KR101998948B1 - Method for amplification of signal in lateral flow assay by using water-soluble coating layer and lateral flow assay device using the method - Google Patents

Abstract

The present invention relates to a method for analyzing an analyte comprising the step of binding an analyte with a conjugate in which a first antibody or a first specific binding substance capable of specifically binding to a first epitope or first binding site of an analyte is bound to a label, ; Binding an analyte to which the conjugate is bound and an immobilized second antibody or a second specific binding substance that specifically binds to a second epitope or second binding site of the analyte; And a signal amplification method in a lateral flow analysis, comprising the step of delaying the signal amplification material coated with a coating layer of a water-soluble substance and reacting the signal amplification material transferred thereto; And a lateral flow analyzer configured to perform the method of the present invention.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for amplifying a signal in a lateral flow using a water-soluble coating layer and a lateral flow analyzer using the same,

The present invention relates to a signal amplification method in the analysis of a lateral flow system for highly sensitive detection of analytes and a lateral flow analysis apparatus using the same. More particularly, the present invention relates to a signal amplification method in a highly sensitive lateral flow analysis that effectively amplifies a signal by forming a structure in which a signal amplifying substance subsequently reacts in a sandwich analysis, and a lateral flow Lateral flow analyzer.

Immunochromatography is a method that can qualitatively and quantitatively inspect analytes in a short period of time using the property that biological substances or chemical substances adhere to each other in a specific manner. Particularly, sandwich immunoassay is an object It is well known that a first antibody capable of binding specifically to a first epitope of an analyte is immobilized on a solid support and a second antibody specific for a second epitope of the analyte is used.

As an apparatus for such analysis, an analytical strip or an analytical apparatus in which the analytical strip is assembled inside the housing is generally used. At this time, when a fluid containing an analyte is applied to one side of the porous strip, the analyte is bound to the antibody including the label and the immobilized antibody while the fluid flows by the capillary phenomenon.

In more detail, a lateral flow device generally has an antibody immobilized on a membrane, which is a porous membrane through which a liquid sample can flow as a capillary phenomenon. On the upstream side of the membrane, a sample pad and a bonding pad are provided. Pads are connected. The sample pad absorbs the liquid sample containing the analyte and ensures uniform flow, and the binding pad is dried with a label attached with an antibody capable of selectively binding to the analyte. The immobilized antibody selectively binding to the analyte and the substance capable of binding to the antibody immobilized on the label are immobilized on the membrane at different positions to form a detection unit and a control unit, respectively. An antibody immobilized on a membrane capable of selectively binding with the analyte and an antibody immobilized on the label are configured to be sandwich-bound to the analyte. The absorbent pad is made of a material capable of absorbing a liquid sample. In such an immunochromatography analyzer, when a liquid sample containing an analyte is dropped on a sample pad, an antibody-labeled antibody having selectivity to the analyte and an antibody immobilized to the membrane are bound in a sandwich form, A visible band is formed at the membrane position.

As a conventional technique, there has been disclosed a signal amplification method of immunochromatography in which a primary conjugate and an antigen are bound and then a secondary conjugate is further combined and finally bound to an antibody immobilized on a membrane. However, There is a problem that it is difficult to measure a sample requiring a higher sensitivity because the sensitivity to be measured is low. In addition, Korean Patent Application No. 2009-0047004 discloses a method of improving the sensitivity by using a signal amplification pad configured not to be in contact with any of the bonding pad and the membrane, but in this case, when the user puts the signal amplification pad on the membrane There is a further need for a process for contacting.

In the lateral flow analysis, the first antibody or the first specific binding substance is bound to the label and the analyte is bound, and the antibody or the immobilized specific binding substance is sandwiched A method in which a signal amplifying material is subsequently reacted to improve the sensitivity of signal amplification without performing additional processing, and a lateral flow analyzing apparatus to which such a method is applied, is widely applied in related fields It is expected to be possible.

Therefore, one aspect of the present invention is to provide a signal amplification method in a lateral flow analysis.

Another aspect of the present invention is to provide a lateral flow analyzer having improved sensitivity using the signal amplification method.

According to one aspect of the present invention, there is provided a method of analyzing an antibody or antigen-binding fragment thereof, which comprises a conjugate in which a first antibody or a first specific binding substance capable of specifically binding to a first epitope or first binding site of an analyte is bound to a label, Combining water; Binding an analyte to which the conjugate is bound and an immobilized second antibody or a second specific binding substance that specifically binds to a second epitope or second binding site of the analyte; And a step of delaying the signal amplifying material in a signal amplifying part coated with a coating layer of a water-soluble substance to react the transferred signal amplifying material.

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According to another aspect of the present invention, there is provided a conjugate in which a first antibody or a first specific binding substance capable of specifically binding to a first epitope or first binding site of an analyte is bound to a label Bonding pads; And a detection site in contact with the binding pad and immobilized with a second antibody or a second specific binding substance that specifically binds to a second epitope or a second binding site of the analyte to which the binding substance is bound, Membrane; And a signal amplification unit formed upstream of the detection unit of the membrane, wherein the signal amplification unit is a lateral flow analysis apparatus, wherein the signal amplification material is a delay transmission signal amplification unit coated with a coating layer of a water-soluble substance.

According to the present invention, there is provided a single step assay kit in which sensitivity is improved by binding a marker to an analyte in the sample, fixing the same on a porous membrane membrane, and subsequently delaying the signal amplification material. Further, according to the present invention, there is no limitation of the signal amplifying material that can be applied, and further work for signal amplification is not required. In particular, when the signal amplifying material and the water soluble substance are manufactured using the supporting method, .

1 is a schematic diagram showing a process of signal amplification by the method of the present invention.
Fig. 2 is a schematic diagram showing each configuration of the immunochromatography apparatus of the present invention. Fig. 10 is a sample pad, 11 is a bonding pad, 12 is a membrane (porous membrane) (Control band), (15) an absorption pad, and (16) and (17) a delay transmission signal amplifying unit, (16) a gold precursor retardation transmitting film, And (17) represents a reducing agent retarding film.
FIG. 3 schematically shows a process of preparing a retardation transfer membrane using a water-soluble substance and introducing it into a lateral flow analysis kit.
4 is a scanning electron micrograph of a nitrocellulose porous membrane after (A) and (B) before polyethylene oxide coating.
Figure 5 shows the results of a commercial lateral flow assay kit for detecting signal amplification troponin i protein following reduction of gold ions without a delayed delivery membrane.
Figure 6 shows the results of a commercial lateral flow assay kit for detecting troponin eye proteins amplifying signal amplification using the delayed delivery membrane of the present invention.

Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. However, the embodiments of the present invention can be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below.

The signal amplification method of the present invention is a method for amplifying a signal in a lateral flow by combining a first antibody or a first specific binding substance and a conjugate composed of a labeling substance with an analyte and then immobilizing a second antibody, The signal is amplified by the signal amplifying substance which is bound to the target binding substance and subsequently the signal amplifying substance is added and reacted so that the signal amplifying substance exhibits a strong labeling reaction around the target with the label as a seed .

Particularly, in the present invention, the signal amplifying material is coated with a coating layer of a water-soluble substance and formed into a signal amplifying unit, and the water-soluble substance is dissolved in a liquid sample containing an analyte, particularly, a liquid sample based on water such as blood And the delayed signal amplifying material is formed to be delayed and delivered during the dissolving time, and the delayed signal amplifying material is subsequently reacted in the signal amplifying unit.

In the present invention, the term "signal amplifying substance" means not only a substance capable of amplifying a signal itself, but also a substance capable of reacting with another substance to generate an amplified signal, . For example, luminol which can produce a luminescent signal can not generate a signal by itself, but when luminescence of H ₂ O ₂ is delayed, a luminescent signal can be generated through a subsequent reaction. Therefore, will be.

In more detail, the signal amplification method in the lateral flow analysis of the present invention comprises a first antibody capable of specifically binding to a first epitope or a first binding site of an analyte or a first specific binding Combining the analyte with a conjugate wherein the substance is bound to a label; Binding an analyte to which the conjugate is bound and an immobilized second antibody or a second specific binding substance that specifically binds to a second epitope or second binding site of the analyte; And a step in which the signal amplifying material reacted with the signal amplifying material is delayed in the signal amplifying unit coated with the coating layer of the water-soluble substance.

The signal amplification method of the present invention can be applied to a lateral flow method, and the lateral flow method includes a vertical flow method and a flow though method. In the case of the vertical flow system, the samples are vertically stacked on the porous membrane arranged in parallel.

In the meantime, the lateral flow system is not limited to the antibody-antigen reaction, and the 'binding site (ligand)' referred to in the present specification refers to a protein ligand, nucleic acid (DNA or RNA) Specific binding substance "includes a protein, a virus phage, a nucleic acid molecule aptamer, hapten (DNP), etc. which can selectively and specifically bind to the binding site, and the like. Biomolecules, and is not particularly limited to those described above.

As used herein, the term "conjugate" refers to a first antibody or a first specific binding substance, and a labeled substance, which is bound to an analyte and flows through the strip, and is bound to a capture second antibody or a second specific binding Lt; / RTI >

The mechanism of the signal amplification method in the lateral flow analysis according to the present invention will be described in more detail with reference to FIG. 1. FIG. 1 illustrates an example of using the antigen-antibody reaction in the lateral flow analysis .

1, a first antibody 1 capable of specifically binding to a first epitope of an analyte, as shown in FIG. 1, Wherein the analyte (4) is bound to the analyte (4) and the analyte bound to the analyte (4) is flowing along the strip and is specifically bound to the second epitope of the analyte The water-soluble substance forming the coating layer is dissolved in the sandwich bonding, and the signal amplifying material therein is attached to the second antibody 5 as a seed, Allows the signal in the lateral flow analysis to be amplified.

The label that can be used in the present invention is not particularly limited as long as it is capable of specifically binding to the first antibody and then amplifying the signal by binding with the signal amplifying material. For example, gold nanoparticles, platinum Nanoparticles, fluorescent molecules, luminescent molecules, and quantum dots.

The signal amplifying substance that can be used in the present invention is not particularly limited as long as it is capable of specifically binding to the label and amplifying the signal, and examples thereof include tetrachloromethane, hydroxylamine, 3,3 ' 5,5'-tetramethylbenzidine, and hydrogen peroxide, and may further be two or more materials that are separately coated and then disposed in contact with each other.

The signal amplification material is preferably contained at a concentration of 1 to 100 mM. If the concentration is less than 1 mM, signal amplification does not occur smoothly. If the concentration exceeds 100 mM, the signal-to-noise ratio of the lateral flow analysis is increased. However, the appropriate concentration of the signal amplifying material may be varied depending on the signal amplification reaction, the type of the reactant, and the type of the solvent, and an appropriate concentration range may be adjusted depending on the reactivity of the signal amplifying material and the type of the solvent.

The coating layer of the water-soluble substance melts when the coating layer of the water-soluble substance comes into contact with the liquid sample containing the analyte to specifically bind the signal-amplifying substance inside thereof to the label. Therefore, May be at least one selected from, for example, polyethylene oxide, polyvinylpyrrolidone, polyvinyl alcohol, and polylactic acid.

For example, the signal amplifying material may be a single material or a combination of a plurality of materials that react with each other to induce signal amplification, and the signal amplifying material may react with two or more substances to induce signal amplification In a combination of a plurality of materials, the two or more substances may each be coated with a coating layer of a water-soluble substance.

The coating composition for forming the coating layer of the water-soluble substance of the present invention preferably has a property of melting upon contact with an aqueous solution, while using a solvent that does not affect other substances such as other signal amplifying substances. For example, in the case of nitrocellulose membranes each carrying tetrachloroauric acid and hydroxylamine, a polyketylene oxime coating composition can be prepared using chloroform as a solvent.

On the other hand, at this time, the coating composition preferably contains the substance which performs the actual coating in an amount of more than 0.1% by weight, preferably not more than 15% by weight, more preferably not more than 0.5% by weight and not more than 10% will be. When the amount of the substance that performs the actual coating in the coating composition is more than 15% by weight, the pores of the porous membrane are filled up to inhibit the diffusion of the solution during the lateral flow analysis, If the content is less than 0.1% by weight, delayed delivery of the signal amplifying material is not achieved. However, the appropriate concentration of the water-soluble substance coating composition may vary depending on the kind of the water-soluble substance, the molar mass, and the kind of the solvent, and the amount of the signal-amplifying substance and the amount of the water-soluble polymer do not influence each other.

The analyte that can be detected by the method of the present invention is not particularly limited as long as it is capable of binding to the first and second antibodies and forming a sandwich-type immunoconjugate by an immunological reaction, that is, an antigen-antibody reaction, It can be applied to environmental pollutants, viruses, etc., including proteins or DNA, environmental hormones and the like.

In the present invention, the antigen and the antibody are not particularly limited as long as they are substances capable of specifically binding to an analyte by an antigen-antibody reaction. When the analyte is an antibody, a substance specifically binding to the analyte can be used as an antigen have. The antigens and the antibodies may adopt known antigens and antibodies depending on the analyte. Furthermore, the reaction of a 'specific binding substance' selectively recognizing an analyte as a 'binding site (ligand)' is considered to be included in an antigen-antibody reaction in a broad sense.

Meanwhile, in the lateral flow analyzer of the present invention, the first antibody or the first specific binding substance capable of specifically binding to the first epitope or the first binding site of the analyte is attached to the label A bonding pad comprising a bonded assembly; And a detection site in contact with the binding pad and immobilized with a second antibody or a second specific binding substance that specifically binds to a second epitope or a second binding site of the analyte to which the binding substance is bound, Membrane; And a signal amplification unit formed upstream of the detection unit of the membrane, wherein the signal amplification unit is a delayed transmission signal amplification unit in which the signal amplification material is coated with a coating layer of a water-soluble substance.

Fig. 2 is a schematic diagram showing an exemplary structure of each configuration in a lateral flow analyzer according to the present invention, wherein (10) is a sample pad, (11) is a bonding pad, (12) 13 denotes a detection unit, 14 denotes a control unit, 15 denotes an absorption pad, and 16 and 17 denote delay transmission signal amplification units.

More specifically, FIG. 2 illustrates a case where the delayed transmission signal amplification unit includes a first delayed transmission signal amplification unit 16 and a second delayed transmission signal amplification unit 17, And the second delay transmission signal amplifying part is coated with a coating layer of a water-soluble substance, and the first delay transmission signal amplifying part and the second delay transmission signal amplifying part are coated with a coating layer of a water- State.

The pads which can be used in the production of the lateral flow analysis apparatus of the present invention are not particularly limited as natural or synthetic materials as porous materials. Preferably, nitrocellulose is used.

The sample pad is a portion where the liquid sample containing the analyte is first absorbed. The sample pad may be formed by using one end of the membrane as it is or by a separate member. The sample pad is absorbed by the capillary phenomenon Thereby moving the analyte to the bonding pad.

The conjugate pad is present in the dried state of the conjugate, and when the liquid sample containing the analyte is absorbed, the conjugate pad acquires fluidity and moves to the membrane. The bonding pad may be made of a material such as glass fiber, cellulose, or the like.

Preferably, the delayed transmission signal amplification unit is formed on the membrane as shown in FIG. 2, in an area between the bonding pad and the detection unit of the membrane. According to the present invention, after the sample is applied to the sample pad, the amplified signal can be obtained without performing any additional operation.

The signal amplification unit may be a single amplification unit or two or more additional amplification units including a plurality of materials for causing signal amplification by reacting two or more materials with each other as shown in FIG. 2 may be disposed in contact with each other, They can be placed in contact with each other or stacked.

The signal amplification unit including a coating layer made of a water-soluble substance is a system in which a liquid sample containing an analyte is absorbed in an absorption pad, and a first antibody and a conjugate composed of a label bind to the antigen and bind to the second antibody, , The coating layer is dissolved by the liquid sample, and subsequently the label is bound to the periphery of the label with the label as a seed to amplify the signal.

The coating layer is formed by a coating composition, and it is preferable that the substance that performs the actual coating is contained in an amount of 0.1 to 15% by weight, more preferably 0.5 to 10% by weight, .

When the content of the substance that performs the actual coating in the coating composition is less than 0.1% by weight, delayed delivery of the signal amplifying material is not realized. When the amount exceeds 15% by weight, the water- There is a problem that the diffusion of the signal amplifying substance is prevented and diffusion is prevented.

The method of preparing the coating layer using the coating composition is not particularly limited. For example, the coating layer may be formed by dropping a signal amplifying material on the membrane and drying the coating layer. The signal amplifying material may be formed by dropping the coating composition on the dried signal amplifying material and then drying it.

On the other hand, when the signal amplifying unit is 2 or more, the signal amplifying material and the coating layer may be formed on two or more membrane pieces as described above, and then they may be disposed on the membrane.

It is preferable that the signal amplifying unit manufactured as described above is disposed so that the surface on which the signal amplifying material and the coating composition are formed is in contact with the membrane.

The step of dropping the signal amplifying material on the membrane, followed by drying, and the step of dropping the coating composition on the dried signal amplifying material, followed by drying, may be performed by a drying method such as vacuum drying at room temperature, At this time, it is preferable that the drying temperature and the decompression pressure are within a suitable range according to the volatility of the solvent of the coating composition, and it can be determined in consideration of the reactivity of the signal amplifying material.

For example, when chloroauric acid is added dropwise in water as a solvent, the drying temperature is preferably less than 50 ^° C. in consideration of the reactivity with nitrocellulose and moisture of the tetrachloromonous acid, and it is preferable to reduce the pressure in the vacuum pump Do. When polyethylene oxide is added dropwise with volatile chloroform as a solvent, it can be sufficiently dried at room temperature and normal pressure.

In the present invention, the membrane is not particularly limited as long as it is a material capable of securing fluidity of the liquid sample, and it is preferably made of a porous membrane. More specifically, the present invention relates to an antibody or a specific binding substance, and a method for immobilizing an enzyme protein and minimizing nonspecific reaction, such as nitrocellulose, cellulose, polyvinylidene fluoride (PVDF), poly (ethylene terephthalate) Hydrophobic porous membranes, such as fibers, nylons, and the like, which can be adjusted to a certain micropore size, can be used.

The membrane may include a detection site on which a second antibody or a second specific binding substance that specifically binds to a second site of the analyte to which the conjugate is bound is immobilized, and a control site, And a control site for controlling the operation of the apparatus. The detector is the part that shows the results for reading the test results. The control unit is configured to identify errors of the gold nanoparticle conjugate and capture antibody or immobilized second specific binding material and to confirm whether or not the substances with mobility are properly reacted to the detection unit / control unit .

The absorbing pad is not particularly limited as long as it can sufficiently absorb the residue remaining after the reaction by the capillary phenomenon, for example, cellulose, cotton. Hydrophilic porous polymer and the like can be used.

Hereinafter, the present invention will be described more specifically by way of specific examples. The following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited thereto.

Example

1. Reagents and equipment

Chloroform, Gold (III) chloride, HAuCl ₄ , hydroxylamine (NH ₂ OH), human serum, and polyethylene oxide (Mv 900,000) Purchased from Aldrich. A porous membrane, Nitrocellulose membrane, was purchased from Millipore. The test detection troponin eye protein (TnI protein) was purchased from Boreal Biotech. The commercial lateral flow analysis kit was purchased from NANOENTECH TnI Rapid Card product by Shin Yang Chemical Co.,

2. The signal amplification unit  Produce

A nitrocellulose membrane is cut into a size of 1 mm X 4 mm in order to prepare a signal amplification part by delayed transmission by coating a water-soluble substance on the signal amplification material.

FIG cropped as 3 to nitro knocking each fall tetrachloride Keumsan 2.5 mmol concentration aqueous solution of 2 microliters and hydroxylamine 5 mmol concentration aqueous solution of 2 microliters of a cellulosic membrane placed in a desiccator using a vacuum pump 25 for two hours, ^o C Dry at temperature.

Two microliters of a 1 wt% polyethylene oxide / chloroform solution is added to the nitrocellulose membrane carrying the gold precursor and the reducing agent, respectively, and dried at 25 ^° C for 3 minutes.

The two-nitrocellulose thus obtained is placed upside down on the porous membrane between the detection zone (detection unit) of the membrane of the lateral flow analysis kit and the bonding pad.

The reason for using chloroform as a coating solvent in this experiment is that it melts nitrocellulose, tetrachloromethane, and hydroxylamine but does not dissolve polyethylene oxide. Through this, it is possible to dissolve carbon tetrachloride and hydroxylamine Can be coated. The signal amplification unit used in the present invention can be manufactured by selecting a solvent in consideration of the solubility of the signal amplifying material, the porous film, and the water-soluble coating material.

3. Lateral flow  analysis Of kit  Check signal amplification effect

The troponin eye solution is prepared in human serum at a concentration of 0.01, 0.1, 1, 10 nanogram per milliliter. Wherein the human serum is a negative control at a concentration of 0 ng per milliliter. The lateral flow analysis kit without the separate treatment and the signal amplification part obtained in the above 2 were placed in the sample injection part of the assembled lateral flow analysis kit as shown in FIGS. 2 and 3, and 120 microliters of each sample was dropped. waiting.

As a result, as shown in FIG. 5 and FIG. 6, the detection limit of the visually identifiable lateral flow analysis kit according to whether the present invention is equipped with the signal amplifying unit is more than one hundred times as large as 0.1 ng per milliliter at 10 ng per milliliter I can confirm that it is improved.

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, but, on the contrary, It will be obvious to those of ordinary skill in the art.

1: first antibody 2: label
3: conjugate 4: analyte
5: Second antibody
10: sample pad 11: bonding pad
12: Membrane 13: Detector
14: Control section 15: Absorption pad
16: Delay transmission The first signal amplification unit
17: delay transmission second signal amplification unit

Claims (14)

Binding an analyte with a conjugate having a first antibody or a first specific binding substance capable of specifically binding to a first epitope or first binding site of an analyte bound to a label;
Binding an analyte to which the conjugate is bound and an immobilized second antibody or a second specific binding substance that specifically binds to a second epitope or second binding site of the analyte; And
The signal amplifying material is delayed in the signal amplifying part coated with the coating layer of the water-soluble substance, and the transferred signal amplifying material reacts
Wherein the signal amplifying material is a combination of a plurality of materials that cause two or more materials to react with each other to induce signal amplification and the two or more materials are coated with a coating layer of a water soluble material, A signal amplification method in a lateral flow analysis included in a signal amplification section.
The method of claim 1, wherein the binding site is at least one selected from the group consisting of a protein ligand, a DNA sequence and an RNA sequence, and the specific binding substance is a protein capable of specifically binding to the binding site , A viral phage, a nucleic acid molecule aptamer, and a hapten (DNP). 10. The signal amplification method according to claim 1,
The signal amplification method according to claim 1, wherein the signal amplifying substance is specifically bound to the label.
2. The method according to claim 1, wherein the analyte is selected from the group consisting of DNA, environmental hormones, and antigenic proteins.
2. The method according to claim 1, wherein the coating layer of the water soluble substance is dissolved in a liquid sample containing the analyte to specifically bind the signal amplifying substance therein to a label, Way.
delete delete A conjugate comprising a first antibody or a first specific binding substance capable of specifically binding to a first epitope or first binding site of an analyte is bound to a label, Bonding pad;
And a detection site in contact with the binding pad and immobilized with a second antibody or a second specific binding substance that specifically binds to a second epitope or a second binding site of the analyte to which the binding substance is bound, Membrane; And
Wherein the signal amplification unit comprises at least two signal amplification units each including a plurality of materials for reacting with each other to induce signal amplification so as to be in parallel contact with each other,
Wherein the signal amplification unit is a delayed transmission signal amplification unit in which the signal amplifying material is coated with a coating layer of a water-soluble substance.
The lateral flow analyzer according to claim 8, wherein the signal amplifying section is formed on the membrane between the bonding pad and the detecting section of the membrane.
delete delete 9. The lateral flow analyzer of claim 8, wherein the analyte is selected from the group consisting of DNA, environmental hormones, and antigenic proteins.
9. A lateral flow analyzer according to claim 8, further comprising a sample pad to which a liquid sample comprising the analyte is applied.
The lateral flow analyzer according to claim 8, further comprising an absorption pad disposed downstream of the membrane and capable of absorbing the liquid sample by capillary action.

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