KR20220007021A - Reagents for immunochromatography including latex beads, linkers and antibodies, and rapid kits comprising the same - Google Patents

Abstract

The present invention relates to a reagent composition for immunochromatography comprising latex beads, linkers, and antibodies, and a rapid kit comprising the same. A diagnostic kit using the latex beads of the present invention introduces, to the latex beads, a first linker and a second linker which have different combinable lengths to antibodies so that the loading rate of antibodies in an antibody-latex bead complex is increased and a physical network is constructed, thereby significantly improving detection efficiency compared to the existing diagnostic kit.

Description

Reagents for immunochromatography including latex beads, linkers and antibodies, and rapid kits comprising the same}

The present invention relates to a reagent for immunochromatography comprising latex beads, a linker, and an antibody, and a rapid kit comprising the same, and more particularly, a first linker, a second linker capable of binding an antibody to an antigen or hormone to be detected It relates to a diagnostic kit using a combined form of latex beads and a diagnostic method using the same.

Immunochromatographic analysis is a method that can qualitatively and quantitatively analyze a trace amount of an analyte in a short time using an antigen-antibody reaction, and includes diagnosis or examination of various diseases, medicine, agriculture, livestock industry, food , military, environment, etc. are used in various fields. Pregnancy diagnostic kits widely used in homes and hospitals use this immunochromatographic analysis method. In such immunochromatographic analysis, an assay strip including a reactant capable of reacting with a target material to be detected and showing a change or a device-type assay device in which the assay strip is mounted in a plastic case is generally used. . A typical assay strip includes a sample (specimen) pad containing a liquid sample; a conjugate pad containing a conjugate in which a labeling material (eg, gold particles, latex beads, etc.) that generates a signal that can be detected with the naked eye or a sensor is conjugated to a ligand such as an antigen or an antibody; a detection pad (mainly a porous membrane pad) having a test line immobilized with a target substance in the sample and/or a binding agent (antibody or antigen) specifically binding to the conjugate and a control line capable of confirming the development of the sample; and a moisture absorption pad finally receiving the liquid sample, and these functional pads are connected in a partially overlapped form in the order listed above and are attached to the solid support and arranged continuously. When the assay strip is mounted inside a plastic case, there is a sample inlet for dropping the sample to the position of the sample pad on the upper part of the case, and a result confirmation window for checking the test result at the location where the binder of the detection pad is fixed. is formed As such, in the immunochromatographic analysis method using the assay strip, when a liquid sample is dropped onto the sample pad, the liquid sample moves through the conjugate pad and the detection pad by capillary action, and is finally accommodated in the moisture absorption pad.

Diagnostic kits using conventional immunochromatography have a limited detection range, resulting in false negative results due to "hook effect". That is, a hook effect is observed when a high analyte concentration in the test sample actually causes a decrease in the assay response signal for the analyte, resulting in the reporting of a false negative or false low concentration result.

The hook effect is based on the saturation curve of an antibody with antigen and occurs when the concentration of analyte in the test sample exceeds the binding capacity of the antibody used in the assay reagent, resulting in incomplete formation of the immune complexes necessary for the generation of the signal. . For example, an excessively high concentration of analyte can simultaneously saturate both capture and detection antibodies used in an immunoassay, resulting in the "hook" or falsely reduced concentration measurement shown in FIG. 1 . In these examples, false negatives or low false positives are reported, which can negatively affect the accuracy of the analyzer and have a devastating effect on patient management.

In order to solve this problem, the present inventors confirmed that when linkers of different lengths are introduced into the latex beads, the loading rate (binding ratio) of the latex beads and the antibody is increased, and the detection efficiency is significantly improved by constructing a physical network shape, and the present invention was completed.

Republic of Korea Patent Publication No. 10-2009-0101823

An object of the present invention is latex beads; a first linker; a second linker; and an antibody; to provide a reagent composition for immunochromatography comprising.

Another object of the present invention is to provide a rapid kit comprising the reagent composition for immunochromatography.

Another object of the present invention is to provide a method for providing information for determining the presence of an antigen or hormone.

Another object of the present invention is to provide a method for quantitative analysis of an antigen or hormone present in a biological sample.

In order to achieve the above object,

The present invention is a latex bead; a first linker; a second linker; and an antibody; wherein the first linker and the second linker have reactive functional groups at both ends, and the main chain length of the second linker is longer than the main chain length of the first linker, One end of the first linker and the second linker is bound to a reactive functional group on the surface of the latex bead, and one end of the remaining first linker and the second linker is bound to the antibody. provides

In addition, the present invention provides a rapid (rapid) kit comprising the reagent composition for immunochromatography.

Furthermore, the present invention provides the steps of contacting a biological sample with an antibody using the kit (step 1); and detecting the binding of the antibody to the antigen or hormone to be detected in the test line (step 2); provides a method for providing information for determining whether an antigen or hormone is present.

In addition, the present invention comprises the steps of injecting a biological sample into the sample pad of the kit and developing the strip (step 1); It provides a method for quantitative analysis of an antigen or hormone present in a biological sample, comprising the step of measuring the intensity of the spectral signal from the test line, and measuring the amount of the antigen or hormone (step 2).

The diagnostic kit using the latex beads of the present invention introduces a first linker and a second linker having different lengths capable of binding to the antibody in the latex beads to increase the loading rate of the antibody in the antibody-latex bead complex and to build a physical network. It has the effect of significantly improving the detection efficiency compared to the diagnostic kit.

1 shows a schematic image of the antibody-latex bead complex 100 into which the first linker 120 and the second linker 130 are introduced.
Figure 2 is a schematic diagram of a rapid kit according to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

Reagent composition for immunochromatography

The present invention is a latex bead; a first linker; a second linker; and an antibody; wherein the first linker and the second linker have reactive functional groups at both ends, and the main chain length of the second linker is longer than the main chain length of the first linker, One end of the first linker and the second linker is bound to a reactive functional group on the surface of the latex bead, and one end of the remaining first linker and the second linker is bound to the antibody. provides

The latex beads include polystyrene, polyvinyltoluene, polystyrene-acrylic acid, polyacrolein, polyacrylate, polyacrylamide, derivatives thereof, etc. It can be used, and any latex beads used in conventional immunochromatography can be used without any restrictions.

As the first linker, PEG (polyethylene glycol) having 1 to 5 repeating units, a peptide having an amino acid sequence of 1 to 5, a compound represented by the following Chemical Formula 1, etc. may be used alone or in combination.

[Formula 1]

In the above formula,

x is an integer from 1 to 5;

As the second linker, the second linker may be PEG (polyethylene glycol) having 6 to 15 repeating units, a peptide having an amino acid sequence of 6 to 15, a compound represented by the following formula (2), alone or in combination. have.

[Formula 2]

In the above formula,

y is an integer from 6 to 15;

In addition, the antibody is not particularly limited as long as it can bind directly to latex beads or indirectly using a linker, and all kinds of antibodies (eg, Corona virus-19 antibody, influenza antibody , pregnancy-related anti-hormonal antibodies, etc.) can be used. As used herein, the term “antibody” refers to a protein molecule serving as a receptor that specifically recognizes an antigen or hormone, and may include polyclonal antibodies, monoclonal antibodies, whole antibodies, antibody fragments, and the like. have.

Hormones in the pregnancy-related anti-hormonal antibody include human chorionic gonadotropin, luteinizing hormone, follicle-stimulating hormone, estradiol, progesterone, It includes testosterone and the like, and any hormone that can be used for diagnosing pregnancy can be used.

The term "reactive functional group" used in the present invention may be any functional group capable of forming a covalent bond through a chemical reaction in organic chemistry, and as an example, an amine group, a carboxyl group, an aldehyde group, an amidine group, a sulfate group, a chloromethyl group, etc. include

As used herein, the term “main chain” refers to a molecular structure constituting the main axis direction of a linker.

As used herein, the term “bond” refers to a covalent bond, an ionic bond, passive adsorption, and the like.

rapid kit

The present invention provides a rapid kit comprising the reagent composition for immunochromatography.

The kit comprises (a) a sample pad for receiving a biological sample;

(b) the antibody, the first linker, the second linker and the antibody-latex bead bound to the reagent composition for immunochromatography comprising a latex bead complex, wherein the antigen or hormone present in the biological sample and the complex a conjugate pad that serves as a site for the antibodies to bind to each other;

(c) a membrane on which a test line to which an antibody to which the antigen or hormone-binding complex binds is immobilized and a control line to which an antibody to which the complex binds are immobilized are printed; and

(d) an absorbent pad;

may be included by sequentially connecting them (see FIG. 2 ).

The conjugate pad is to include a reagent composition for immunochromatography including the complex 100 to which the antibody 140, the first linker 120, the second linker 130, and the latex bead 110 are bound. can

The membrane may be configured to immobilize an antibody to which the antigen or hormone-bound complex binds and a control antibody. The membrane is not particularly limited thereto, but preferably a nitrocellulose membrane to which an antibody can be bound, a polyvinylidene fluoride (PVDF) membrane, or the like may be used.

The antibody of (b) and the antibody of (c) may bind to different sites of the antigen or hormone to be detected.

The latex bead 110 is used for the purpose of improving the binding surface area of the antibody, and in the present invention, a first linker 120 and a second linker 130 having different lengths to the latex bead 110 are one step further. ) to increase the antibody loading rate (binding ratio) of the antibody-latex bead complex 100 and build a physical network shape to directly bind an antibody that binds an antigen or hormone to the surface of an existing latex bead, compared to the detection efficiency aspect may have a beneficial effect on

The biological sample may include tissue, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine isolated from a patient suspected of having a disease or a woman of childbearing age.

The kit may include a biological sample development solution. The buffer solution contained in the developing solution is not particularly limited thereto, as long as it exhibits the effect of assisting the sample delivery and the antigen or hormone-antibody binding reaction, but borate buffer solution, Tris buffer solution, phosphate buffer solution, etc. may be used. In addition, the biological sample developing solution may include BSA, albumin, Triton X-100, Tween 20, casein, etc. capable of inhibiting a non-specific reaction.

Assay systems for use in the kit include, but are not limited to, ELISA plates, dip-stick devices, immunochromatographic methods, radiation division immunoassay devices, flow-through devices, and the like. Preferably, a diagnostic kit in the form of a strip or a device using an immunochromatographic assay (ICA) may be used.

A method of providing information for determining the presence of an antigen or hormone

The present invention comprises the steps of contacting a biological sample with an antibody using the rapid kit (step 1); and detecting the binding of the antibody to the antigen or hormone to be detected in the test line (step 2); provides a method for providing information for determining whether an antigen or hormone is present.

Quantitative analysis method

The present invention provides a method for quantitative analysis of an antigen or hormone present in a biological sample using the kit.

The method comprises the steps of injecting a biological sample into a sample pad of the rapid kit and developing it on a strip (step 1); and measuring the intensity of the spectral signal from the test line to measure the amount of the antigen or hormone (step 2).

Here, the spectral signal may be used without any restrictions as long as it is a conventional spectrometer.

Hereinafter, the present invention will be described in more detail through examples. These Examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these Examples.

<Example 1> Preparation of Immunochromatography Rapid Kit

1-1. Preparation of latex beads with linkers of different lengths attached

400nm polystyrene particles (latex beads) containing a carboxyl group at the terminal, a _{peptide having a molecular formula of H 2} N(CH ₂ ) ₃ NH ₂ (first linker), a peptide having a molecular formula of H ₂ N(CH ₂ ) ₁₀ NH ₂ After preparing the (second linker), the first linker and the second linker were attached to the latex beads using a carbodiimide method to prepare latex beads having different lengths of linkers attached thereto.

Specifically, a carbodiimide reagent was added to a 12.5 mM sodium phosphate buffer at pH 5 containing the polystyrene particles (latex beads) and incubated at room temperature for 2-3 minutes. , The mixture was added to a 0.2 M sodium phosphate buffer at pH 8 containing the first linker and the second linker, and reacted at room temperature for 24 hours. Then, the reaction mixture was dialyzed with 0.1 M sodium phosphate of pH 8 to remove linkers not attached to the latex beads, thereby obtaining latex beads having different lengths of linkers attached thereto.

1-2. Of the reagent composition for immunochromatography comprising latex beads Produce

Anti-I-hCG antibody (Monoclonal anti-hCG) was added to MES buffer (2-(N-mophorino)ethane sulfonic acibiffer) to prepare 250 μg/600 μl MES of HCG antibody. Meanwhile, 200 μl of a 2% latex solution obtained by adding the latex beads prepared in Example 1-1 to MES buffer was prepared, and this latex solution was slowly added to the monoclonal antibody, reacted at 25° C. for 2.5 hours, and then 22,000 g The unbound hCG antibody was removed by centrifugation, and the precipitate was released with a phosphate buffer (pH 7.2) containing 4% bovine serum albumin. In this state, the reaction was carried out at 25° C. for 2 hours or more, and the region where the antibody was not bound to the latex was bound with bovine serum albumin, and then centrifuged at 22,000 g and washed with a phosphate buffer. The washed latex was diluted with a phosphate buffer to a final content of 1% to prepare a first conjugate solution.

Next, a second conjugate solution to which the latex beads prepared in Example 1-1 and mouse immunoglobulin (Mouse IgG) were bound was prepared in the same manner as above.

1-3. Preparation of Immunochromatographic Rapid Kit

A. Preparation of Membrane Containing Test Line (T) and Control Line (C)

Two analysis lines were dispensed on a nitrocellulose membrane. A nitrocellulose membrane was laminated on a plastic card using a laminator.

에서 2일(48시간) 동안 건조시켰다.Specifically, as a test line, a monoclonal anti-human chorionic gonadotropin antibody (moclonal anti-hCG, Medix, Finland), and as a control line, a goat anti-mouse immunoglobulin antibody (antimouse antibody from goat, arista, USA) were dispensed in a straight line at regular intervals using an automatic dispenser and adsorbed, then 18~22

dried for 2 days (48 hours).

Here, the membrane laminated on the plastic card is dispensed with a width of 300 mm x length of 1 mm, and it is cut in units of 4 mm. The furnace area was set to 4 mm2.

B. Preparation of conjugate pad

The pad was pre-treated by fully wetting the pad with Tris buffer (10 mM, pH 8.5) containing 0.5% PVA (polyvinylalcohol) and 0.5% BSA (Bovine Serum Albumin) and drying it completely in a dryer.

Then, the first conjugate solution and the second conjugate solution of Example 1-2 were dispensed on the pretreated conjugate pad, dried completely, and cut to an appropriate size.

C. Preparation of sample pads

A glass fiber pad was sufficiently wetted with 0.08 M borate buffer containing 0.05% Triton X-100, dried completely in a dryer, and cut to an appropriate size.

D. Preparation of absorbent pad

The moisture was completely dried in the dryer and used as it is without any treatment, and it was prepared by cutting it to an appropriate size.

E. Fabrication of Immunochromatographic Strips

The sample pad, the conjugate pad, the membrane, and the moisture absorption pad prepared through each of the above processes were sequentially assembled to prepare an immunochromatographic strip. Specifically, one end of the conjugate pad is attached to overlap one end of the sample pad, one end of the membrane is attached to the other end of the conjugate pad to overlap, and one end of the moisture absorption pad is overlapped with the other end of the membrane. attached as much as possible.

F. Case assembly

After putting the immunochromatographic strip into the fixed position of the strip of the plastic lower case, insert the sample inlet and the upper case with the result confirmation window, and press it with a press to assemble.

<Comparative Example 1> Preparation of Immunochromatography Rapid Kit

A rapid kit was prepared in the same manner as in Example 1 except that only the first linker was used in Example 1-1.

<Experimental Example 1> Immunochromatographic strip analysis according to hCG concentration

Using the rapid kits prepared in Example 1 and Comparative Example 1, respectively, experiments were conducted as follows to determine the accuracy of quantitative analysis of human chorionic gonadotropin (hCG) as a pregnancy diagnostic marker according to latex beads.

Specifically, spiking of human chorionic gonadotropin (hCG) standards in non-pregnant urine at concentrations of 0mIU/ml, 100mIU/ml, 200mIU/ml, 400mIU/ml, 800mIU/ml, 1600mIU/ml, 3200mIU/ml to prepare a standard sample. After dripping 100 μl of the prepared standard sample solution onto the sample pad of the rapid kit, it was developed for 5 minutes. Next, the signal intensity displayed on the test line was quantitatively analyzed with a spectrometer, and the result was compared with the concentration of a standard sample to evaluate the accuracy of quantitative analysis.

Quantitative accuracy percentage (%) hCG concentration (mIU/ml) Comparative Example 1 Example 1 0 98 99 100 71 95 200 74 98 400 75 96 800 83 96 1600 79 94 3200 81 98

As a result, as shown in Table 1, it was found that the accuracy of quantitative analysis was significantly improved when linkers of different lengths were attached to the latex beads.

<Experimental Example 2> False negative evaluation by high concentration hCG

Experiments were conducted as follows to determine whether a sample containing a high concentration of human chorionic gonadotropin (hCG) was false-negative using the rapid kits prepared in Example 1 and Comparative Example 1, respectively.

Specifically, a standard sample was prepared by spiking human chorionic gonadotropin (hCG) standards in non-pregnant urine at concentrations of 100,000mIU/ml, 200,000mIU/ml, 400,000mIU/ml, and 800,000mIU/ml. After dispensing 100 μl of the standard sample solution on the sample pad of the rapid kit and developing it for 5 minutes, the inspection results were visually determined. The intensity of the signal appearing in the test line (T) and the control line (C) was visually confirmed, and the signal intensity of the test line was relatively evaluated based on the intensity of the control line as ++. The false negative evaluation result of Example 1 is shown in Table 2, and the false negative evaluation result of Comparative Example 1 is shown in Table 3.

hCG concentration (mIU/ml) 100,000 200,000 400,000 800,000 Inspection line (T) strength +++ +++ +++ ++ Control line (C) intensity ++ ++ ++ ++

hCG concentration (mIU/ml) 100,000 200,000 400,000 800,000 Inspection line (T) strength ++ + +/- - Control line (C) intensity ++ ++ ++ ++

+++: strong detection signal++: good detection signal

+: Weak detection signal

+/-: almost no detection signal

-: not detected

As a result, in the case of the rapid kit of Comparative Example 1, in the sample containing 400,000 mIU/ml or more of hCG, the band was faintly colored or did not appear at all, confirming the presence of false negatives (Table 3).

However, in the case of the rapid kit of Example 1, even in a sample containing hCG at a concentration of 800,000 mIU/ml, color development was visually discernible (Table 2).

As such, in the case of diagnosis using the immunochromatographic reagent composition comprising the latex beads with linkers of different lengths of the present invention, the detection efficiency is remarkably improved as well as the hook effect by constructing a physical network shape. By reducing it, it is possible to accurately diagnose disease or pregnancy even in a sample containing a high concentration of antigen.

So far, with respect to the present invention, the preferred embodiments have been looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is particularly indicated in the claims rather than the foregoing description, and all differences within an equivalent scope should be construed as being included in the present invention.

100: antibody-latex bead complex
110: latex beads
120: first linker
130: second linker
140: antibody

Claims (11)

latex beads;
a first linker;
a second linker; and
antibody;
Including a complex comprising,
The first linker and the second linker have reactive functional groups at both ends, and the main chain length of the second linker is longer than the main chain length of the first linker,
One end of the first linker and the second linker is bound to a reactive functional group on the surface of the latex bead, and one end of the remaining first linker and the second linker is bound to the antibody,
A reagent composition for immunochromatography.
According to claim 1,
The latex beads are selected from polystyrene, polyvinyltoluene, polystyrene-acrylic acid, polyacrolein, polyacrylate, polyacrylamide and derivatives thereof. A reagent composition for immunochromatography, characterized in that it is a polymer material.
According to claim 1,
The antibody is a corona virus-19 (Corona virus-19) antibody, influenza antibody, or a reagent composition for immunochromatography, characterized in that the pregnancy-related anti-hormonal antibody.
4. The method of claim 3,
Hormones in the pregnancy-related anti-hormonal antibody include human chorionic gonadotropin, luteinizing hormone, follicle-stimulating hormone, estradiol, progesterone and A reagent composition for immunochromatography, comprising at least one selected from the group consisting of testosterone.
According to claim 1,
The first linker is a reagent composition for immunochromatography, characterized in that at least one of polyethylene glycol (PEG) having 1 to 5 repeating units, a peptide having an amino acid sequence of 1 to 5, and a compound represented by Formula 1 below .
[Formula 1]

(In Formula 1,
x is an integer from 1 to 5.)
According to claim 1,
The second linker is a reagent composition for immunochromatography, characterized in that at least one of polyethylene glycol (PEG) having a repeating unit of 6 to 15, a peptide having an amino acid sequence of 6 to 15, and a compound represented by the following Chemical Formula 2 .
[Formula 2]

(In Formula 1,
y is an integer from 6 to 15.)
A rapid kit comprising the composition of any one of claims 1 to 6.
8. The method of claim 7,
The rapid kit,
(a) a sample pad for receiving a biological sample;
(b) a conjugate pad comprising the reagent composition for immunochromatography comprising the complex of claim 1, wherein the antigen or hormone present in the biological sample and the antibody of the complex bind to each other;
(c) a test line on which an antibody binding to the antigen or hormone-bound complex is immobilized and a control line on which an antibody binding to the complex is immobilized, respectively, printed on a membrane; and
(d) an absorbent pad;
Rapid kit, characterized in that it comprises sequentially connected.
9. The method of claim 8,
The antibody of (b) and the antibody of (c) are a kit, characterized in that it binds to different sites of the antigen or hormone to be detected.
The method of any one of claims 7 to 9, comprising the steps of contacting a biological sample with an antibody (step 1); and
Detecting the binding of the antigen or hormone to be detected with the antibody in the test line (step 2);
A method of providing information for determining the presence of an antigen or hormone.
10. A method comprising: injecting a biological sample into the sample pad of any one of claims 7 to 9 and developing it on a strip (step 1); and
Including, by measuring the intensity of the spectral signal from the test line, measuring the amount of the antigen or hormone (step 2);
A method for quantitative analysis of an antigen or hormone present in a biological sample.

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