KR20120029549A - Lateral flow assay device with rapid result and improved sensitivity - Google Patents

Abstract

PURPOSE: A lateral flow immunoassay device is provided to quickly absorb samples and to enhance signal sensitivity. CONSTITUTION: A lateral flow immunoassay device is used for quantitative or qualitative analysis. The device comprises: a sample pad(10) for applying whole blood sample containing analyte; an erythrocyte separation pad(11) for separating erythrocytes by pore size; a conjugate pad(12) containing a binder; and a membrane(13) having detection site(20) in which a second binder is fixed. The sample pad is chemically used for isolating erythrocytes from the whole blood samples containing polyvalent cation.

Description

Lateral flow assay device with rapid result and improved sensitivity

The present invention relates to an improved lateral flow immunoassay device for quantitatively or qualitatively measuring analytes in whole blood samples, which rapidly absorbs the sample and provides excellent red blood cell separation and Delayed sample flow increases the reaction time of binding of the target analyte, allowing rapid test results and improved lateral flow immunoassay devices with improved signal sensitivity.

Development of diagnostic methods and diagnostic apparatuses, which are performed by qualitative or quantitative analysis of trace substances in biopsies such as blood or urine, are in progress. Since the first introduction of radioimmunoassay (RIA) using radioisotopes in the 1950s, enzyme immunoassay (ELISA) was developed and developed in the 70s and 80s. ELISA immunoassay is now one of the most used methods and has become an essential tool in medical and life science research.

However, typical immunodiagnostic methods, including RIA and ELISA, usually quantify a single analyte per sample using expensive analyzers in a complex multi-step process. Therefore, it is not easy to use in a small hospital, emergency room, home, etc. which are not equipped with such facilities or facilities. Diagnostic products designed to compensate for these weaknesses are simple diagnostic kits using immunochromatography methods.

Representative types of immunochromatography assays include lateral flow assays. Looking at the structure of the kit of this side flow analysis type, a sample pad to which a sample is applied, a conjugation pad or a release pad coated with a detection antibody, and a development in which an antibody antigen reaction occurs after the sample is moved and separated A membrane (primarily nitrocellulose) or strip, and an absorption pad for continuous movement of the sample.

The detection antibody is bound to, for example, colloidal gold particles for display. Latex beads or carbon particles may be used instead of gold particles. Diagnostic kits for lateral flow analysis are usually designed to detect analytes in the form of sandwiches. That is, the analyte in the liquid sample is applied to the sample pad and begins to move, first reacting with a detection antibody that is unfixedly coated on the release pad (or conjugation pad) to continue development in the form of an antigen-antibody conjugate. do. As it moves, it reacts once more with the capture antibody immobilized on the developing membrane (membrane) to form a sandwich-type complex. Since the capture antibody is immobilized on the developing membrane, if antigen-antibody reactions continue, accumulation of the complex takes place in terms of the immobilization of the capture antibody. Since proteins are transparent to the naked eye, the formation and relative amounts of complexes are determined by the amount of gold particles attached.

This lateral flow analysis method is widely used in a variety of fields, such as pregnancy diagnosis, cancer diagnosis, microbial detection, but can be diagnosed very easily, but since the judgment is made with the naked eye, it is difficult to determine the exact amount. In particular, it is not easy to accurately diagnose the case where the judgment should be made near the cut-off value.

Conventional lateral flow quantitative assay strips, including all products known in the literature or actually on the market, have low sensitivity and are generally used as a means for qualitative rather than quantifying analytes. Therefore, there is a need for an analysis method that can be quantified with higher sensitivity.

Furthermore, red blood cells contained in whole blood present several disadvantages in the immunoassay by lateral flow. In particular, red blood cells interfere with the fluid flow required for a reaction to occur in the immunoassay device, and furthermore, the color of the red blood cells creates a background effect around the control line, which reduces the sensitivity of the detector.

As described above, techniques for isolating red blood cells from plasma in whole blood samples include (i) filtering the mixture through a solid absorbent element to which one or more specific binding pair members are bound to remove aggregated red blood cells. Techniques for combining with erythrocyte binders, (ii) passing whole blood through a glass microfiber filter with or without a coagulant introduced, (iii) preventing red blood cells from passing through and detecting or visualizing signals on anhydrous test strips The use of a blocking or exclusion layer of polysaccharide material to prevent interference, and (iv) the use of a polymer having a polyvalent cation surface to which red blood cells bind, not plasma.

However, there is a need for a lateral flow analysis device that can more easily and efficiently separate red blood cells and increase sensitivity.

Accordingly, an aspect of the present invention is to provide a lateral flow immunoassay device having a high test sensitivity as well as rapid redox cell separation and increased binding reaction time of a target analyte.

According to one aspect of the present invention, a lateral flow immunoassay device for quantitatively or qualitatively measuring analytes in whole blood samples, wherein a whole blood sample comprising an analyte is Sample pads applied; Red blood cell separation pad for separating red blood cells by pore size; A junction pad that binds specifically to the analyte and is provided with a diffuseable conjugate comprising a first binder conjugated with a primary label; And a detection site including a detection site to which a second binder specifically binding to the analyte is immobilized, and an average pore size of the sample pad, the red blood cell separation pad, and the junction pad. A lateral flow immunoassay device is provided, wherein the relationship of P1>P2> P3 is established, respectively, as P1, P2 and P3.

The sample pad preferably contains a polyvalent cation to chemically separate red blood cells from a whole blood sample.

It is preferable that the average diameter of the pore of the said sample pad is more than 8 micrometers, and 15 micrometers or less.

The average diameter of the pores of the red blood cell separation pad is preferably 6 to 8 ㎛.

It is preferable that the average diameter of the pore of the said bonding pad is 2-6 micrometers or less.

The flow immunoassay device preferably includes a second conjugation pad that further contains a secondary marker that binds to the primary marker and amplifies the signal.

The marker is preferably a gold nanoparticle, a chromogenic enzyme or a fluorescent substance.

The analytes are antibodies, antigens, nucleic acid aptamers, haptens, antigenic proteins, DNA, DNA-binding proteins, hormones, tumor-specific markers. ) And tissue-specific markers.

The first binder and the second binder are preferably selected from antibodies, antigens, nucleic acid aptamers, haptens, antigenic proteins, DNA, DNA-binding proteins and hormone-receptors.

The membrane preferably further comprises a control site for error checking.

The lateral flow immunoassay device preferably further comprises an absorption pad capable of absorbing the whole blood sample by capillary action.

In the lateral flow immunoassay device according to the present invention, the sample is rapidly absorbed as the average pore size of the sample pad, the red blood cell separation pad, and the conjugation pad is sequentially reduced, and the separation of the red blood cells is excellent, thereby increasing the signal sensitivity, and the conjugation pad. The average pore size of is smaller than the upstream sample pads and erythrocyte separation pads to delay the flow of the sample and thus increase the binding reaction time of the target analyte, thereby improving signal strength at the detector.

1 illustrates each pad constituting an exemplary lateral flow immunoassay device of the present invention.
2 illustrates an exemplary lateral flow immunoassay device of the present invention assembled.
3 illustrates a lateral flow immunoassay device of the present invention comprising a base member 15.

According to the present invention, with the rapid sample absorption of the sample pad, the separation of red blood cells is excellent, the signal sensitivity to the analyte is increased, and the reaction time of the binding of the target analyte increases as the sample flow is delayed at the junction pad. A lateral flow immunoassay device is provided in which signal strength is enhanced.

Target analytes that may be used in the lateral flow immunoassay device of the present invention are, for example, whole blood samples containing antigens, red blood cells, etc., and the term “analyte” as used herein refers to one or more epitopes. Or a compound or composition that is detected or measured with a binding site or ligand.

Analytes include antibodies, antigens, nucleic acid aptamers, toxins, organic compounds, proteins, peptides, microorganisms, amino acids, antigenic proteins, nucleic acids (DNA and RNA), and DNA-binding proteins. , Hormones, steroids, vitamins, drugs or metabolites of any of the above substances or antibodies thereto, including but not limited to certain antigenic substances, hapten, antibodies, macromolecules and combinations thereof, hormones, Tumor-specific markers and tissue-specific markers.

1 shows each pad constituting the lateral flow immunoassay device of the present invention. Sample pad 10 to which a whole blood sample including an analyte is applied is applied to red blood cells by a pore size. A red blood cell separation pad 11 for separating, a bonding pad 12 that specifically binds to an analyte and comprises a conjugate composed of a first binder bound to a primary label, and to the analyte. The membrane 13 including the detection site 20 to which the second binder specifically binding is immobilized, and the control site 21 for error checking, and the whole blood sample are capillary. And an absorbent pad 14 that can absorb.

Figure 2 shows the structure in which they are assembled, and Figure 3 shows the structure further comprising a base member 15 under the lateral flow immunoassay device strip of the present invention as described above.

In this case, the binder specifically binds to the analyte to qualitatively or quantitatively identify the analyte by the marker. When the first binder specifically binds to the first epitope of the analyte, the second binder may Preference is given to using what specifically binds to another second epitope.

The first binder and the second binder may be selected from antibodies, antigens, nucleic acid aptamers, haptens, antigen proteins, DNA, DNA-binding proteins, and hormone-receptors, but are not limited thereto.

In immune analysis by lateral flow, red blood cells contained in whole blood present several disadvantages. In particular, red blood cells impede the flow of fluid necessary for the reaction to occur in the immunoassay device, and furthermore, due to the color of the red blood cells, a background effect is generated around the detection unit 20 of the membrane 13, thereby lowering the sensitivity of the detection unit.

In this regard, the lateral flow immunoassay device of the present invention is characterized in that the pore sizes of the sample pad 10, the erythrocyte separation pad 11, and the junction pad 12 are sequentially reduced, that is, the sample pad, When the average pore sizes of the erythrocyte separation pad and the junction pad are P1, P2, and P3, respectively, a relationship of P1>P2> P3 is established. This structure allows the sample pad to rapidly absorb the sample and then physically separate the red blood cells from the red blood cell separation pad.

The red blood cell separation pad 11 has an average pore size suitable for separating red blood cells, that is, the average pore diameter of the red blood cell separation pad 11 is generally about 6 to 8 μm. Silver is preferably about 8 μm or less, preferably 6 μm or less, but is not particularly limited so long as it is an appropriate range for physically separating red blood cells. However, when the average pore diameter of the red blood cell separation pad exceeds 8 μm, red blood cells cannot be efficiently separated, and when the average pore diameter of the red blood cell separation pad becomes too small, it may affect the flow of the sample. This may be excessively extended. Therefore, the average pore diameter of the red blood cell separation pad 11 is most preferably about 6 to 8 μm.

On the other hand, the sample pad 10 disposed on the upstream side of the red blood cell separation pad 11 may be made of a material having a porosity sufficient to receive and contain the whole blood sample to be analyzed. Fine pore membranes of cellulose materials such as paper, paper, cellulose, cellulose derivatives such as cellulose acetate, nitrocellulose, glass fibers, naturally occurring cotton, fabrics such as nylon, or porous gels, but are not limited to these no.

The average pore diameter of the sample pad 10 is preferably greater than 8 µm and 15 µm or less, but is not particularly limited as long as it exceeds 8 µm. If the average pore diameter of the sample pad 10 is 8 μm or less, the sample may not be rapidly absorbed. If the average pore diameter is more than 15 μm, foreign substances included in the sample may not be filtered out.

On the other hand, the sample pad 10 is preferably configured to chemically separate the red blood cells from the whole blood sample including a polyvalent cation.

The polyvalent cation is poly-L-lysine hydrobromide, poly-L-arginine hydrochloride, poly-L-histidine, poly (lysine, alanine) 3: 1 hydrobromide, poly (lysine, alanine) 2: 1 hydrobromide, It is preferably selected from the group consisting of poly (lysine, alanine) 1: 1 hydrobromide, poly (lysine, tryptophan) 1: 4 hydrobromide and poly (diallyldimethylammonium chloride), and a sample in such a polyvalent cation solution The pad may be immersed and then dried to prepare a sample pad including the polyvalent cation, but the present invention is not limited thereto, and any method known in the art may be used.

The bonding pad 12 of the present invention is disposed in contact with the red blood cell separation pad 11, and the bonding pad also has porosity to receive blood from the sample pad 10. In this case, the blood is blood in which red blood cells are chemically and physically separated by the sample pad 10 and the red blood cell separation pad 11. In addition, the conjugate pad 12 binds specifically to an analyte, preferably a conjugate consisting of a first antibody specifically bound to a first epitope and bound to a primary label. It is included. Accordingly, the analyte and the conjugate bind to the conjugate pad 12 to form a first immuno-complex.

That is, as used herein, "cojugate" refers to a detectable label bound to a specific attachment component, such as an antibody, wherein the binding between the specific attachment component and the label is covalent or non-covalent. It can be a binding, and nucleic acid hybridization can be included. Such labeling materials result in the production of detectable signals that are directly or indirectly related to the amount of analyte in the test sample.

On the other hand, the diameter of the pore of the bonding pad 12 of the present invention is preferably less than 2 to 6 ㎛ average, if the average diameter of the pore is less than 2 ㎛ there is a problem that the flow of the sample is excessively slow or clogged, If it is 6 μm or more, there is a problem in that the flow delay of the sample cannot be performed. That is, the bonding pad 12 of the present invention is characterized in that it serves to extend the reaction time appropriately by delaying the flow of the sample.

In the analysis of the sample entering the sample pad 10, not all of the analytes are actually attached to the first antibody bound to the label in the conjugation pad 12, and there are still analytes that are not bound. do. Therefore, it is desirable to increase the amount of binding of analyte and conjugate not yet bound as described above for higher sensitivity analysis. According to the present invention, the average pore diameter of the bonding pad 12 disposed subsequent to the red blood cell separation pad 11 is made smaller than the average pore diameter of the two upstream pads, specifically 2 to less than 6 μm of blood. Configure to delay the diffusion flow. Thus, the flow of the sample is delayed by the confinement pads with limited pore diameter, which increases the reaction time for binding the conjugate to the analyte, thereby increasing the formation of the first immune conjugate. Accordingly, the intensity of the immune response signal in the detector 20 may be increased to obtain more accurate measurement results, and the final test quality and accuracy may be improved.

Labeling materials that can be used in the present invention may be gold nanoparticles, color development enzymes or fluorescent materials, it is preferable to use gold nanoparticles. The gold nanoparticles are typically red in color and have an absorption coefficient of approximately 510-540 nm.

Meanwhile, the binder, which is a specific attachment component included in the conjugate, is selected to be specifically attached to the analyte, and typically, but not limited to, an antibody, antigen, antibody, nucleic acid aptamer, in an immune response, Hapten, antigenic protein, DNA, DNA-binding protein and hormone-receptor or complex thereof and the like. For example, when an antibody is used, it may be selected from a monoclonal antibody, a polyclonal antibody, a recombinant protein or a chimeric antibody.

The membrane (13) is a material coated with particles of nitrocellulose (Nitrocellulose) to maintain a sufficient reaction time between antigen antibodies at a lateral flow flow rate of 180 sec / cm fluidity of the liquid sample There is no particular limitation as long as it is a material capable of securing the membrane, and the membrane includes a detection site 20 to which a second antibody that specifically binds to a second site of the analyte to which the primary conjugate is bound is immobilized. As a control for this, it may further include a control site (21) for determining the presence or absence of a reaction due to an error. The detection part shows the result for reading the test result. The control unit is configured to check the error of the gold nanoparticle conjugate and the capture antibody, and is to check whether the mobile materials are properly reacted to the detector / control unit without error.

The lateral flow immunoassay device of the present invention may further comprise an absorbing pad 14, which is not particularly limited as long as it can sufficiently absorb the residue remaining after the reaction by capillary action.

Looking at the mechanism of the immunochromatography signal amplification method according to the present invention in more detail, when the whole blood sample is introduced into the sample pad 10, the red blood cells are physically separated by the pore size in the red blood cell separation pad 11, at this time conjugation The flow is delayed by the pad 12 to form a sufficient reaction time. The conjugation pad 12 contains a primary conjugate in which a first antibody capable of specifically binding to the first epitope of the analyte is bound to the first labeling substance so that it is specifically associated with the introduced analyte. The analyte to which the primary conjugate is bound flows along the strip and binds to an immobilized second antibody that specifically binds to the second epitope of the analyte at the detector of membrane 13 to form a sandwich bond. do.

On the other hand, the flow immunoassay device of the present invention may contain a secondary marker that additionally binds to the primary marker and amplifies the signal, in which case it will specifically bind to the primary conjugate subsequently to the flow described above. A further third antibody may comprise an additional conjugation pad comprising a secondary conjugate to which the second labeling substance is bound so that the signal of the immunochromatography can be amplified.

Furthermore, the flow immunoassay device of the present invention can be configured to analyze multiple analytes in a whole blood sample, in which case a fourth antibody and a marker that are specifically attached to another analyte are conjugated to one another and proliferately attached. Other additional conjugates may be contained within the conjugate pad, in which case the membrane 13 further comprises a second detector disposed further, wherein the second detector is specifically attached to the additional analyte. The fifth antibody to be immobilized can be immobilized.

Meanwhile, the analyte may include antibodies, antigens, nucleic acid aptamers, haptens, antigen proteins, DNA, DNA-binding proteins, hormones, and tumor-specific markers. markers and tissue-specific markers.

In addition, the first binder, the second binder is selected from antibodies, antigens, nucleic acid aptamers, haptens, antigenic proteins, DNA, DNA binding proteins and hormone-receptors.

Hereinafter, the present invention will be described in more detail with reference to Examples, which are intended to illustrate the present invention, and the scope of the present invention is not limited by these Examples.

Example 1 Preparation of Lateral Flow Immunoassay Device According to the Invention

1. Synthesis of Conjugates

0.1 mL of 0.1 M boric acid buffer (pH 8.5) was added to 1 mL of gold nanoparticle colloidal solution (BBInternational, 10 nm), and 10 mg of 1 mg / mL of the first antibody was reacted for 30 minutes. One conjugate was prepared. As the first antibody, 4T21, 560 (HyTest) may be used for immunoassay against troponin I, and M012607 (Fitzgerald) may be used for immunoassay against myoglobin.

2. Preparation of Lateral Flow Immunoassay Device

A capture antibody (second antibody) dissolved in PBS using a dispenser system (Zeta Co.) after attaching a nitrocellulose membrane (Millipore, 180 sec Nitrocellulose) and an absorption pad (Millipore) to a plastic base member (Millipore) A 1 mg / mL solution and a 1 mg / mL solution of Goat anti-mouse IgG antibody (Sigma, M8642) dissolved in PBS as a control were lined at 6 cm / sec each to the membrane. A detection site and control lines were formed. The membrane was dried and then cut into 3 mm intervals in a cutter.

The second antibody, which is the capture antibody, may use a troponin capture antibody (Hytest) for immunoassay against troponin I, and a myoglobin capture antibody M09983110 (Fitzgerald) for immunoassay against myoglobin.

The sample pad (Millipore) was immersed in 0.5% Tween 20, 5% sucrose, 5% dextran 0.05% sodium azide aqueous solution, dried, cut to 10 X 3 mm, pore size Was used having a diameter of 10 μm.

Erythrocyte separation pad was used GFC (Millipore Co., Ltd.), pore size of 7 ㎛ diameter was used.

The bonding pad was cut by GFC (Millipore Co., Ltd.) to 5 x 3 mm and dried by applying 5 μL of the conjugate in 1. and a pore size of 4 μm was used.

Bond pads, sample pads and erythrocyte separation pads were assembled to the plastic pads to which the membrane and absorbent pads were assembled, similar to FIG. 3.

Comparative Example 1: Preparation of the Same Large Pore Size Lateral Flow Immunoassay Device

A lateral flow immunoassay device was prepared in the same manner as in Example 1 except that the average pore size of the junction pad, the sample pad, and the red blood cell separation pad were equalized to 10 μm.

As a result, although the test measurement speed was faster than that of Example 1, it was confirmed that the red blood cell separation ability was low and the amount of primary immune conjugates generated in the junction pad was small, so that the signal intensity at the detection unit was small.

Comparative Example 2: Fabrication of Identical Small Pore Size Lateral Flow Immunoassay Devices

A lateral flow immunoassay device was prepared in the same manner as in Example 1 except that the pore sizes of the junction pad, sample pad, and erythrocyte separation pad were equalized to 4 μm.

As a result, it was confirmed that the sample was not absorbed well in the sample pad, and the sample measurement speed was slowed.

Comparative Example 3: Preparation of Lateral Flow Immunoassay Device with Different Pore Sizes

A lateral flow immunoassay device was prepared in the same manner as in Example 1 except that the pore sizes of the sample pad, erythrocyte separation pad, and junction pad were sequentially increased to 4 μm, 7 μm, and 10 μm, respectively.

As a result, in this case, it was confirmed that the sample was not absorbed well in the sample pad, and the amount of the first immune body generated in the bonding pad was small, so that the signal at the detector was low in intensity.

Experimental Example 1: Confirmation of the sensitivity of the lateral flow immunoassay device

100 μl whole blood pipettes were introduced into the sample pads of Examples 1 and Comparative Examples 1 to 3, respectively, and the time and signal sensitivity of the signal appearing in the detector were measured.

In Example 1, a detectable signal appeared in the detection unit after 2 minutes and 50 seconds, and exhibited an analysis sensitivity of 0.5 ng / mL.

On the other hand, in the case of Comparative Example 1, a detectable signal appeared in the detection unit after 1 minute 40 seconds, and showed an analysis sensitivity of 2.0 ng / mL.

In Comparative Example 2, a detectable signal appeared in the detector after 5 minutes and exhibited an assay sensitivity of 0.7 ng / mL.

In Comparative Example 3, a detectable signal appeared after 3 minutes and 30 seconds, and showed an analysis sensitivity of 1.5 ng / mL.

In view of the above results, according to the lateral flow immunoassay device of the present invention, it is confirmed that the test time is fast, and the separation of red blood cells is excellent and the binding reaction time of the target is increased, thereby improving signal sensitivity.

10: sample pad 20: detection unit
11: red blood cell separation pad 21: control unit
12: bonding pad
13: membrane
14: absorption pad
15: base member

Claims (11)

A lateral flow immunoassay device for quantitatively or qualitatively measuring analytes in whole blood samples,
A sample pad to which a whole blood sample comprising the analyte is applied;
Red blood cell separation pad for separating red blood cells by pore size;
A junction pad that binds specifically to the analyte and is provided with a diffuseable conjugate comprising a first binder conjugated with a primary label; And
Membranes including a detection site to which a second binder specifically binding to the analyte is immobilized are arranged in sequence,
The relationship of P1>P2> P3 is established when the average pore sizes of the sample pad, the erythrocyte separation pad and the junction pad are P1, P2 and P3, respectively.
The device of claim 1, wherein the sample pad comprises a polyvalent cation to chemically separate erythrocytes from a whole blood sample.
The lateral flow immunoassay device of claim 1, wherein the average diameter of the pores of the sample pad is greater than 8 μm and no greater than 15 μm.
The lateral flow immunoassay device of claim 1, wherein the average diameter of the pores of the erythrocyte separation pad is 6 to 8 μm.
The device of claim 1, wherein the average diameter of the pores of the bond pad is less than 2-6 μm.
The lateral flow immunoassay device of claim 1, wherein the flow immunoassay device comprises a second junction pad further containing a secondary marker that binds to the primary marker and amplifies the signal.
The lateral flow immunoassay device of claim 1, wherein the marker is gold nanoparticles, chromogenic enzymes, or fluorescent material.
The method of claim 1, wherein the analyte is an antibody, antigen, nucleic acid aptamer, hapten, antigen protein, DNA, DNA-binding protein, hormone, tumor-specific. Lateral flow immunoassay device, characterized in that it is selected from the group consisting of a marker (tumor-specific marker) and a tissue-specific marker.
The lateral flow immunity of claim 1, wherein the first binder and the second binder are selected from antibodies, antigens, nucleic acid aptamers, haptens, antigenic proteins, DNA, DNA-binding proteins and hormone-receptors. Analysis device.
The device of claim 1, wherein the membrane further comprises a control site for error checking.
The device of claim 1, further comprising an absorption pad capable of absorbing the whole blood sample by capillary action.

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