Bladder cancer diagnostic kit using NMP22 as biomarker

Abstract

The present invention relates to a bladder cancer diagnostic kit using Nuclear Matrix Protein 22 (NMP22) as a biomarker, the kit comprising: a support corresponding to a base body of a diagnostic strip accommodated and installed in an inner space of a diagnostic kit body; a sample pad which is attached to the upper part of the support and into which a sample, corresponding to urine of a subject to be diagnosed, and a buffer are injected; test films attached to the upper part of the support by being sequentially connected to the sample pad; a test line which is coated on the test films and to which an anti-NMP22 antibody is fixed; and a control line which is coated on the test films and positioned spaced apart from the test line, and to which a Chicken IgY antibody is fixed, wherein the buffer includes a first complex in which Europium (Eu)-based fluorescent beads and an anti-Chicken IgY antibody are bound, and a second complex in which Europium (Eu)-based fluorescent beads and an anti-NMP22 antibody are bound.

Description

Bladder cancer diagnostic kit using NMP22 as a biomarker

[0001] The present invention relates to a bladder cancer diagnostic kit using NMP22 as a biomarker.

[0002] Bladder cancer diagnostic technologies are broadly classified into imaging tests, cytological examinations, and biomarker detection methods. Each method is used to accurately diagnose bladder cancer, and recently, these diagnostic technologies are evolving in the form of diagnostic kits that can be conveniently used outside of hospitals.

[0003] Since bladder cancer has a high treatment efficacy when detected early, there is a growing need for non-invasive yet highly accurate diagnostic technologies. Accordingly, detection technologies utilizing various biomarkers are playing an important role, and in particular, biomarkers such as NMP22 (Nuclear Matrix Protein 22) are establishing themselves as important indicators in the diagnosis of bladder cancer.

[0004] Among these, technologies related to bladder cancer diagnosis using biomarkers are developing into a useful method for early diagnosis and patient monitoring because they can be performed non-invasively and conveniently by checking for the presence of specific biomarkers in urine to determine the presence of cancer. Representative examples include NMP22, BTA, and FISH tests. In particular, NMP22 is a nuclear matrix protein that is detected at high concentrations in the urine of bladder cancer patients and is utilized as a major biomarker for bladder cancer.

[0005] Bladder cancer diagnostic technology in the form of diagnostic kits, which is being developed based on such biomarker detection methods, miniaturizes and automates existing laboratory-based detection methods to enable rapid detection of bladder cancer biomarkers in urine, thereby making diagnosis possible without visiting a hospital. Recently developed diagnostic kits possess high sensitivity and specificity, allowing for more efficient bladder cancer diagnosis.

[0006] In this regard, prior art for providing a bladder cancer diagnostic kit configured to quantitatively determine pathological changes in bladder cancer by providing multiple detection lines for NMP22, UBC, and BTA biomarkers, and capable of increasing sensitivity and specificity by using quantum dots of different sizes for each biomarker, includes Chinese Registered Patent Publication CN 108593922, "Joint detection kit for measuring bladder cancer and preparation method thereof" (hereinafter referred to as "prior art").

[0007] However, in the case of existing bladder cancer diagnostic kit technologies utilizing NMP22 as a biomarker, including conventional technologies, interference between other fluorescent substances may occur during fluorescence detection, making accurate signal detection difficult. Furthermore, the detection sensitivity required for the actual diagnosis of bladder cancer is still low, resulting in problems of reduced diagnostic accuracy and reliability.

[0008] The present invention was created to solve the above problems, and the objective of the present invention is to provide a diagnostic kit that minimizes interference of fluorescent signals by specifying the compositional characteristics of the components in the buffer injected into the sample pad after mixing with the sample, including the components dispensed to the detection line and control line within the strip, and to enhance the level of diagnostic accuracy and reliability by increasing detection sensitivity when performing quantitative analysis by detecting the concentration of NMP22, a bladder cancer biomarker, in urine.

[0009] To achieve the above objective, the bladder cancer diagnostic kit utilizing NMP22 as a biomarker according to the present invention comprises: a support corresponding to the base body of a diagnostic strip installed in the internal space of the diagnostic kit body; a sample pad attached to the upper part of the support, into which a specimen corresponding to the urine of the diagnostic subject and a buffer are injected; a detection film attached to the upper part of the support in a manner sequentially connected to the sample pad; and a detection (test) line formed by coating on the detection film, on which an anti-NMP22 (Nuclear Matrix Protein 22) antibody is immobilized. and a control line formed by coating at a position spaced apart from the detection line of the detection membrane and having a Chicken IgY antibody immobilized thereon; wherein the buffer comprises a first complex in which a Europium (Eu)-based fluorescent bead is combined with an anti-Chicken IgY antibody and a second complex in which a Europium (Eu)-based fluorescent bead is combined with an anti-NMP22 (Nuclear Matrix Protein 22) antibody.

[0010] Here, the detection membrane is formed such that the control line and the detection line are sequentially formed based on the sample pad side, the anti-NMP22 antibody included in the detection line is prepared at a concentration of 1.8 mg / mL to 2.2 mg / mL, and the Chicken IgY antibody included in the control line is prepared at a concentration of 0.8 mg / mL to 1.2 mg / mL.

[0011] In addition, the europium-based fluorescent beads combined in the first and second complexes included in the buffer have an excitation wavelength of 330 nm to 340 nm and an emission wavelength of 610 nm to 620 nm.

[0012] In addition, the first complex included in the buffer is prepared by treating europium-based fluorescent beads with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to prepare them in a state capable of antibody binding, and then reacting 1.3 to 1.7 parts by weight of anti-Chicken IgY antibody with 10 parts by weight of the europium-based fluorescent beads prepared in a state capable of antibody binding through stirring.

[0013] In addition, the second complex included in the buffer is prepared by treating europium-based fluorescent beads with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to prepare them in a state capable of antibody binding, and then reacting 0.8 to 1.2 parts by weight of anti-NMP22 antibody with 10 parts by weight of the europium-based fluorescent beads prepared in a state capable of antibody binding through stirring.

[0014] In addition, the first complex included in the buffer is provided in an amount of 0.8 to 1.2 parts by volume relative to the total volume of the sample and buffer injected into the injection pad, which is 100 parts by volume, and the second complex included in the buffer is provided in an amount of 0.8 to 1.2 parts by volume relative to the total volume of the sample and buffer injected into the injection pad, which is 100 parts by volume.

[0015] According to the present invention, by specifying the compositional characteristics of complexes formed by the binding of Europium (Eu)-based fluorescent beads and specific antibodies in a buffer injected into a sample pad after mixing with a sample, including components dispensed to the detection line and control line within the strip, interference of fluorescent signals is minimized, and a diagnostic kit can be provided that enhances the level of diagnostic accuracy and reliability by increasing detection sensitivity when performing quantitative analysis by detecting the concentration of NMP22, a bladder cancer biomarker, in urine.

[0016] FIG. 1 is a cross-sectional view illustrating the structure of a diagnostic strip of a bladder cancer diagnostic kit using NMP22 of the present invention as a biomarker.

[0017] FIG. 2 is a diagram illustrating the process of forming a complex by binding an antibody to a europium-based fluorescent bead included in a buffer injected into a sample pad in a bladder cancer diagnostic kit using NMP22 of the present invention as a biomarker.

[0018] Figure 3 is a graph showing the results of analyzing the change in fluorescence intensity according to NMP22 concentration using the bladder cancer diagnostic kit of the present invention that utilizes NMP22 as a biomarker.

[0019] Figure 4 is a graph showing the results of quantifying fluorescence intensity according to NMP22 concentration using the bladder cancer diagnostic kit of the present invention that utilizes NMP22 as a biomarker.

[0020] Preferred embodiments of the present invention will be described in more detail with reference to the attached drawings, provided that technical details that are already well known are omitted or compressed for the sake of brevity.

[0021] 1. Description of a bladder cancer diagnostic kit using NMP22 as a biomarker

[0022] Referring to FIGS. 1 and 2, the bladder cancer diagnostic kit (100) using NMP22 as a biomarker according to the present invention is formed such that a diagnostic strip, as shown in FIG. 1, is received and installed within a separate receiving space provided inside the entire housing of the diagnostic kit (100), which corresponds to the body of the diagnostic kit.

[0023] More specifically, the diagnostic strip received and installed in the body of the diagnostic kit includes a support (110), a sample pad (120), a detection film (130), an absorption pad (140), a control line (150), and a detection line (160).

[0024] Here, a sample injection port is formed on one side of the upper part of the diagnostic kit body to provide an open space into which a sample and buffer can be injected into the sample pad (120) of the diagnostic strip.

[0025] In addition, a transparent diagnostic result observation window may be formed on one side of the upper part of the diagnostic kit body to expose the detection result through the control line (150) and detection line (160) within the detection film (130) to the outside.

[0026] In addition, the diagnostic strip is configured to be received and installed within the body of the diagnostic kit, and to directly perform a diagnosis using a lateral flow immunochromatographic assay (LFIA) of the injected specimen and buffer.

[0027] First, the support body (110) provides a base body to which a sample pad (120), a detection film (130), an absorption pad (140), a control line (150), and a detection line (160) are attached to the top and connected sequentially, as shown in FIG. 1.

[0028] Specifically, as shown in FIG. 1, a sample pad (120), a detection membrane (130), and an absorption pad (140) are attached in sequence to the upper part of the support (110), starting with the sample pad (120), and the detection membrane (130) and the absorption pad (140) are connected in sequence so that the sample injected into the sample pad (120) can move through the detection membrane (130) and finally reach the absorption pad (140) by capillary action.

[0029] Additionally, a control line (150) and a detection line (160) are formed sequentially on the detection film (130), and the detection line (150) is located on the sample pad (120) side and the control line (160) is located on the absorption pad (140) side to form a sequential structure.

[0030] First, the sample pad (120) is attached to the upper part of the support (110) and is an area into which a sample containing ferritin and a buffer are injected, and is located at the foremost end of the path of movement according to capillary action of the sample containing ferritin and the buffer.

[0031] As an example, the sample pad (120) can be made in a 20mm size using ‘Grade 8964, AHLSTROM’ and bonded to the detection film (130) in a structure that overlaps by 1mm.

[0032] Here, the material injected into the sample pad (120) consists of a sample corresponding to the urine of the diagnostic subject and a buffer.

[0033] Additionally, the buffer injected into the sample pad (120) includes a first complex in which a europium (Eu)-based fluorescent bead is combined with an anti-Chicken IgY antibody and a second complex in which a europium (Eu)-based fluorescent bead is combined with an anti-NMP22 (Nuclear Matrix Protein 22) antibody.

[0034] It is preferable that the europium-based fluorescent beads bound to antibodies in the first complex and the second complex, respectively, included in the buffer have an excitation wavelength of 330 nm to 340 nm and an emission wavelength of 610 nm to 620 nm.

[0035] This is because it allows for cleaner measurement of the light emitted by fluorescent beads by utilizing the fact that there is no signal interference, as the light of the excitation wavelength does not affect the synchrotron radiation by forming a wide Stokes shift, thereby reducing interference between other fluorescent materials and enabling reliable results.

[0036] Most preferably, the europium-based fluorescent beads bound to antibodies in the first complex and the second complex, respectively, included in the buffer, provide excitation at a wavelength of 333 nm and emission at a wavelength of 613 nm to produce a wide Stokes shift between 333 nm and 613 nm.

[0037] The structure in which a Europium (Eu)-based fluorescent bead corresponding to the first complex among these buffer complexes and an anti-Chicken IgY antibody are combined undergoes a process as shown in Fig. 2, and the antibody is stably bound to the surface of the bead through the 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide chemical reaction.

[0038] The preparation process of the first complex included in this buffer is prepared by treating europium-based fluorescent beads with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to prepare them in a state capable of antibody binding, and then reacting 1.3 to 1.7 parts by weight (most preferably 1.5 parts by weight) of anti-Chicken IgY antibody with 10 parts by weight of the europium-based fluorescent beads prepared in a state capable of antibody binding through stirring.

[0039] Specifically, the manufacturing process of the first complex included in the buffer proceeds in the following steps: Step 1-1, preparing europium-based fluorescent beads; Step 1-2, preparing the prepared europium-based fluorescent beads for binding; Step 1-3, binding with an antibody; Step 1-4, stabilizing the complex; and Step 1-5, washing and storage treatment.

[0040] Here, in the preparation process of the first complex included in the buffer, step 1-1 is carried out by taking 100 µl of Fluore-Max Fluorescent Beads (9347-0520-010150, Thermo Fisher Scientific) stock solution with a concentration of 10 mg / ml, adding 1 mL of MES Buffer (pH 6.1), centrifuging for 15 minutes under conditions of a rotation speed of 14,500 rpm and a temperature of 4°C to remove the supernatant, and finally adding 900 µl of MES buffer and sonicating to evenly disperse the particles.

[0041] Subsequently, in the first step of the preparation process of the first complex included in the buffer, 80 µl of 50 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 20 µl of 500 mM N-hydroxysuccinimide are added to the europium-based fluorescent beads corresponding to the Fluore-Max Fluorescent Beads prepared through the first step, and stirred at a rotation speed of 180 rpm for 60 minutes at room temperature (25°C to 35°C) to prepare the mixture to a state capable of antibody binding.

[0042] Next, in the first step of the preparation process of the first complex contained in the buffer, 150 μg of Goat anti-Chicken IgY antibody (AGGAC-0500, Atrista Biologicals Inc) is mixed per 1 mg of europium-based fluorescent beads prepared in a state capable of binding to antibodies through the first step, and then MES (2-(N-morpholino)ethanesulfonic acid) buffer is added to make the total volume 1 mL, and the mixture is stirred for 2 hours under conditions of a rotation speed of 180 rpm and a temperature of 30°C so that the fluorescent beads bind to the surface of the anti-Chicken IgY antibody to form the first complex.

[0043] Here, an appropriate compositional condition is maintained in which 1.3 to 1.7 parts by weight of anti-Chicken IgY antibody react with 10 parts by weight of europium-based fluorescent beads prepared in a state capable of antibody binding. This is because if the anti-Chicken IgY antibody reacts in an amount less than 1.3 parts by weight relative to 10 parts by weight of europium-based fluorescent beads prepared in a state capable of antibody binding, the amount of antibody bound to the light beads is insufficient, which may cause the signal intensity in the control line to drop, and if it reacts in an amount exceeding 1.7 parts by weight, non-specific binding increases, causing the signal in the control line to become excessively strong, which may induce a false positive reaction.

[0044] Furthermore, in the preparation process of the first complex included in the buffer, step 1-4 involves adding the first complex, which was formed by combining through step 1-3, to 100 µl of 0.1 M glycine and stirring for 30 minutes under conditions of a rotation speed of 180 rpm and a temperature of 30°C to ensure stabilization.

[0045] Finally, in the manufacturing process of the first complex included in the buffer, step 1-5 involves centrifuging the product of step 1-4 for 15 minutes at a rotation speed of 14,500 rpm and a temperature of 4°C to remove the supernatant, then adding 1 mL of MES buffer, performing sonication, and then repeating the centrifugation process under the same conditions to discard the supernatant.

[0046] In addition, the process of adding 1 mL of washing solution, performing sonication, and then repeating centrifugation under the same conditions at least twice to wash the particles is followed by dispersing them in 1 mL of washing solution through sonication, and then storing them in a temperature environment of 4°C.

[0047] And the structure in which the Europium (Eu)-based fluorescent bead corresponding to the second complex in the buffer and the anti-Chicken IgY antibody are combined undergoes a process as shown in Fig. 2, and the antibody is stably bound to the surface of the bead through the 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide chemical reaction.

[0048] The process of preparing the second complex included in this buffer is as follows: europium-based fluorescent beads are prepared in a state capable of antibody binding by treating them with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and then 0.8 to 1.2 parts by weight (most preferably 1.0 parts by weight) of anti-NMP22 antibody is reacted with 10 parts by weight of the europium-based fluorescent beads prepared in a state capable of antibody binding through stirring.

[0049] Specifically, the manufacturing process of the second complex included in the buffer proceeds in the following steps: Step 2-1, preparing europium-based fluorescent beads; Step 2-2, preparing the prepared europium-based fluorescent beads for binding; Step 2-3, binding with an antibody; Step 2-4, stabilizing the complex; and Step 2-5, washing and storage treatment.

[0050] Here, in the process of manufacturing the second complex included in the buffer, step 2-1 is carried out by taking 100 µl of Fluore-Max Fluorescent Beads (9347-0520-010150, Thermo Fisher Scientific) stock solution with a concentration of 10 mg / ml, adding 1 mL of MES Buffer (pH 6.1), centrifuging for 15 minutes at a rotation speed of 14,500 rpm and a temperature of 4°C to remove the supernatant, and finally adding 900 µl of MES buffer and sonicating to evenly disperse the particles.

[0051] Subsequently, in the second step-2 of the preparation process for the second complex included in the buffer, 80 µl of 50 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 20 µl of 500 mM N-hydroxysuccinimide are added to the europium-based fluorescent beads corresponding to the Fluore-Max Fluorescent Beads prepared through the second step-1, and stirred at a rotation speed of 180 rpm for 60 minutes at room temperature (25°C to 35°C) to prepare the mixture to a state capable of antibody binding.

[0052] Next, in the second step of the preparation process for the second complex included in the buffer, 100 μg of anti-NUMA1 (NMP22) antibody (MB2026863, MyBioSource) is mixed per 1 mg of europium-based fluorescent beads prepared in a state capable of binding to the antibody through the second step, and then MES (2-(N-morpholino)ethanesulfonic acid) buffer is added to make the total volume 1 mL, and the mixture is stirred for 2 hours under conditions of a rotation speed of 180 rpm and a temperature of 30°C so that the fluorescent beads bind to the surface of the anti-NMP22 antibody to form the second complex.

[0053] Here, an appropriate compositional condition is maintained in which 0.8 to 1.2 parts by weight of anti-NMP22 antibody react with 10 parts by weight of europium-based fluorescent beads prepared in a state capable of antibody binding. This is because if the anti-NMP22 antibody reacts in an amount less than 0.8 parts by weight relative to 10 parts by weight of europium-based fluorescent beads prepared in a state capable of antibody binding, it cannot generate a sufficient signal in the detection line, which may result in reduced detection sensitivity, and if it reacts in an amount exceeding 1.2 parts by weight, non-specific binding increases, causing the signal intensity in the detection line to become excessively high, which increases the likelihood of false positives.

[0054] Furthermore, in the process of preparing the second complex included in the buffer, step 2-4 involves adding the second complex, which was formed by combining through step 2-3, to 100 µl of 0.1 M glycine and stirring for 30 minutes under conditions of a rotation speed of 180 rpm and a temperature of 30°C to ensure stabilization.

[0055] Finally, in the manufacturing process of the second complex included in the buffer, step 2-5 involves centrifuging the product of step 2-4 for 15 minutes at a rotational speed of 14,500 rpm and a temperature of 4°C to remove the supernatant, then adding 1 mL of MES buffer, performing sonication, and then repeating the centrifugation process under the same conditions to discard the supernatant.

[0056] In addition, the process of adding 1 mL of washing solution, performing sonication, and then repeating centrifugation under the same conditions at least twice to wash the particles is followed by dispersing them in 1 mL of washing solution through sonication, and then storing them in a temperature environment of 4°C.

[0057] It is preferable that the first complex and the second complex prepared in this embodiment be each prepared in an amount of 0.8 to 1.2 parts by volume relative to the total volume of 100 parts by volume of the sample and buffer, and then mixed together and injected into the sample pad (120).

[0058] According to the embodiment, 1 µl of the first complex and 1 µl of the second complex can be mixed with 98 µl of a sample corresponding to the urine of the subject to diagnosis and injected into the sample pad (120).

[0059] This is because if the first complex is injected in an amount of less than 0.8 parts by volume relative to the total volume of the sample and buffer of 100 parts by volume, the signal intensity in the control line is insufficient, making the reference signal of the control line unclear and causing difficulty in interpreting the results, and if it is injected in an amount exceeding 1.2 parts by volume, it generates a non-specific signal in the control line, reducing the differentiation between the control line and the detection line and potentially distorting the actual diagnostic results.

[0060] In addition, if the second complex is injected in an amount of less than 0.8 parts by volume relative to the total volume of the sample and buffer of 100 parts by volume, the presence of a low concentration of NMP22 biomarker weakens the signal, thereby reducing detection sensitivity and increasing the possibility of false negatives in the diagnostic results; and if it is injected in an amount exceeding 1.2 parts by volume, a non-specific signal may occur in the detection line due to the excessive amount of the second complex, and a positive reaction may appear even when the actual result is negative, which increases the possibility of false positives.

[0061] Next, the detection membrane (130) is attached to the upper part of the support (110) in a manner sequentially connected after the sample pad (120) as shown in FIG. 1, and is a porous membrane including a control line (150) and a detection line (160).

[0062] Here, the detection membrane (130) is a porous membrane made of a polymer material such as NC (nitrocellulose), PVDF (polyvinylidene fluoride), (charge modified) Nylon, or PES (Polyethersulfone), and depending on the polymer material of the detection membrane (124) provided, the binding form of the antibody can be applied as electrostatic binding or hydrophobic binding.

[0063] Here, the detection line (160) is formed by coating on the detection membrane (130), and an anti-NMP22 (Nuclear Matrix Protein 22) antibody having a concentration of 1.8 mg / mL to 2.2 mg / mL is fixedly installed.

[0064] As an example, a substance prepared at a concentration of 2.0 mg / mL using an anti-NUMA1 (NMP22) antibody (MBS2111277, MyBioSource) stock solution and a 1x PBS (pH 7.2) dilution solution is dispensed at a speed of 4 cm / sec using a dispenser device onto the upper surface of a nitrocellulose membrane (Nitrocellulose membrane, CN140, Satorius) to form a detection line (160).

[0065] Additionally, the control line (150) is formed by application at a position spaced apart from the detection line (160) of the detection membrane (130), and a Chicken IgY antibody having a concentration of 0.8 mg / mL to 1.2 mg / mL is fixedly installed.

[0066] As an example, a substance prepared at a concentration of 1.0 mg / mL using a stock solution of Chicken IgY antibody (ABGAC-0500, Atrista Biologicals Inc) and a 1x PBS (pH 7.2) dilution solution is dispensed at a speed of 4 cm / sec at a distance of 3.0 mm from the detection line (160) using a dispenser device to form a control line (150).

[0067] Finally, after the dispensing for forming such a control line (150) and detection line (160) is completed, the samples are stored for 24 hours in a dehumidifier with a humidity environment of 10% or less for use.

[0068] Finally, the absorbent pad (140) is attached to the upper part of the support (110) in a manner that is sequentially connected after the detection membrane (130) as shown in FIG. 1, and the components of the residual sample and buffer that have been removed from the detection membrane (130) are absorbed, and it is attached in a structure that overlaps the nitrocellulose membrane by 1 mm with the TN-666 (Woorichem) 18 mm standard.

[0069] In addition, the absorbent pad (140) serves to provide power for various moving substances, including the sample, due to capillary action.

[0070] The bladder cancer diagnostic kit (100) using NMP22 of the present invention, which has such compositional and structural characteristics, applies a sample prepared as urine to the diagnostic kit together with a buffer to derive a quantitative analysis of NMP22 levels through fluorescent bead-based luminescence analysis, thereby minimizing interference of fluorescent signals and increasing detection sensitivity when performing quantitative analysis by detecting the concentration of NMP22, a bladder cancer biomarker, in urine, thereby enhancing the level of diagnostic accuracy and reliability.

[0071] 2. Description of the quantitative analysis performance of a bladder cancer diagnostic kit using NMP22 as a biomarker

[0072] Below, in order to verify that the bladder cancer diagnostic kit (100) using NMP22 as a biomarker according to the present invention possesses excellent quantitative analysis performance through the previously described structural features, we intend to compare and explain the results of the intensity test of the marker substance according to the sample concentration for each sample and the derivation of a standard quantitative curve (calibration curve) based thereon.

[0073] First, when a urine sample is applied as a sample to the bladder cancer diagnostic kit (100) according to the present invention having the structural features described above to perform ferritin diagnosis, a Chicken IgY antibody (ABGAC-0500, Atrista Biologicals Inc) with a concentration of 1.0 mg / mL is immobilized on the control line (150) of the detection membrane (130) in the diagnostic strip, and an anti-NMP22 (Nuclear Matrix Protein 22) antibody (MBS2111277, MyBioSource) with a concentration of 2.0 mg / mL is immobilized on the detection line (160).

[0074] Next, NMP22 recombinant protein (MBS2023384, MyBioSource) was diluted to 50, 25, 10, 1.0, and 0.1 ng / mL using 1xPBS solution, and 1 µl of the first complex prepared by following the previously described steps 1-1 through 1-4 based on mixing 150 µg of Goat anti-Chicken IgY antibody (AGGAC-0500, Atrista Biologicals Inc) per 1 mg of europium-based fluorescent beads corresponding to Fluore-Max Fluorescent Beads (9347-0520-010150, Thermo Fisher Scientific), and anti-NUMA1 (NMP22) per 1 mg of europium-based fluorescent beads corresponding to Fluore-Max Fluorescent Beads (9347-0520-010150, Thermo Fisher Scientific) Based on mixing 100 μg of antibody (MB2026863, MyBioSource), 1 μl of the second complex prepared by following steps 2-1 to 2-4 described above was mixed with 98 μl of NMP22 recombinant protein at each concentration and injected into sample pads (120) to perform fluorescence signal analysis using a fluorescence signal analyzer, and the results are as shown in Table 1 and Figure 3 below.

[0075] Control Line Expression Level Detection Line Expression Level NMP22 0.1 ng / ㎖ 58.5 22.3 NMP22 1.0 ng / ㎖ 61.2 42.9 NMP22 10 ng / ㎖ 61.1 10 2.3 NMP22 25 ng / ㎖ 58.9 14 9.4 NMP22 50 ng / ㎖ 58.8 16 3.5

[0076] As shown in Table 1 and Figure 3, the intensity of the fluorescent labeling substance on the detection line (130) of the detection film (130) in the diagnostic strip was analyzed using a fluorescent signal analysis device (BioTek Instruments’ Cytation™ 5 Cell Imaging Multi-Mode Reader) comprising a CMOS image sensor, LED, light sensor, dicronic mirror, and bandpass filter. As a result, it was found that the intensity of the control line appeared relatively constant at all NMP22 concentrations and maintained an intensity between about 55 and 65, serving as a standard to confirm that the experiment was performed normally.

[0077] In addition, the expression level of the detection line increases in dependence on the NMP22 concentration. As the NMP22 concentration increases, the fluorescence intensity appears stronger, showing the highest intensity particularly at 50 ng / mL. This indicates that the diagnostic kit emits a strong fluorescent signal from the Test Line depending on the NMP22 concentration, and that the signal intensity increases as the concentration increases.

[0078] Furthermore, by examining the results of deriving a standard calibration curve using the graph showing the intensity of the labeling substance on the detection line (150) of the detection membrane (130) in the diagnostic strip of the bladder cancer diagnostic kit (100) according to the present invention according to the NMP22 concentration shown in FIG. 3, as illustrated in FIG. 4, it can be more clearly determined that the quantification of high-concentration samples is possible.

[0079] Here, the X-axis in the result graph of Fig. 4 represents the concentration (ng / mL) of NMP22 recombinant protein, showing that the fluorescence intensity detected at the Test Line changes as this concentration increases. The Y-axis represents the fluorescence intensity measured at the Detection Line, which increases as the NMP22 concentration increases, but reaches a saturation state above a certain concentration and no longer increases linearly.

[0080] Through this, it can be seen that in the case of the bladder cancer diagnostic kit (100) according to the present invention, the shape of the standard quantitative curve shows a pattern in which the fluorescence intensity increases proportionally as the concentration increases within the range of NMP22 concentrations from 0.1 ng / ml to 50 ng / ml, and quantitative analysis is possible.

[0081] The embodiments disclosed in this invention are intended to illustrate, not limit, the technical concept of the invention, and the scope of the technical concept of the invention is not limited by these embodiments. The scope of protection shall be interpreted by the claims below, and all technical concepts within an equivalent scope shall be interpreted as being included within the scope of rights of this invention.

[0082] <Explanation of Symbols>

[0083] 100: Bladder cancer diagnostic kit

[0084] 110: Support 120: Sample pad

[0085] 130: Detection film 140: Absorbent pad

[0086] 150 : Control line 160 : Detection line

Claims

1. A support corresponding to the base body of a diagnostic strip that is received and installed in the internal space of the diagnostic kit body; A sample pad attached to the upper part of the above support, into which a specimen corresponding to the urine of the diagnostic subject and a buffer are injected; A detection film attached to the upper part of the support in a manner sequentially connected to the sample pads above; A detection (test) line formed by coating on the above detection film and having an anti-NMP22 (Nuclear Matrix Protein 22) antibody immobilized thereon; and A control line formed by coating at a position spaced apart from the detection line of the detection membrane, on which a Chicken IgY antibody is immobilized; comprising The above buffer is characterized by comprising a first complex in which a europium (Eu)-based fluorescent bead is conjugated with an anti-Chicken IgY antibody, and a second complex in which a europium (Eu)-based fluorescent bead is conjugated with an anti-NMP22 (Nuclear Matrix Protein 22) antibody. Bladder cancer diagnostic kit using NMP22 as a biomarker.

2. In Paragraph 1, The detection film has the control line and the detection line formed sequentially based on the sample pad side, and The anti-NMP22 antibody included in the above detection line is prepared at a concentration of 1.8 mg / mL to 2.2 mg / mL, and The Chicken IgY antibody included in the above control line is characterized by being prepared at a concentration of 0.8 mg / mL to 1.2 mg / mL. Bladder cancer diagnostic kit using NMP22 as a biomarker.

3. In Paragraph 2, The europium-based fluorescent beads combined within the first and second complexes included in the buffer are characterized by having an excitation wavelength of 330 nm to 340 nm and an emission wavelength of 610 nm to 620 nm. Bladder cancer diagnostic kit using NMP22 as a biomarker.

4. In Paragraph 3, The first complex included in the buffer is characterized by being prepared by treating europium-based fluorescent beads with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to prepare them to a state capable of antibody binding, and then reacting 1.3 to 1.7 parts by weight of an anti-Chicken IgY antibody with 10 parts by weight of the europium-based fluorescent beads prepared to a state capable of antibody binding through stirring. Bladder cancer diagnostic kit using NMP22 as a biomarker.

5. In Paragraph 3, The second complex included in the buffer is characterized by being prepared by treating europium-based fluorescent beads with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to prepare them to a state capable of antibody binding, and then reacting 0.8 to 1.2 parts by weight of an anti-NMP22 antibody with 10 parts by weight of the europium-based fluorescent beads prepared to a state capable of antibody binding through stirring. Bladder cancer diagnostic kit using NMP22 as a biomarker.

6. In Paragraph 3, The first complex included in the buffer is provided in an amount of 0.8 to 1.2 parts by volume relative to the total volume of the sample and buffer injected into the injection pad, which is 100 parts by volume, and The second complex included in the buffer is characterized by being provided in an amount of 0.8 to 1.2 parts by volume relative to the total volume of 100 parts by volume of the sample and buffer injected into the injection pad. Bladder cancer diagnostic kit using NMP22 as a biomarker.

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