A new immunodiagnostic device that distinguishes between normal and abnormal pregnancy by measuring the distribution ratio of beta core fragment hCG

Abstract

The present invention relates to an immune device for determining the presence or absence of pregnancy or abnormal pregnancy, and more specifically, to an immune device capable of recognizing both intact hCG and βcf hCG. R This immunological device contains a multi-linked anti-hCG monoclonal antibody, and can separately detect intact hCG and βcf hCG contained in the sample, including two detection lines and a control line.By quantifying the ratio of the color intensity of βcf hCG to the color intensity of intact hCG and βcf hCG (the distribution ratio of βcf hCG), it can accurately and quickly determine the number of weeks of pregnancy, normal pregnancy, ectopic pregnancy, and abnormal pregnancy including blighted oocytes.

Description

[Technical Field]

[0001] The present invention relates to an immunoassay device capable of distinguishing between normal and abnormal pregnancy and an analytical method using the same, more specifically, to an immunoassay device and an analytical method using the same for distinguishing between normal and abnormal pregnancy by measuring the distribution ratio of βcf hCG (Beta core fragment hCG) among various hCG proteins secreted in the urine of pregnant women. The core principle of the immunoassay device is to use a novel multimeric anti-hCG monoclonal antibody, which can commonly recognize and bind to only intact hCG and βcf hCG among the various hCG proteins present in the urine of pregnant women, as a mobile phase bound to a label, and to specifically separate intact hCG and βcf hCG in a stationary phase. [Background technology]

[0002] Human chorionic gonadotropin (hCG) is a glycoprotein hormone produced during pregnancy. hCG is produced in the placental villous cytotrophoblast and stimulates the continuous production of progesterone during early pregnancy, maintaining implantation until the 10th week of pregnancy when placental function is complete.

[0003] Intact hCG (I-hCG), the active form in vivo, has a molecular weight of approximately 37 kDa and consists of 244 amino acids. Intact hCG is broadly divided into two subunits, alpha (α) and beta (β), with the alpha subunit containing 92 amino acids and the beta subunit containing 145 amino acids. Intact hCG is generally present in the blood during pregnancy and is the main structure that exhibits biological activity. In addition to the active form, various dissociation and degradation products of hCG are found in blood and urine, including nicked hCG (N-hCG), free beta hCG, free alpha hCG, and beta-core fragment hCG.

[0004] Beta core fragment hCG (βcf hCG) is the predominant hCG protein present in the urine of pregnant women, with a molecular weight of approximately 14 kDa. Intact hCG is predominantly secreted at week 4 of the last menstrual period (LMP), and βcf hCG gradually increases from week 5 of LMP, becoming more abundant than intact hCG at week 5 and 6 of LMP (John Walter Larsen et al., 2015).

[0005] In pregnant women, intact hCG is predominant in blood (98%), whereas βcf hCG increases to a maximum of 80% in urine as pregnancy progresses, becoming more prevalent than intact hCG. Conventional pregnancy test kits using intact hCG as a marker qualitatively diagnose intact hCG in urine to determine pregnancy. However, in a study of 11,760 women diagnosed with pregnancy using conventional pregnancy test kits, 22 women (approximately 0.2%) reported false negatives. The blood concentrations of intact hCG in these women were measured at a maximum of 268,022.5 IU / mL, suggesting that conventional urine pregnancy test kits suffer from false negatives due to the hook effect. The underlying cause of these false negatives was found to be excess βcf hCG in urine (Richard T. Griffey et al., 2013).

[0006] False-negative results in urine pregnancy tests can have serious consequences in emergency department (ED) situations. For example, if a female patient is actually pregnant and a false-negative result is obtained when a general pregnancy test kit is used to test her urine, it could lead to a medical error, as the patient may be treated without considering the fetus.

[0007] On the other hand, ectopic pregnancy accounts for 9% of all mortality rates among pregnant women in the first trimester, increasing the mortality rate among pregnant women, and the prevalence of ectopic pregnancy is gradually increasing worldwide.

[0008] Under these circumstances, the inventors have conducted intensive research to solve the fundamental hook effect and develop a method for distinguishing between normal and abnormal pregnancies. As a result, they have developed a novel multimeric anti-hCG monoclonal antibody that can commonly recognize intact hCG and βcf hCG, and have solved the hook effect by specifically separating and detecting intact hCG and βcf hCG in urine samples. They have also confirmed that the ratio of the color intensity of βcf hCG to the color intensity of intact hCG and βcf hCG (βcf hCG distribution ratio) can be quantified to accurately and quickly distinguish between the number of weeks of pregnancy, normal pregnancy, ectopic pregnancy, and abnormal pregnancy including blighted oocytes, thereby completing the present invention. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention has been made in consideration of the above circumstances, and its purpose is to provide an immunological device and an analytical method that can specifically separate and detect βcf hCG and intact hCG contained in the urine of pregnant women and accurately measure the distribution ratio of βcf hCG, thereby maintaining normal pregnancy and distinguishing between abnormal pregnancies in the early stages of pregnancy.

[0010] The immunological device of the present invention is an immunological device for determining pregnancy or abnormal pregnancy, comprising: i) a specimen region for accommodating a test sample to be analyzed; ii) a conjugate region linked to the specimen region and containing a multimeric anti-hCG monoclonal antibody conjugated to a probe substance; iii) a signal detection region connected to the conjugate region and including a first detection line to which an anti-βcf hCG antibody capable of specifically binding only to βcf hCG is immobilized, a second detection line to which an anti-intact hCG antibody capable of specifically binding only to intact hCG is immobilized, and a control line; and iv) a moisture-absorbing region located downstream of the signal detection region for absorbing the test sample after the signal detection reaction has completed.

[0011] The analytical method of the present invention is a method for analyzing information necessary for distinguishing between pregnancy and abnormal pregnancy, comprising the following steps: i) using a multimeric anti-hCG monoclonal antibody that commonly recognizes βcf hCG and intact hCG to bind in a manner that reflects the distribution ratio of βcf hCG and intact hCG in the test sample to be analyzed; ii) measuring the color intensity of βcf hCG and intact hCG; and iii) distinguishing between pregnancy and abnormal pregnancy based on the distribution ratio of βcf hCG color intensity. [Means for solving the problem]

[0012] The advantages and features of the present invention, as well as methods for achieving them, will become apparent from the following detailed description of the embodiments in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, and may be embodied in various different forms. However, the present embodiments are provided to complete the disclosure of the present invention and to allow those skilled in the art to fully understand the scope of the present invention, and the present invention is defined only by the scope of the claims.

[0013] The terms used in this specification are for the purpose of describing the embodiments and are not intended to limit the present invention. In this specification, the singular includes the plural unless otherwise specified. The terms "comprises" and / or "comprising" used in this specification do not exclude the presence or addition of one or more other elements other than the elements listed. The same reference numerals refer to the same elements throughout this specification, and "and / or" includes each and every combination of one or more of the listed elements. Even if "first," "second," etc. are used to describe various elements, these elements are not limited by these terms. These terms are used merely to distinguish one element from another. Therefore, it goes without saying that a first element referred to below may also be a second element within the technical spirit of the present invention.

[0014] Unless otherwise defined, all terms (including technical and scientific terms) used in this specification are used in the sense that they can be commonly understood by those skilled in the art to which the present invention belongs. Furthermore, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless they are clearly and specifically defined.

[0015] The present invention provides an immunological device and analytical method that can specifically separate and detect βcf hCG and intact hCG contained in the urine of pregnant women using a polyvalent anti-hCG monoclonal antibody that commonly recognizes intact hCG and βcf hCG, and accurately measure the distribution ratio of βcf hCG, thereby maintaining normal pregnancy and distinguishing between abnormal pregnancies in the early stages of pregnancy.

[0016] The multimeric anti-hCG monoclonal antibody used in the present invention, which commonly recognizes intact hCG and βcf hCG, has the characteristic of accurately reflecting the distribution ratio of intact hCG and βcf hCG according to the number of weeks of pregnancy, and is therefore useful for more accurately and efficiently distinguishing between normal and abnormal pregnancies by analyzing this distribution ratio.

[0017] The present invention aims to accurately distinguish between normal and abnormal pregnancies. This cannot be achieved by simply measuring the concentrations of intact hCG and βcf hCG using antibodies to intact hCG and βcf hCG, but can be achieved by measuring the proportions of intact hCG and βcf hCG reacting with the intact hCG and βcf hCG present in a sample. When antibodies to intact hCG and βcf hCG are used, there is a limit to the concentrations of intact hCG and βcf hCG that the antibodies can recognize, making it difficult to accurately determine the concentrations and proportions of intact hCG and βcf hCG. On the other hand, when a monoclonal antibody that commonly recognizes intact hCG and βcf hCG of the present invention is used, even though there is a limit to the recognizable values ​​of intact hCG and βcf hCG, the proportions of intact hCG and βcf hCG reacting with the intact hCG and βcf hCG present in a sample can be measured, allowing for more accurate prediction of the concentrations and concentration ratios of intact hCG and βcf hCG.

[0018] The present invention does not use monoclonal antibodies corresponding to intact hCG and βcf hCG, but uses a monoclonal antibody that recognizes both intact hCG and βcf hCG, because this allows the total contents of intact hCG and βcf hCG in a sample to be proportionally determined.

[0019] By using the polyvalent anti-hCG monoclonal antibody of the present invention, an accurate distribution ratio can be derived, and a normal or abnormal pregnancy can be accurately determined.

[0020] The abnormal pregnancy of the present invention may be, but is not limited to, an ectopic pregnancy or a blighted ovum.

[0021] An ectopic pregnancy is one in which the fertilized egg implants somewhere other than inside the uterus, where it normally does. The implantation site of an ectopic pregnancy can be an abnormal site within the uterus or a site outside the uterus. Most ectopic pregnancies occur in the fallopian tubes, but ovarian, peritoneal, or cervical pregnancies are rare. It is one of the most common emergencies, accounting for approximately 1-2% of all pregnancies.

[0022] Blighted ovum refers to a condition in which the placenta is developed but no fetus is visible on an ultrasound image. In the case of blighted ovum, the placenta can grow for a certain period of time without an embryo, but ultimately ends in a spontaneous abortion, with lower abdominal pain and bleeding followed by the expulsion of the conception material into the uterus.

[0023] The present invention also provides an immunological device for determining pregnancy or abnormal pregnancy, comprising: i) a specimen region for accommodating a test sample to be analyzed; ii) a conjugate region linked to the specimen region and containing an anti-hCG monoclonal antibody of an anti-intact hCG / βcf hCG multimeric bond conjugated to a probe substance; iii) a signal detection region linked to the specimen region and including a first detection line to which an anti-βcf hCG antibody that specifically binds only to βcf hCG is immobilized, a second detection line to which an anti-intact hCG antibody that specifically binds only to intact hCG is immobilized, and a control line; and iv) a moisture-absorbing region located downstream of the signal detection region for absorbing the test sample after the signal detection reaction has completed.

[0024] The sample region i) preferably contains a liquid sample such as urine from a pregnant woman, but any sample that is expected to contain intact hCG and / or βcf hCG can be used.

[0025] The sample region may further have a filtering function to further improve selectivity for the analyte or to minimize the influence of interfering substances that may be contained in the sample. If necessary, an auxiliary region containing a substance that can increase the reaction between the analyte and the conjugate or eliminate the influence of interfering substances may be further provided upstream of the sample region.

[0026] The probe material of the conjugate region of ii) may be one or more selected from the group consisting of gold nanoparticles, silver nanoparticles, quantum dot nanoparticles, carbon nanoparticles, latex beads / fluorescent nanoparticles, cellulose nanoparticles, magnetic nanoparticles, silica nanoparticles, polymer beads, fluorescent substances, luminescent substances, dye beads, and proteins, but is not limited thereto.

[0027] In a specific embodiment of the present invention, the probe material may be, but is not limited to, colloidal gold nanoparticles.

[0028] The signal detection region (iii) is a medium in which the mobile phase and the test sample are developed, and the mobile phase and the test sample can migrate by capillary action through the porous membrane of the signal detection region. The signal detection region may be, but is not limited to, any one selected from the group consisting of a well plate made of nitrocellulose, cellulose, polyethylene, polyethersulfone, polystyrene, polycarbonate, polymethylmethacrylate, nylon, PVDF, polyvinyl resin, or polystyrene resin, and a glass slide. In a specific embodiment of the present invention, the signal detection region may be, but is not limited to, a nitrocellulose membrane having pores of 5 to 15 μm.

[0029] According to one specific embodiment of the present invention, the signal detection region may include a first detection line to which an anti-βcf hCG antibody is immobilized, a second detection line to which an anti-intact hCG antibody is immobilized downstream of the first detection line, and a control line downstream of the second detection line. According to another embodiment of the present invention, the signal detection region may include a second detection line to which an anti-intact hCG antibody is immobilized, a first detection line to which an anti-βcf hCG antibody is immobilized downstream of the second detection line, and a control line downstream of the first detection line.

[0030] The anti-intact hCG antibody specifically recognizes active intact hCG, which is composed of two subunits, α and β, and the anti-βcf hCG antibody specifically recognizes beta core fragment hCG (βcf hCG).

[0031] The present invention is an immunological device consisting of two detection lines, which quantitatively or qualitatively analyzes the color intensity of the detection lines that detect βcf hCG and intact hCG in a sample, and is characterized in that it can accurately determine the number of weeks of pregnancy, normal pregnancy, and abnormal pregnancy including ectopic pregnancy by quantifying and comparing the distribution ratio of βcf hCG using the following formula derived from the present invention.

[0032] <expression> JPEG2025530936000002.jpg20170

[0033] In a specific embodiment of the present invention, if the ratio of the color intensity of βcf hCG to the color intensity of βcf hCG and intact hCG according to the above formula increases by 50-60% or more at 5-6 weeks LMP, it can be determined to be a normal pregnancy, if it is 5-15% or less, it can be determined to be an ectopic pregnancy, and if it is 20-30% or less and maintained until 6 weeks and 6 days, it can be determined to be a blighted egg.

[0034] The control line refers to a portion of the test sample that gives a constant signal regardless of the concentration of intact hCG or βcf hCG.

[0035] The moisture-absorbing region of iv) may include an absorbent dispersed in the pores of the porous support or adsorbed or coated on the fiber threads of the porous support, and may further include, but is not limited to, a porous film layer on the upper surface of the porous support.

[0036] In the present invention, when the multimeric anti-hCG monoclonal antibody conjugate present in the conjugate region reacts with a sample such as urine from a pregnant woman, it reacts according to the concentration ratio of intact hCG to βcf hCG present in the sample, and the reacted conjugate migrates to the membrane side. At the first detection line on the membrane where the anti-βcf hCG monoclonal antibody is immobilized, a sandwich complex is formed specifically with only βcf hCG among the hCG that reacts with the conjugate, and at the second detection line where the anti-intact hCG monoclonal antibody is immobilized, a sandwich complex is formed specifically with only intact hCG among the hCG that reacts with the conjugate.

[0037] The immunoassay device of the present invention may include a solid support at its bottom. The solid support may be made of any one material selected from the group consisting of, but not limited to, nitrocellulose, nylon, polyvinylidene fluoride (PVDF), glass, and plastic. By attaching a pad, membrane, etc. to the solid support, the durability of the strip can be improved, and handling and storage can be simplified. Furthermore, the attachment of additional external devices can be facilitated.

[0038] Examples of plastic materials that can be used as the solid support include, but are not limited to, polypropylene film, polyester film, polycarbonate film, and acrylic film.

[0039] The immunoassay device of the present invention may further include an analytical device. The analytical device may evaluate the color intensity of the first and second detection lines. The analytical device may include a reader coupled to a software program capable of evaluating the intensity, color, luminescence, and / or fluorescence of the analyte complex formed at the first and second detection lines. The reader may compare the intensity, color, luminescence, and / or fluorescence with a preprogrammed threshold and provide a digital output based on the comparison. According to one embodiment of the present invention, the analytical device uses a reflectance reader based on a CCD camera or laser light source and equipped with analytical software, enabling high-accuracy discrimination between normal and abnormal pregnancies, including ectopic pregnancies. The analytical device may further include a display providing information related to abnormal pregnancies, including ectopic pregnancies.

[0040] The immunological device of the present invention can determine the gestational age at which the gestational sac and fetus can be observed. In a specific embodiment of the present invention, if the ratio of the color intensity of βcf hCG to the color intensity of βcf hCG and intact hCG increases by 50-60% or more at 5-6 weeks LMP, the gestational sac and fetus can be observed in normal pregnancy, but if it is less than 5-15%, the gestational sac cannot be observed in ectopic pregnancy.

[0041] In another specific embodiment of the present invention, the ratio of the color intensity of βcf hCG to the color intensity of βcf hCG and intact hCG, i.e., βcf hCG / (βcf hCG+intact hCG), was compared and it was confirmed that it was significantly lower in abnormal pregnancies, including ectopic pregnancies, than in normal pregnancies.

[0042] The present invention also provides a kit, wherein the immune device is additionally fixed within a device.

[0043] The kit has a lower device equipped with a guide and a strip support, and an upper device equipped with a sample insertion port and result confirmation windows at positions corresponding to the first detection line, the second detection line, and the control line.

[0044] The upper and lower devices may be manufactured using a common plastic material, such as, but not limited to, polycarbonate, acrylonitrile butadiene styrene (ABS), etc.

[0045] The present invention also provides a method for analyzing information necessary for determining pregnancy or abnormal pregnancy, comprising the following steps: i) measuring the amount or concentration of βcf hCG and intact hCG from a test sample to be analyzed; and ii) determining pregnancy or abnormal pregnancy based on the ratio of the amount or concentration of βcf hCG to the amount or concentration of βcf hCG and intact hCG, wherein the amount or concentration is measured using a polyvalent anti-hCG monoclonal antibody that commonly recognizes βcf hCG and intact hCG.

[0046] The amount or concentration can be measured by the intensity of the color development, which is proportional thereto.

[0047] If the ratio of the amount or concentration of βcf hCG to the amount or concentration of βcf hCG and intact hCG exceeds 50% at 5 weeks and 6 days of LMP, it is determined to be a normal pregnancy, and if it is 30% or less at 5 weeks and 6 days, it can be determined to be an abnormal pregnancy such as an ectopic pregnancy or a blighted ovum, but is not limited to these.

[0048] Specifically, the present invention provides a method for interpreting information necessary for determining pregnancy or abnormal pregnancy, including the steps of: i) applying a sample to the immunological device and reacting intact hCG and βcf hCG in the sample with a multimeric anti-hCG monoclonal antibody conjugated to a probe material; ii) confirming the reaction of βcf hCG and intact hCG in the sample through a first detection line and a second detection line, wherein βcf hCG and intact hCG are detected by the color intensity generated by the presence of a sandwich complex at each detection line; and iii) determining that a pregnancy is normal if the ratio of the color intensity of the first detection line to the first and second detection lines (the ratio of the color intensity of βcf hCG to the color intensities of βcf hCG and intact hCG) increases by 50-60% or more at 5-6 weeks LMP, i.e., 5 weeks and 6 days LMP, and that a pregnancy is ectopic or blighted ovum if it is 30% or less.

[0049] The color intensity is a color intensity depending on the concentration of βcf hCG and intact hCG, and it has been confirmed that the concentration and the color intensity are proportional to each other in a specific embodiment of the present invention.

[0050] The present invention also provides a method for determining pregnancy or abnormal pregnancy using the immunochromatography strip.

[0051] The method for determining pregnancy or abnormal pregnancy includes the steps of: i) applying a sample to the immunological device and reacting intact hCG and βcf hCG present in the sample with a multimeric anti-hCG monoclonal antibody conjugated to a probe material; ii) confirming the reaction of βcf hCG and intact hCG in the sample through the first and second detection lines, where βcf hCG and intact hCG are detected by the color intensity generated by the presence of the sandwich complex at each detection line; and iii) determining a normal pregnancy if the ratio of the color intensity of the first detection line to the first and second detection lines (the ratio of the color intensity of βcf hCG to the color intensities of βcf hCG and intact hCG) exceeds 50% at 5 weeks and 6 days of LMP, and determining an abnormal pregnancy if it is 30% or less at 5 weeks and 6 days of LMP. [Effects of the Invention]

[0052] The present invention provides a novel immunodiagnostic device that measures the distribution ratio of beta core fragment hCG (βcf hCG) to distinguish between normal and abnormal pregnancy. An increase in the distribution ratio of βcf hCG confirmed by this method indicates normal fetal implantation and development, which has the effect of alleviating anxiety in early pregnant women. Furthermore, if the distribution ratio of βcf hCG significantly decreases, prompt measures and treatment can be expected in cooperation with a hospital, and it is useful for providing pregnant women with information on maintaining a normal pregnancy. [Brief explanation of the drawings]

[0053] [Figure 1] FIG. 1 is a schematic diagram showing the reaction principle resulting from the concentration ratio of intact hCG and βcf hCG in a sample of the present invention and an example of an immunochromatography strip. [Figure 2] FIG. 1 is a graph showing the correlation between the color intensity (intensity) depending on the amount or concentration of intact hCG and βcf hCG. [Figure 3] FIG. 1 shows changes in the distribution of intact hCG and βcf hCG during normal pregnancy. [Figure 4]FIG. 1 shows changes in the distribution of intact hCG and βcf hCG during ectopic pregnancy. [Figure 5] This is a graph comparing the color intensity ratio of βcf hCG / (intact hCG+βcf hCG) measured in a normal pregnancy group and an ectopic pregnancy group by gestational age. [Figure 6] FIG. 1 shows the results of clinical evaluation of normal pregnancy samples using the diagnostic device of the present invention. [Figure 7] FIG. 1 shows the results of clinical evaluation of normal pregnancy samples using the diagnostic device of the present invention. [Figure 8] FIG. 1 shows the results of clinical evaluation of ectopic pregnancy samples using the diagnostic device of the present invention. [Figure 9] FIG. 1 shows the results of clinical evaluation of ectopic pregnancy samples using the diagnostic device of the present invention. [Figure 10] This figure shows the correlation between the color intensity and the concentration ratio when intact hCG alone, βcf hCG alone, and a mixed intact hCG and βcf hCG positive sample were prepared and reacted at different concentrations using a kit prepared according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0054] The present invention will be described in more detail with reference to the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, and includes modifications of the technical ideas equivalent thereto.

[0055] Example 1. Preparation of immunochromatographic strips 1-1. Fabrication of membrane with first detection line, second detection line, and control line Three different antibodies were dispensed onto a nitrocellulose membrane: anti-βcf hCG monoclonal antibody was dispensed onto the first detection line as the βcf hCG antibody, anti-intact hCG monoclonal antibody was dispensed onto the second detection line as the intact hCG antibody, and goat anti-mouse IgG was dispensed onto the control line, which was then dried.

[0056] 1-2. Conjugate pad fabrication A first conjugate solution was prepared by conjugating colloidal gold nanoparticles with a multivalent anti-hCG monoclonal antibody that recognizes both intact hCG and βcf hCG. A second conjugate solution was prepared by conjugating colloidal gold nanoparticles with mouse immunoglobulin (mouse IgG). The first and second conjugate solutions were dispensed onto pretreated conjugate pads, allowed to dry completely, and then cut to the appropriate size.

[0057] 1-3. Preparation of sample pad The sample pad was thoroughly immersed in a sample pad pretreatment solution containing a buffer and a preservative, and after being completely dried, it was cut to an appropriate size and prepared.

[0058] 1-4. Making an absorbent pad Dry absorbent pads were prepared by cutting them to the appropriate size.

[0059] 1-5. Preparation of immunochromatography strips The membrane, conjugate pad, sample pad and moisture absorption pad prepared through the above steps are assembled according to the structure shown in FIG.

[0060] That is, the sample pad was attached so as to overlap one end of the conjugate pad, one end of the detection pad was attached so as to overlap the other end of the conjugate pad, and the other end of the detection pad and one end of the moisture absorption pad were attached so as to overlap each other.

[0061] In FIG. 1, the immunochromatographic strip comprises the following structure:

[0062] 1: Sample pad 2: Conjugate pad 3: Nitrocellulose membrane 4: Absorption pad 5: First detection line (Test line 1) where anti-βcf hCG monoclonal antibody is immobilized 6: Test line 2, where anti-intact hCG monoclonal antibody is immobilized 7: Control line immobilized with goat anti-mouse immunoglobulin

[0063] 1-6. Analyzer The color development or brightness sensitivity of the detection line was quantified using a reflectance reader equipped with analysis software based on a CCD camera or laser light source, and used to be quantified as signal intensity.

[0064] 1-7. Device assembly The manufactured immunochromatography strip for detecting normal pregnancy, gestational age, and abnormal pregnancy including ectopic pregnancy and blighted eggs is placed in the strip fixing position of the plastic lower device, and then inserted into the upper device which has a sample insertion port and a result confirmation window, and then assembled.

[0065] Example 2: Measurement of changes in color intensity depending on the concentration of intact hCG and βcf hCG in normal pregnancy groups and abnormal pregnancy groups including ectopic pregnancy While conventional methods have been based on the concentration ratio, the pregnancy diagnosis device of the present invention checks and determines pregnancy based on color intensity. The color intensity is measured uniformly using any device that measures color intensity, and the measured value is converted into a numerical value.

[0066] Measurement was performed using an analyzer that is a component of the pregnancy diagnosis device of the present invention, and it was confirmed that the concentration and color intensity were proportional (Figure 2).

[0067] 2-1. Measurement of the change in color intensity according to the concentration of intact hCG and βcf hCG in normal pregnancy groups We measured the change in color intensity depending on the concentration of intact hCG and βcf hCG in the normal pregnancy group. In the normal pregnancy group, intact hCG begins to be detected from day 4 of the 3rd week of LMP and increases until the 5th week. This allows us to determine whether or not a pregnancy is early. βcf hCG begins to be detected from the latter half of the 4th week of LMP, increases rapidly from the 5th week, and remains at a high concentration from the 6th week onwards (Figure 3).

[0068] Furthermore, when βcf hCG was detected at 5 weeks LMP, ultrasound examination confirmed the presence of a gestational sac (the bag that contains the baby).From 6 weeks LMP, intact hCG decreased, while βcf hCG remained constant, resulting in a reversal phenomenon, and ultrasound examination confirmed the presence of the fetus.

[0069] 2-2. Measurement of the change in color intensity according to the concentration of intact hCG and βcf hCG in abnormal pregnancies, including ectopic pregnancies When changes in intact hCG and βcf hCG concentrations were measured in the ectopic pregnancy group, it was confirmed that intact hCG was detectable at 5 weeks LMP, while βcf hCG was not detected or was detected at very low concentrations at 5 weeks LMP (Figure 4).

[0070] As a result, in normal pregnancies at 4 to 7 weeks LMP, the concentration of intact hCG increased and then decreased, and the concentration of βcf hCG gradually increased and remained constant, whereas in ectopic pregnancies, βcf hCG was either not detected or was detected at very low concentrations, which was confirmed as a significant result in distinguishing between normal and ectopic pregnancies.

[0071] On the other hand, in the case of blighted eggs, βcf hCG remained low at 30% or less from 5 weeks and 6 days of LMP without increasing, and ultrasound observation confirmed the gestational sac, but the fetus and heartbeat were not detected.

[0072] Example 3. Measurement of the βcf hCG / (intact hCG+βcf hCG) color intensity ratio in normal pregnancy groups and abnormal pregnancy groups including ectopic pregnancy In order to use the results of Example 2 to distinguish between normal pregnancy groups and abnormal pregnancy groups, including ectopic pregnancy, based on the gestational age, the intensity ratio of βcf hCG / (intact hCG+βcf hCG) at each gestational age was measured and compared for the normal pregnancy group and the abnormal pregnancy group.

[0073] 3-1. Verification of the measurement principle and derivation of the formula for the distribution ratio (%) of βcf hCG The pregnancy diagnostic device of the present invention comprises a "mobile phase" that reacts with a sample and migrates to the stationary phase, and a "stationary phase" that can separate and detect intact hCG and βcf hCG present in the sample. In the present invention, the mobile phase corresponds to the conjugate region of the pregnancy diagnostic device, and the stationary phase corresponds to the signal detection region including the first detection line, the second detection line, and the control line.

[0074] The mobile phase is a dried version of an indicator capable of expressing the concentration intensity of a sample, i.e., gold particles, nanobeads, fluorescent or phosphorescent, to which is bound a multimeric anti-hCG monoclonal antibody, a novel antibody developed by the present inventors, capable of recognizing both intact hCG and βcf hCG.

[0075] That is, the antibody bound to the label is characterized by its ability to commonly recognize and react with intact hCG and βcf hCG.

[0076] An anti-βcf hCG monoclonal antibody is fixed to the first detection line of the stationary phase, and only βcf hCG is detected in the sample. An anti-intact hCG monoclonal antibody is fixed to the second detection line, and only intact hCG is detected in the sample.

[0077] As a result, the antibody reacts primarily with the conjugate according to the concentration ratio of intact hCG and βcf hCG contained in the sample and migrates to the stationary phase, and each sample is separated and detected according to the characteristics of the antibody immobilized on the stationary phase.

[0078] On the other hand, the formula for calculating the distribution ratio (%) of βcf hCG was derived from the color intensity ratio of βcf hCG / (Intact hCG+βcf hCG), which enabled more clear measurement and analysis.

[0079] 3-2. Measurement of the color intensity ratio of βcf hCG / (Intact hCG + βcf hCG) in normal pregnancy groups and abnormal pregnancy groups, including ectopic pregnancy, according to gestational age Based on the color intensity corresponding to the detected concentration ratio of intact hCG and βcf hCG measured in the normal pregnancy group and ectopic pregnancy group by gestational age, the distribution ratio (%) of βcf hCG according to the color intensity ratio of βcf hCG / (intact hCG + βcf hCG) was determined using the following formula.

[0080] <expression> JPEG2025530936000003.jpg20170

[0081] As a result, as shown in Table 1 and Figures 5 to 9 below, in normal pregnancies where the fertilized egg implants normally in the uterus and the fetus develops, the distribution ratio of βcf hCG was approximately 10-20% at 5 weeks and 0 days of LMP, increased to 50% at 5 weeks and 6 days of LMP, 70% at 6 weeks of LMP, and maintained at 80% from 8 weeks of LMP onwards. In contrast, in ectopic pregnancies where implantation occurs abnormally, the distribution ratio of βcf hCG remained below 10% at 5 to 6 weeks of LMP, with no observed increase, and no gestational sac (the bag that contains the baby) was observed on ultrasound images.

[0082] JPEG2025530936000004.jpg50170**Percentages for each week are based on the median value.

[0083] In addition, in the case of blighted eggs, there was no increase from LMP 5 weeks 6 days, and the distribution ratio of βcf hCG was confirmed to be maintained below 30%. Ultrasound observation confirmed the gestational sac, but the fetus and heart sounds were not confirmed.

[0084] As a result, the pregnancy diagnosis device of the present invention distinguishes between normal pregnancy groups and abnormal pregnancy groups, including ectopic pregnancy, based on the color intensity ratio of βcf hCG / (intact hCG+βcf hCG) and the distribution ratio (%) of βcf hCG, based on the color intensity corresponding to the detected concentrations of intact hCG and βcf hCG, and determines the time when a gestational sac can be confirmed, allowing for more accurate and rapid discrimination with a single measurement.

[0085] Example 4. Verification of kit performance (specificity) and analysis of correlation of color intensity Using the kit prepared according to the present invention, intact hCG alone, βcf hCG alone, and a mixed intact hCG and βcf hCG positive specimens were prepared at different concentrations and reacted. The results are shown in FIG.

[0086] Intact hCG was diluted to 100, 600, and 800 mIU / mL concentrations in a negative standard sample to prepare positive samples, and the reaction was performed. As a result, the first detection line (βcf hCG) did not develop color, and the color intensity at each concentration was confirmed using the second detection line (intact hCG).

[0087] βcf hCG was diluted to concentrations of 0.03, 0.5, and 5 pmol / mL in a negative standard sample, and these were prepared as positive samples and reacted. As a result, the second detection line (intact hCG) did not develop color, and the color intensity at each concentration was confirmed using the first detection line (βcf hCG).

[0088] The intact hCG and βcf hCG were mixed and diluted with the negative standard sample to concentrations of 100mIU / mL (intact hCG) + 0.03pmol / mL (βcf hCG), 800mIU / mL (intact hCG) + 0.5pmol / mL (βcf hCG), and 600mIU / mL (intact hCG) + 5pmol / mL (βcf hCG), respectively, to prepare positive samples, and the reaction was performed. As a result, both the first and second detection lines showed color intensity corresponding to each concentration, and the color intensity at each concentration was equal to that obtained when intact hCG alone or βcf hCG alone was treated.

[0089] These results verify that the multimeric anti-hCG monoclonal antibody used in the present invention reacts specifically with intact hCG and βcf hCG without any interference, and suggest that the color intensity increases in a quantitative correlation with the concentration of intact hCG and βcf hCG mixed in the sample, enabling accurate measurement of the distribution ratio.

[0090] Although the embodiments of the present invention have been described above, the present invention is not limited to the above embodiments, and can be implemented in various different forms. Those skilled in the art will understand that the present invention can be embodied in other specific forms without changing the technical spirit or essential characteristics of the present invention. Therefore, the above-described embodiments should be understood as illustrative in all respects and not restrictive.

Claims

1. i) a sample area containing the test sample to be analyzed; ii) a conjugate region linked to the sample region and containing a multimeric anti-hCG monoclonal antibody conjugated to a probe substance that commonly recognizes intact hCG and βcf hCG; iii) a signal detection region capable of completely separately detecting intact hCG and βcf hCG bound to the conjugate, the signal detection region comprising a first detection line immobilized with a monoclonal antibody capable of forming a complex specifically only with βcf hCG, a second detection line immobilized with a monoclonal antibody capable of forming a complex specifically only with intact hCG, and a control line; iv) An immunological device for determining pregnancy or abnormal pregnancy, comprising a moisture-absorbing region located downstream of the signal detection region, which absorbs the test sample after the signal detection reaction has been completed.

2. The immunological device of claim 1 , further comprising an analytical device.

3. The immune device according to claim 1 or 2, wherein the immune device determines pregnancy or abnormal pregnancy based on the ratio of the color intensity of βcf hCG to the color intensities of βcf hCG and intact hCG measured at the detection line, which is calculated by the following formula:

4. The immune device of claim 1, characterized in that the probe material ii) is one or more selected from the group consisting of gold nanoparticles, silver nanoparticles, quantum dot nanoparticles, carbon nanoparticles, latex beads / fluorescent nanoparticles, cellulose nanoparticles, magnetic nanoparticles, silica nanoparticles, polymer beads, fluorescent substances, luminescent substances, dyes, and proteins.

5. The immune device according to claim 1, characterized in that the signal detection region (iii) is any one selected from the group consisting of nitrocellulose, cellulose, polyethylene, polyethersulfone and nylon.

6. The immune device described in claim 1, characterized in that the moisture-absorbing region iv) includes an absorbent dispersed in the pores of the porous support or adsorbed or coated on the fiber threads of the porous support.

7. The immune device according to claim 1, wherein the immune device can determine the number of weeks of pregnancy at which the gestational sac can be observed by the color development of the first detection line.

8. A method for analyzing information necessary to distinguish between pregnancy and abnormal pregnancy, comprising the steps of: i) measuring the amount or concentration of βcf hCG and intact hCG from a test sample to be analyzed; ii) determining pregnancy or abnormal pregnancy based on the ratio of the amount or concentration of βcf hCG to the amounts or concentrations of βcf hCG and intact hCG, wherein the amount or concentration is measured using a polyclonal anti-hCG monoclonal antibody that commonly recognizes βcf hCG and intact hCG.

9. 9. The method of claim 8, wherein the amount or concentration is measured by a proportional color intensity.

10. The method according to claim 8 or 9, wherein if the ratio of the color intensity of βcf hCG to the color intensities of βcf hCG and intact hCG exceeds 50% at 5 weeks and 6 days of LMP, the pregnancy is judged to be normal, and if it is 30% or less, the pregnancy is judged to be abnormal, including ectopic pregnancy and blighted oocyte.

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