A novel immunodiagnostic device that distinguishes between normal and abnormal pregnancies 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 discriminating normal and abnormal pregnancies during pregnancy and an analysis method using the same. More specifically, the present invention relates to an immunoassay device that measures the distribution ratio of βcf hCG (Beta core framgment hCG) among various hCGs secreted from the urine of pregnant women to discriminate normal and abnormal pregnancies, and an analysis method using the same. The core principle of the immunoassay device is that it can commonly recognize and bind only Intact hCG and βcf hCG among the various hCG proteins present in the urine of pregnant women. ru It is to use a mobile phase in which a multiple-binding anti-hCG monoclonal antibody is bound to a label and specifically separate Intact hCG and βcf hCG on a stationary phase.

Background Art

[0002] Human chorionic gonadotropin (hCG) is a glycoprotein hormone produced during pregnancy. hCG is produced by the trophoblast of the placenta, continuously produces progesterone in the early stage of pregnancy, and functions to maintain implantation until 10 weeks of pregnancy when the placenta's function is completed.

[0003] The intact form of hCG (Intact hCG; I-hCG), which is the active form in the body, has a molecular weight of about 37 kDa and is composed of 244 amino acids. Intact hCG is roughly divided into two subunits, alpha (α) and beta (β). The alpha-subunit contains 92 amino acids, and the beta-subunit contains 145 amino acids. Intact hCG generally exists in the blood during pregnancy and is the main structure showing biological activity. In addition to the active form, various dissociation and degradation products of hCG are found in blood and urine, and these exist as nicked hCG (N-hCG), free β-hCG, free α-hCG, beta core fragment hCG, etc.

[0004] Beta-core fragment hCG (βcf hCG) is an hCG protein primarily found in the urine of pregnant women, with a molecular weight of approximately 14 kDa. Intact hCG is predominantly secreted during the fourth week of the LMP (Last Menstrual Period), and from the fifth week of the LMP, βcf hCG gradually increases, becoming more dominant than intact hCG from week 5 and 6 or week 6 (John Walter Larsen et al., 2015).

[0005] In pregnant women, the distribution of hCG protein shows that intact hCG is predominantly present in the blood (98%), but in urine, βcf hCG increases to a maximum of 80% as pregnancy progresses, becoming more dominant than intact hCG. Conventional pregnancy diagnostic kits that use intact hCG as a marker determine the presence or absence of pregnancy by qualitatively diagnosing intact hCG in the urine. However, when 11,760 women who were diagnosed as pregnant were tested with conventional pregnancy diagnostic kits, 22 women (approximately 0.2%) were found to have false negative results. The blood concentration of intact hCG in these women was measured at a maximum of 268,022.5 IU / mL, suggesting that conventional urine pregnancy diagnostic kits suffer from the problem of false negatives due to the hook effect. It was revealed that the underlying cause of these false negatives is the excessive presence of βcf hCG in the urine (Richard T. Griffey et. al., 2013).

[0006] False negative results in urine pregnancy tests can have serious consequences in emergency department (ED) settings. For example, if a female patient is pregnant but a standard pregnancy test using urine yields a false negative result, it could lead to medical errors such as treating the patient without considering the fetus.

[0007] On the other hand, ectopic pregnancies account for 9% of all maternal mortality in women at the first trimester, increasing the overall mortality rate among pregnant women, and the prevalence of ectopic pregnancies is steadily rising worldwide.

[0008] Against this backdrop, the inventors diligently conducted research to resolve the fundamental hook effect and develop a method to distinguish between normal and abnormal pregnancies. As a result, they discovered a method that can recognize both intact hCG and βcf hCG. ru By developing a multi-binding anti-hCG monoclonal antibody, specifically separating and detecting intact hCG and βcf hCG in urine samples to resolve the Hooke phenomenon, and quantifying the ratio of βcf hCG color intensity to that of intact hCG and βcf hCG (the distribution ratio of βcf hCG), we confirmed that it is possible to accurately and rapidly distinguish between gestational age, normal pregnancy, ectopic pregnancy, and abnormal pregnancy including blighted ovum, thus completing the present invention. [Overview of the project] [Problems that the invention aims to solve]

[0009] The present invention has been made in view of the above circumstances, and its object is to provide an immunoassay device and 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 enabling the maintenance of a normal pregnancy and the identification of abnormal pregnancies in the early stages of pregnancy.

[0010] The present invention relates to an immunoassay apparatus for determining pregnancy or abnormal pregnancy, comprising: i) a sample area for containing a test sample to be analyzed; ii) a conjugate area linked to the sample area and containing a multi-binding anti-hCG monoclonal antibody conjugated to a probe material; iii) a signal detection area connected to the conjugate area and including a first detection line immobilized with an anti-βcf hCG antibody that can specifically bind only to βcf hCG, a second detection line immobilized with an anti-Intact hCG antibody that can specifically bind only to Intact hCG, and a control line; and iv) a moisture absorption area located downstream of the signal detection area for absorbing the test sample after the signal detection reaction has been completed.

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

[0012] The advantages and features of the present invention, and methods for achieving them, will become clear with reference to the embodiments described below in detail with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below and can be embodied in a variety of different forms. These embodiments are provided to complete the disclosure of the present invention and to allow a person ordinary in the art to fully understand the scope of the invention, and the present invention is defined only by the scope of the claims.

[0013] The terms used herein are for illustrative purposes only and are not intended to limit the invention. In this specification, singular terms include plural terms 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 components in addition to those mentioned. Throughout the specification, the same reference numerals indicate the same component, and “and / or” includes each of the components mentioned and all combinations of one or more of them. Even if terms such as “first,” “second,” etc., are used to describe a variety of components, these components are not limited by these terms. These terms are used simply to distinguish one component from another. Accordingly, it goes without saying that the first component mentioned below may also be the second component within the technical concept of the invention.

[0014] Unless otherwise defined, all terms used herein (including technical and scientific terms) are used in the sense that they would be commonly understood by an ordinary person skilled in the art to which the present invention pertains. Furthermore, terms defined in commonly used dictionaries shall not be interpreted ideally or excessively unless explicitly defined otherwise.

[0015] The present invention provides an immunoassay device and analysis method that can distinguish between maintaining a normal pregnancy and abnormal pregnancies in early pregnancy by using a multi-binding anti-hCG monoclonal antibody that recognizes both intact hCG and βcf hCG in common, specifically separating and detecting βcf hCG and intact hCG contained in the urine of pregnant women, and accurately measuring the distribution ratio of βcf hCG.

[0016] The multi-binding anti-hCG monoclonal antibody used in this invention, which recognizes both Intact hCG and βcf hCG, accurately reflects the distribution ratio of Intact hCG and βcf hCG according to the gestational week. Therefore, by analyzing this distribution ratio, it can be used to more accurately and efficiently distinguish between normal and abnormal pregnancies.

[0017] The present invention aims to accurately distinguish between normal and abnormal pregnancies. This is difficult to achieve simply by measuring the concentrations of Intact hCG and βcf hCG using individual antibodies for Intact hCG and βcf hCG, and can be achieved by measuring the proportion of Intact hCG and βcf hCG present in the sample that react with these antibodies. When using individual antibodies for Intact hCG and βcf hCG, 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 concentration and proportion of Intact hCG and βcf hCG. On the other hand, when using the monoclonal antibody of the present invention that recognizes both Intact hCG and βcf hCG, even if there is a limit to the Intact hCG and βcf hCG values ​​that can be recognized, the proportion of Intact hCG and βcf hCG present in the sample that reacts with these antibodies is measured, allowing for a more accurate prediction of the concentration and concentration ratio of Intact hCG and βcf hCG.

[0018] This invention uses a monoclonal antibody that recognizes both Intact hCG and βcf hCG, rather than using monoclonal antibodies that correspond to Intact hCG and βcf hCG respectively. This is because the total content of Intact hCG and βcf hCG in the sample can be confirmed proportionally.

[0019] By using the multi-binding anti-hCG monoclonal antibody of the present invention, accurate distribution ratios can be derived, enabling accurate determination of whether a pregnancy is normal or abnormal.

[0020] The abnormal pregnancy described in this invention may be, but is not limited to, an ectopic pregnancy or a blighted ovum.

[0021] Ectopic pregnancy refers to a pregnancy in which a fertilized egg implants in a location other than the uterus, where it normally implants. The implantation site in an ectopic pregnancy can be an abnormal location within the uterus or a location outside the uterus. Most ectopic pregnancies occur in the fallopian tubes, but rarely they can occur in the ovaries, peritoneum, or cervix. It is one of the most common emergencies, accounting for about 1-2% of all pregnancies.

[0022] The term "blighted ovum" refers to a case where the placental tissue is developed on ultrasound, but no fetus is visible. In the case of a blighted ovum, the placenta can develop for a certain period without an embryo, but ultimately it ends in a spontaneous miscarriage, with the fertilized material being expelled from the uterus after lower abdominal pain and bleeding.

[0023] Furthermore, the present invention provides an immunoassay device for determining pregnancy or abnormal pregnancy, comprising: i) a sample area for containing a test sample to be analyzed; ii) a conjugate area linked to the sample area and containing an anti-hCG monoclonal antibody with multiple binding of anti-intact hCG / βcf hCG conjugated to a probe material; iii) a signal detection area linked to the conjugate area and connected to the sample area, including a first detection line on which an anti-βcf hCG antibody that specifically binds only to βcf hCG is immobilized, a second detection line on which an anti-intact hCG antibody that specifically binds only to Intact hCG is immobilized, and a control line; and iv) a moisture-absorbing area located downstream of the signal detection area for absorbing the test sample after the signal detection reaction has been completed.

[0024] The sample area i) is preferably used to contain a liquid sample such as urine from a pregnant woman, but any sample 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 present in the sample. If necessary, an auxiliary region may be further provided upstream of the sample region, containing a substance that can increase the reaction between the analyte and the conjugate or eliminate the influence of interfering substances.

[0026] The probe substance in the conjugate region of ii) above 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 substance may be colloidal gold nanoparticles, but is not limited thereto.

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

[0029] According to a 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 is an antibody that specifically recognizes active intact hCG, which has two subunits, α and β, bound to it, and the anti-βcf hCG antibody is an antibody that specifically recognizes beta-core fragment hCG (βcf hCG).

[0031] The present invention is an immunoassay device consisting of two detection lines, which quantitatively or qualitatively analyzes the color intensity of the detection lines that sense βcf hCG and intact hCG in a sample, and quantifies and compares the distribution ratio of βcf hCG using the following formula derived from the present invention, thereby accurately distinguishing between gestational age, normal pregnancy, and abnormal pregnancy, including ectopic pregnancy.

[0032] <expression> JPEG0007838072000001.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 between weeks 5 and 6 of 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 maintained at 20-30% or less until 6 weeks and 6 days, it can be determined to be a blighted oven.

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

[0035] The moisture-absorbing region in iv) above may include an absorbent dispersed in the pores of the porous support or adsorbed or coated onto the fibers 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, the multi-binding anti-hCG monoclonal antibody conjugate present in the conjugate region reacts with a sample such as urine from a pregnant woman, reacting according to the concentration ratio of Intact hCG and βcf hCG present in the sample, and the reacted conjugate migrates to the membrane side. In the first detection line on the membrane immobilized with anti-βcf hCG monoclonal antibody, a sandwich complex is specifically formed only with βcf hCG among the hCGs that reacted with the conjugate, and in the second detection line immobilized with anti-Intact hCG monoclonal antibody, a sandwich complex is specifically formed only with Intact hCG among the hCGs that reacted with the conjugate.

[0037] The immunoassay device of the present invention may include a solid support base at the bottom. The solid support base may, but is not limited to, be made of any one material selected from the group consisting of nitrocellulose, nylon, polyvinylidene fluoride (PVDF), glass, and plastic. Since the device is manufactured by attaching pads, membranes, etc., to the solid support base, the durability of the strip can be improved, and handling and storage can be facilitated. In addition, the attachment of additional external devices can be facilitated.

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

[0039] The immunoassay apparatus of the present invention may further include an analyzer. The analyzer can evaluate the color intensity of a first detection line and a second detection line. The analyzer may include a reader coupled with a software program that can evaluate the intensity, color, emission, and / or fluorescence of the analyte complex formed on the first detection line and the second detection line. The reader can compare the intensity, color, emission, and / or fluorescence with a pre-programmed threshold and provide a digital output based on the comparison. According to one embodiment of the present invention, the analyzer uses a reflectance reader equipped with analysis software on a CCD camera or laser light source substrate, which was able to distinguish between normal pregnancies and abnormal pregnancies, including ectopic pregnancies, with high accuracy. The analyzer may further include a display that provides information related to abnormal pregnancies, including ectopic pregnancies.

[0040] The immunotherapy 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 LMP 5-6 weeks, the gestational sac and fetus can be observed in a normal pregnancy, while if it is 5-15% or less, the gestational sac cannot be observed in an ectopic pregnancy.

[0041] In another specific embodiment of the present invention, a comparison of the measured values ​​of βcf hCG / (βcf hCG + Intact hCG), i.e., the ratio of the color intensity of βcf hCG to the color intensity of Intact hCG, revealed that it was significantly lower in abnormal pregnancies, including ectopic pregnancies, compared to normal pregnancies.

[0042] Furthermore, the present invention provides a kit characterized in that the immunization device is additionally immobilized within the device.

[0043] The kit is characterized in that the lower device is equipped with a guide and a strip support, and the upper device is equipped with a sample input port and result confirmation windows positioned to correspond to the first detection line, the second detection line, and the control line.

[0044] The upper and lower devices can be manufactured using ordinary plastic materials, such as, but are not limited to, polycarbonate and acrylonitrile butadiene styrene (ABS).

[0045] Furthermore, the present invention 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 intact hCG, wherein the amount or concentration is measured using a multi-binding anti-hCG monoclonal antibody that recognizes both βcf hCG and intact hCG.

[0046] The aforementioned amount or concentration can be measured by a color intensity proportional to it.

[0047] If the ratio of the amount or concentration of βcf hCG to the amount or concentration of intact hCG exceeds 50% at 5 weeks and 6 days of LMP, it is determined to be a normal pregnancy. 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 blighted ovum, but it is not limited to this.

[0048] Specifically, the present invention provides a method for interpreting information necessary to determine pregnancy or abnormal pregnancy, including the steps of: i) applying a sample to the immunizer so that Intact hCG and βcf hCG in the sample react with a multi-bound anti-hCG monoclonal antibody conjugated to a probe material; ii) confirming the reaction of βcf hCG and Intact hCG in the sample using a first detection line and a second detection line, wherein βcf hCG and Intact hCG are sensed by the color intensity generated by the presence of a sandwich complex in each detection line; and iii) determining that if the ratio of the color intensity of the first detection line to the color intensity of the second detection line (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 LMP 5-6 weeks, i.e., LMP 5 weeks 6 days, it is determined to be a normal pregnancy, and if it is 30% or less, it is determined to be an ectopic pregnancy or blighted ovum.

[0049] The aforementioned color intensity is the color intensity corresponding to the concentrations of βcf hCG and Intact hCG, and it was confirmed that the concentration and color intensity are proportional in one specific embodiment of the present invention.

[0050] Furthermore, the present invention provides a method for determining pregnancy or abnormal pregnancy using the immunochromatographic strip.

[0051] The method for determining pregnancy or abnormal pregnancy includes the steps of: i) applying the sample to the immunizer so that Intact hCG and βcf hCG present in the sample react with a multi-binding anti-hCG monoclonal antibody conjugated to a probe material; ii) confirming the reaction of βcf hCG and Intact hCG in the sample using a first detection line and a second detection line, wherein βcf hCG and Intact hCG are sensed by the color intensity generated by the presence of a sandwich complex in each detection line; and iii) determining that a normal pregnancy is determined if the ratio of the color intensity of the first detection line to the color intensity of the second detection line (the ratio of the color intensity of βcf hCG to the color intensity of βcf hCG and Intact hCG) exceeds 50% at 5 weeks and 6 days of LMP, and determining that an abnormal pregnancy is determined if it is 30% or less at 5 weeks and 6 days. [Effects of the Invention]

[0052] This invention provides a novel immunodiagnostic device that measures the distribution ratio of beta-core fragment hCG (βcf hCG) to distinguish between normal and abnormal pregnancies. An increase in the βcf hCG distribution ratio confirmed by this device indicates that the fetus has implanted and developed normally, which helps to alleviate anxiety in early-stage pregnant women. Furthermore, if the βcf hCG distribution ratio decreases significantly, prompt action and treatment in cooperation with hospitals can be expected, which helps to provide pregnant women with information on maintaining a normal pregnancy. [Brief explanation of the drawing]

[0053] [Figure 1] This is a schematic diagram illustrating the reaction principle resulting from the concentration ratio of intact hCG and βcf hCG in the sample of the present invention, and an example of its implementation using an immunochromatographic strip. [Figure 2] This figure shows the correlation between the intensity of color development of intact hCG and βcf hCG at different amounts or concentrations. [Figure 3] This figure shows the changes in the distribution of intact hCG and βcf hCG during a normal pregnancy. [Figure 4]This figure shows the changes in the distribution of intact hCG and βcf hCG during ectopic pregnancy. [Figure 5] This figure shows the measured and compared ratios of βcf hCG / (Intact hCG + βcf hCG) color intensity in normal pregnancy groups and ectopic pregnancy groups at different gestational weeks. [Figure 6] This figure shows the results of a clinical evaluation of normal pregnancy samples using the diagnostic device of the present invention. [Figure 7] This figure shows the results of a clinical evaluation of normal pregnancy samples using the diagnostic device of the present invention. [Figure 8] This figure shows the results of a clinical evaluation of ectopic pregnancy specimens using the diagnostic device of the present invention. [Figure 9] This figure shows the results of a clinical evaluation of ectopic pregnancy specimens using the diagnostic device of the present invention. [Figure 10] This figure shows the correlation between the color intensity and the respective concentration ratios when positive samples of Intact hCG alone, βcf hCG alone, and a mixture of Intact hCG and βcf hCG were prepared at different concentrations using the kit manufactured according to the present invention and reacted. [Modes for carrying out the invention]

[0054] The contents of the present invention will be described in more detail below through the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, but also includes equivalent modifications of the technical idea.

[0055] Example 1. Preparation of immunochromatographic strips 1-1. Fabrication of a membrane with a first detection line, a second detection line, and a control line formed on it. Three different antibodies were dispensed onto nitrocellulose membranes. The first detection line contained anti-βcf hCG monoclonal antibody as the βcf hCG antibody, the second detection line contained anti-intact hCG monoclonal antibody as the intact hCG antibody, and the control line contained goat anti-mouse immunoglobulin. After dispensing, the membranes were dried.

[0056] 1-2. Fabrication of a conjugate pad A first conjugate solution was prepared by conjugating colloidal gold nanoparticles with a multi-binding 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 into pre-treated conjugate pads, dried completely, and then cut to appropriate sizes.

[0057] 1-3. Preparation of sample pads The sample pads were thoroughly immersed in a pretreatment solution containing buffering and preservatives, dried completely, and then cut to the appropriate size for preparation.

[0058] 1-4. Production of moisture-absorbing pads I prepared the moisture-absorbing pads by cutting them to the appropriate size while they were still dry.

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

[0060] Specifically, 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-absorbing pad were attached so as to overlap each other.

[0061] In Figure 1, the immunochromatographic strip includes the following structure.

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

[0063] 1-6. Analyzer A reflectance reader equipped with analysis software on a CCD camera or laser light source substrate was used to quantify the color development or brightness sensitivity of the detection line, and to quantify it as signal strength.

[0064] 1-7. Device Assembly After placing the manufactured immunochromatographic strips for normal pregnancy, gestational age, and abnormal pregnancy detection (including ectopic pregnancy and blighted ovum) into the strip fixing position of the plastic lower device, the device is inserted into the upper device, which has a sample input port and a result confirmation window, and then assembled.

[0065] Example 2. Measurement of changes in color intensity according to the concentrations of Intact hCG and βcf hCG in the normal pregnancy group and the abnormal pregnancy group including ectopic pregnancy. Conventionally, diagnoses were made based on concentration ratios, but the pregnancy diagnostic device of the present invention makes a determination by confirming the color intensity. The color intensity was measured uniformly using all instruments that measure color intensity, and this was quantified.

[0066] The measurement was performed using an analytical device that constitutes the pregnancy diagnostic device of the present invention, and it was confirmed that the concentration and color intensity are proportional (Figure 2).

[0067] 2-1. Measurement of changes in color intensity according to the concentration of intact hCG and βcf hCG in the normal pregnancy group. The change in color intensity in response to the concentrations of intact hCG and βcf hCG was measured in the normal pregnancy group. In the normal pregnancy group, intact hCG is detected from week 3 and 4 of the late pregnancy period (LMP) and increases until week 5. This allows for testing whether or not an early pregnancy has occurred. βcf hCG is detected from the latter half of week 4 of the LMP, increases rapidly from week 5, and is maintained at a high concentration from week 6 onward (Figure 3).

[0068] Furthermore, when βcf hCG was detected at LMP 5 weeks, the gestational sac (the sac surrounding the baby) could be confirmed by ultrasound. From LMP 6 weeks onward, intact hCG decreased while βcf hCG remained constant, resulting in a reversal phenomenon, and the fetus could be confirmed by ultrasound.

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

[0070] As a result, in normal pregnancies between 4 and 7 weeks of LMP, intact hCG levels increased and then decreased, while βcf hCG levels gradually increased and remained stable. In contrast, in ectopic pregnancies, βcf hCG was either undetectable or detected at very low concentrations. This was confirmed as a significant result for distinguishing between normal and ectopic pregnancies.

[0071] On the other hand, in cases of blighted ovum, βCF hCG levels remained low, below 30% from 5 weeks and 6 days of LMP, and while a gestational sac was visible on ultrasound, the fetus and heartbeat could not be detected.

[0072] Example 3. Measurement of βcf hCG / (Intact hCG + βcf hCG) intensity ratio in the normal pregnancy group and the abnormal pregnancy group including ectopic pregnancy. To use the results of Example 2 above to distinguish between gestational age, normal pregnancy group, and abnormal pregnancy group including ectopic pregnancy, the intensity ratio of βcf hCG / (Intact hCG + βcf hCG) according to the 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 calculating the distribution ratio (%) of βcf hCG The pregnancy diagnostic device of the present invention consists of a "mobile phase" that reacts with a sample and moves to a 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 a first detection line, a second detection line, and a control line.

[0074] The mobile phase contains an indicator that can express the concentration intensity of the sample, namely gold particles, nanobeads, fluorescence, or phosphorescence. 、 This is a dried product to which multiple-conjugated anti-hCG monoclonal antibodies capable of recognizing both intact hCG and βcf hCG are bound.

[0075] In other words, the antibody bound to the label is characterized by its ability to recognize and react with both Intact hCG and βcf hCG.

[0076] The first detection line of the stationary phase is immobilized with anti-βcf hCG monoclonal antibody, allowing only βcf hCG to be detected from the sample, while the second detection line is immobilized with anti-intact hCG monoclonal antibody, allowing only intact hCG to be detected from the sample.

[0077] As a result, the Intact hCG and βcf hCG contained in the sample react with the conjugate in a primary manner according to their concentration ratio and move to the stationary phase. Then, according to the characteristics of the antibodies immobilized on the stationary phase, each sample is separated and detected.

[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 allowed for clearer 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 pregnancies, at different gestational weeks. Based on the color intensity corresponding to the detected concentration ratio of intact hCG and βcf hCG measured in the normal pregnancy group and the ectopic pregnancy group at different gestational weeks, the distribution ratio (%) of βcf hCG according to the color intensity ratio of βcf hCG / (intact hCG + βcf hCG) was confirmed using the following formula.

[0080] <expression> JPEG0007838072000002.jpg20170

[0081] As a result, as shown in Table 1 and Figures 5-9 below, in the case of a normal pregnancy in which 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 0 days of LMP, increased to 50% at 5 weeks 6 days of LMP, reached 70% at 6 weeks of LMP, and was maintained at a rate of 80% from 8 weeks of LMP onward. On the other hand, in the case of an ectopic pregnancy in which implantation occurs abnormally, the distribution ratio of βcf hCG was maintained at 10% or less at 5-6 weeks of LMP, and no increase was observed. Furthermore, it was confirmed that the gestational sac (the sac surrounding the baby) was not observed on ultrasound.

[0082] JPEG0007838072000003.jpg50170**The percentages for each week are shown based on the median value.

[0083] Furthermore, in cases of blighted ovum, LMP did not increase from 5 weeks and 6 days, and the distribution ratio of βcf hCG was confirmed to be maintained at 30% or less. Ultrasound examination revealed a gestational sac, but the fetus and heartbeat could not be detected.

[0084] As a result, the pregnancy diagnostic device of the present invention distinguishes between normal pregnancies and abnormal pregnancies, including ectopic pregnancies, based on the color intensity (intensity) corresponding to the detected concentrations of Intact hCG and βcf hCG, using the color intensity ratio of βcf hCG / (Intact hCG + βcf hCG) and the distribution ratio (%) of βcf hCG, and determines the timing when a gestational sac can be confirmed. This distinction can be made more accurately and quickly with a single measurement.

[0085] Example 4. Verification of kit performance (specificity) and analysis of correlation with color intensity. Figure 10 shows the results of preparing and reacting positive samples of Intact hCG alone, βcf hCG alone, and a mixture of Intact hCG and βcf hCG at different concentrations using the kit produced according to the present invention.

[0086] Positive samples were prepared by diluting Intact hCG in negative standard samples to concentrations of 100, 600, and 800 mIU / mL, respectively, and then reacted. The first detection line (βcf hCG) did not develop color, while the second detection line (Intact hCG) was used to confirm the color intensity at each concentration.

[0087] βcf hCG was diluted in negative standard samples to concentrations of 0.03, 0.5, and 5 pmol / mL, respectively, and prepared as positive samples. When these samples were reacted, 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 aforementioned Intact hCG and βcf hCG were mixed and diluted in a negative standard sample to concentrations of 100 mIU / mL (Intact hCG) + 0.03 pmol / mL (βcf hCG), 800 mIU / mL (Intact hCG) + 0.5 pmol / mL (βcf hCG), and 600 mIU / mL (Intact hCG) + 5 pmol / mL (βcf hCG), respectively, to prepare positive samples. When these samples were reacted, both the first and second detection lines showed color intensity corresponding to each concentration, and the color intensity at each concentration was the same as that obtained when treating with Intact hCG alone or βcf hCG alone.

[0089] These results verify that the multi-binding anti-hCG monoclonal antibody used in the present invention reacts specifically with Intact hCG and βcf hCG without interference phenomena. Furthermore, the color intensity increases quantitatively in accordance with the concentrations of mixed Intact hCG and βcf hCG in the sample, suggesting that accurate distribution ratios can be measured.

[0090] Although embodiments of the present invention have been described above, the present invention is not limited to the embodiments described above and can be manufactured in a variety of different forms. A person with ordinary skill in the art to which the present invention belongs will understand that the invention can be implemented in other specific forms without changing the technical idea or essential features of the present invention. Accordingly, the embodiments described above should be understood in all respects as illustrative and not limiting.

Claims

1. i) A sample area for containing the test sample to be analyzed, ii) A conjugate region that is linked to the sample region and contains a multi-binding anti-hCG monoclonal antibody that commonly recognizes Intact hCG and βcf hCG conjugated to the probe material, iii) A signal detection region that can completely separate and detect Intact hCG and βcf hCG bound in reaction to the conjugate, and is characterized by including a first detection line on which a monoclonal antibody that can specifically form a complex only with βcf hCG is immobilized, a second detection line on which a monoclonal antibody that can specifically form a complex only with Intact hCG is immobilized, and a control line, iv) Includes a moisture-absorbing region located downstream of the signal detection region that absorbs the test sample after the signal detection reaction has finished, An immunoassay device for determining pregnancy or abnormal pregnancy, wherein if the ratio of the color intensity of βcf hCG to the color intensity of βcf hCG and intact hCG measured by the detection line, derived by the following formula 1, 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, it is determined to be an abnormal pregnancy, including ectopic pregnancy and blighted ovum. <Formula 1>

2. The immunoassay apparatus according to claim 1, further comprising an analytical device.

3. The immunoassay apparatus according to claim 1, characterized in that the probe material in 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.

4. The immunoassay apparatus according to claim 1, characterized in that the signal detection region of iii) is one selected from the group consisting of nitrocellulose, cellulose, polyethylene, polyethersulfone, and nylon.

5. The immunosuppressant according to claim 1, characterized in that the moisture-absorbing region of iv) above includes an absorbent dispersed in the pores of the porous support or adsorbed or coated onto the fibers of the porous support.

6. The immunization device according to claim 1, characterized in that it can determine the gestational age at which the gestational sac can be observed by the color development of the first detection line.

7. Methods for analyzing information necessary to determine pregnancy or abnormal pregnancy, including the following stages: i) A step of measuring the amount or concentration of βcf hCG and intact hCG from the sample to be analyzed, ii) A step in 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 multi-binding anti-hCG monoclonal antibody that recognizes both βcf hCG and Intact hCG. The method is characterized in that if the ratio of the color intensity of βcf hCG to the color intensity of βcf hCG and Intact hCG derived by formula 1 below 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, it is determined to be an abnormal pregnancy including ectopic pregnancy and blighted ovum, the amount or concentration is measured using a multi-binding anti-hCG monoclonal antibody that recognizes both βcf hCG and Intact hCG. <Formula 1>

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