the form of three dots arranged in a row at 1 cm spaced from the right side of the measurement pad. 16F12 capable of binding (antigen-antibody reaction) was arranged at a concentration of 1 mg / ml, three dots were arranged in one row, five rows in total, 15 dots were arranged in a zigzag form, immobilized, and dried at room temperature.

Signal developing substance is Nature Physical Science (1973), 241, 20-22 was used to synthesize 30 nm gold nanoparticles, Sensors and The detector (secondary antibody) 12C4 was bound to gold nanoparticles by referring to Actuators B (2010), 146, 368-372.

Here, the synthesis method of the detector and the gold nanoparticles is as follows. The pH of the gold nanoparticles was titrated to 8.5 using 0.1 M K ₂ CO ₃ buffer. 0.8 ml of 1 mg / ml 12C4 antibody was added to the titrated 10 ml gold nanoparticle solution, followed by slow stirring at room temperature for about one hour. Thereafter, 1 ml of 10 mg / ml BSA solution was added and treated for 30 minutes to block the surface of the gold nanoparticles to which the detector was not bound. Then, the gold nanoparticles to which the detector was bound were rotated at 12000g for 30 minutes at 4 ° C. using an ultra-high speed centrifuge, and the supernatant was separated to remove unreacted residue. Then, 10 ml of 50 mM PBS to which 1% (w / v) BSA was added was added to make a suspension and centrifuged once more. Finally, after removing all the supernatant generated through centrifugation, 5 ml of the same buffer solution was added to prepare a gold nanoparticle solution in which the detector concentrated twice was combined.

In order to measure the COMP concentration in the COMP sample passed through the conjugate pad and moved to the measurement pad, each of the gold nanoparticle solutions in which the COMP sample and the detector were combined with the different concentrations prepared as described above was 1: 1 (v: v). 10 μl was added dropwise to the left end of each measurement pad, followed by immunochromatography from the measurement pad to the mobile phase of the sample, and the signal development point of each measurement pad was measured. It was confirmed.

10 μl was added dropwise to the left end of the measurement pad prepared by first reacting the gold nanoparticle solution to which the detector was bound with 1: 1 (v: v). Immunochromatography was performed to confirm signal development.

As a result, in the case of the buffer solution to which COMP was not added, it was confirmed that only a specific binding between the second trap and the detector was shown to have a negative response.

As a result of confirming the signal according to the concentration of COMP in six COMP samples diluted two-fold, as shown in FIG. 5, the first complex formed by the antigen-antibody reaction between the COMP in the sample and the detector was combined with the first trap. A positive reaction was observed, and as the concentration of the antigen COMP increased, the number of signal emission points and the emission lines increased proportionally.

As described above, in the assay strip of the present invention, the number of signal color points appearing in the test part of the measurement pad increases proportionally with the increase or decrease of the concentration of the target antigen in the sample. This makes it possible to quickly and easily diagnose the infection of various diseases, and through the quantitative analysis according to the concentration of antigens, anyone in the field can readily determine the seriousness of the disease and confirm the prognosis of the disease. .

In particular, diseases through disease markers that maintain a certain level in normal people, such as glycated hemoglobin (HbA1c), diabetes diagnosis markers, Cartilage oligomeric matrix protein (COMP), and tumor markers (PSA, CA19-9), etc. It can be useful for diagnosis.

As a result, the utilization of the disease diagnosis field, which requires concentration measurement, has been increased, and the market expansion effect in the lateral flow assay field can be expected.

In addition, the assay strip of the present invention, unlike the lateral flow assay kit according to the prior art, can directly check the concentration of the antigen by eye without separate analysis equipment and complicated diagnostic process, thereby improving convenience and portability.

1: sample pad
2: conjugate pad
3: measuring pad
4: absorbent pad
5: base member
6,6-1: First captor
7,7-1: second captor
8: coloring particles
9: detector
10: blocker

Claims (5)

The sample pad, the conjugate pad, the measurement pad, and the absorbent pad are laterally arranged on the base member and configured to be in contact between adjacent pads,
The test unit of the measurement pad is formed by immobilizing a plurality of traps, each capable of binding to the target antigen, in front of the control unit, and quantitative concentration measurement of the antigen is possible by counting the colored traps among the traps of the test unit.
Each trap of the inspection unit is formed in a spot shape, and the inspection unit forms a plurality of capture lines aligned along the width direction of the measurement pad to form a single capture line, and the capture lines are configured in a plurality of rows. It is formed in a shape arranged spaced along the longitudinal direction of the pad, characterized in that the capturing arrangement arranged in the rear row with respect to the width direction of the measuring pad in the inspection unit spaced apart to the left and right both sides of the catcher arranged in the front row Lateral flow assay strips for analysis. delete delete The method according to claim 1,
The capture line consisting of a plurality of rows are arranged in a zigzag form arranged alternately up and down along the width direction of the measuring pad lateral flow assay strip capable of quantitative analysis. delete

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