Novel immunodiagnostic device for determining normal and abnormal pregnancies by measuring distribution ratio of beta core fragment hcg

Abstract

The present invention relates to an immune device for determining pregnancy or abnormal pregnancy and, more specifically, to an immune device including a novel multi-binding anti-hCG monoclonal antibody capable of commonly recognizing intact hCG and βcf hCG, wherein the immune device includes two detection lines and a control line to separately detect intact hCG and βcf hCG contained in a specimen, and 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) are digitalized, thereby accurately and quickly determining gestational age, normal pregnancy, and abnormal pregnancy including ectopic pregnancy, and blight ovum.

Description

A novel immunodiagnostic device that determines normal and abnormal pregnancies by measuring the distribution ratio of beta-core fragment HCG.

[0001] The present invention relates to an immunological device capable of determining normal and abnormal pregnancy during pregnancy and an interpretation method using the same, and more particularly, to an immunological device capable of determining 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 a pregnant woman, and an interpretation method using the same. The core principle of the immunological device is to use a novel multi-conjugated anti-hCG monoclonal antibody capable of commonly recognizing and binding only to intact hCG and βcf hCG among various hCG proteins present in the urine of a pregnant woman as a mobile phase bound to a marker, and to specifically separate intact hCG and βcf hCG, respectively, in a stationary phase.

[0002] Human chorionic gonadotropin (hCG) is a glycoprotein hormone produced during pregnancy. Produced by the chorionic trophoblasts of the placenta, hCG stimulates continued progesterone production in early pregnancy, supporting implantation until the 10th week of pregnancy, when placental function is fully developed.

[0003] Intact hCG (I-hCG), the active form in the body, has a molecular weight of approximately 37 kDa and consists of 244 amino acids. Intact hCG is largely composed of 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 biologically active structure. 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 beta hCG (free β-hCG), free alpha hCG (free α-hCG), and beta core fragment hCG (β-core fragment hCG).

[0004] Beta core fragment hCG (βcf hCG) is the hCG protein predominantly present in pregnant women's urine and has a molecular weight of approximately 14 kDa. Intact hCG is predominantly secreted at 4 weeks of LMP (Last Menstrual Period), and from 5 weeks of LMP, βcf hCG begins to gradually increase and is secreted in a concentration superior to intact hCG at 5 weeks and 6 days or after 6 weeks (John Walter Larsen et al., 2015).

[0005] In pregnant women, intact hCG protein distribution is predominantly present in the blood at 98%, but in urine, βcf hCG increases by up to 80% as the gestational age progresses, presenting a predominant concentration compared to intact hCG. Conventional pregnancy diagnostic kits using intact hCG as a marker determine pregnancy by qualitatively diagnosing intact hCG present in urine. However, in a test of 11,760 women diagnosed as pregnant using existing pregnancy diagnostic kits, 22 women (approximately 0.2%) were confirmed as false negatives, and the intact hCG blood concentration in these women was measured as high as 268,022.5 IU / mL, indicating that conventional urine pregnancy diagnostic kits have a false negative problem due to the Hook effect. The fundamental cause of this has been revealed to be due to the excessive presence of βcf hCG in urine (Richard T. Griffey et al., 2013).

[0006] A false-negative urine pregnancy test can have serious consequences in emergency department (ED) settings. For example, if a female patient is pregnant but receives a false-negative result from a standard urine pregnancy test, this could lead to medical errors, such as treatment without considering the fetus.

[0007] Meanwhile, ectopic pregnancy accounts for 9% of all deaths in pregnant women during the first trimester, increasing the mortality rate of pregnant women, and the prevalence of ectopic pregnancy is gradually increasing worldwide.

[0008] Against this backdrop, the present inventors conducted extensive research to resolve the fundamental hook effect and develop a method for distinguishing between normal and abnormal pregnancies, and as a result, developed a novel multi-conjugated anti-hCG monoclonal antibody that can commonly recognize both intact hCG and βcf hCG, and resolved the hook effect by specifically separating and detecting intact hCG and βcf hCG in urine samples, and confirmed that 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) can be quantified to accurately and quickly distinguish gestational weeks, normal pregnancy, ectopic pregnancy, and abnormal pregnancy including dead ovum, thereby completing the present invention.

[0009] The purpose of the present invention is to provide an immune device and an analysis method capable of maintaining a normal pregnancy and determining an abnormal pregnancy in the early stage of pregnancy by specifically separating and detecting βcf hCG and intact hCG contained in the urine of a pregnant woman and accurately measuring the distribution ratio of βcf hCG.

[0010] The immunological device of the present invention is an immunological device for determining pregnancy or abnormal pregnancy, comprising: i) a sample region for receiving a test sample to be analyzed; ii) a conjugate region linked to the sample region and including a multi-conjugated 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 on which an anti-βcf hCG antibody capable of specifically binding only to βcf hCG is immobilized, a second detection line on which an anti-intact hCG antibody capable of specifically binding only to intact hCG is immobilized, and a control line; and iv) an absorption region located downstream of the signal detection region for absorbing a test sample for which a signal detection reaction has been completed.

[0011] The interpretation method of the present invention is a method for interpreting information necessary for determining pregnancy or abnormal pregnancy, comprising the following steps: i) a step of combining a multi-binding anti-hCG monoclonal antibody that commonly recognizes βcf hCG and Intact hCG to reflect the distribution ratio of βcf hCG and Intact hCG in a test sample to be analyzed; ii) a step of measuring the intensity of βcf hCG and Intact hCG color development; and iii) a step of determining pregnancy or abnormal pregnancy based on the intensity distribution ratio of βcf hCG color development.

[0012] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the embodiments described in detail below together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below and may be implemented in various different forms. These embodiments are provided solely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the present invention, and the present invention is defined solely by the scope of the claims.

[0013] The terminology used herein is for the purpose of describing embodiments only and is not intended to limit the present invention. In this specification, the singular also includes the plural unless specifically stated otherwise. As used herein, the terms "comprises" and / or "comprising" do not exclude the presence or addition of one or more other components in addition to the mentioned components. Like reference numerals refer to like components throughout the specification, and "and / or" includes each and any combination of one or more of the mentioned components. Although "first", "second", etc. are used to describe various components, these components are not limited by these terms. These terms are only used to distinguish one component from another. Therefore, it should be understood that a first component mentioned below may also be a second component within the technical spirit of the present invention.

[0014] Unless otherwise defined, all terms (including technical and scientific terms) used herein may be used in their common sense to those skilled in the art to which the present invention pertains. Furthermore, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise.

[0015] The present invention provides an immune device and an analysis method capable of maintaining a normal pregnancy and determining an abnormal pregnancy in the early stage of pregnancy by specifically separating and detecting βcf hCG and intact hCG contained in the urine of a pregnant woman using a multi-conjugated anti-hCG monoclonal antibody that commonly recognizes intact hCG and βcf hCG and accurately measuring the distribution ratio of βcf hCG.

[0016] The multivalent anti-hCG monoclonal antibody commonly recognizing intact hCG and βcf hCG used in the present invention has the characteristic of accurately reflecting the distribution ratio of intact hCG and βcf hCG according to the gestational week, and is useful for more accurately and efficiently determining normal and abnormal pregnancy based on the interpretation of the distribution ratio.

[0017] The present invention aims to accurately distinguish between normal and abnormal pregnancy. This is difficult to achieve by simply measuring the concentration of intact hCG and βcf hCG using each antibody for intact hCG and βcf hCG, and can be achieved by measuring the ratio of reactions with intact hCG and βcf hCG present in a sample. When using each antibody for intact hCG and βcf hCG, there is a limit to the concentration of intact hCG and βcf hCG that the antibody can recognize, so it is rather difficult to accurately determine the concentration of intact hCG and βcf hCG and its ratio. On the other hand, when using a monoclonal antibody of the present invention that commonly recognizes intact hCG and βcf hCG, even if there is a limit to the value of intact hCG and βcf hCG that can be recognized, the concentration and concentration ratio of intact hCG and βcf hCG can be more accurately predicted because the ratio of reactions with intact hCG and βcf hCG present in the sample is measured.

[0018] The present invention does not use monoclonal antibodies corresponding to Intact hCG and βcf hCG, respectively, but uses a monoclonal antibody that commonly recognizes Intact hCG and βcf hCG. This is because the total content of Intact hCG and βcf hCG in a sample can be confirmed proportionally.

[0019] By using the multi-conjugated anti-hCG monoclonal antibody of the present invention, it is possible to accurately determine normal or abnormal pregnancy by deriving an accurate distribution ratio.

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

[0021] An ectopic pregnancy refers to a pregnancy in which the fertilized egg implants in a location other than the uterus, the normal implantation site. The implantation site of an ectopic pregnancy can be an abnormal location within the uterus or somewhere else. Most ectopic pregnancies occur in the fallopian tubes, but less common cases include ovarian, peritoneal, or cervical pregnancies. It is one of the most common medical emergencies, accounting for approximately 1-2% of all pregnancies.

[0022] The above-mentioned aborted ovum refers to a case where placental tissue develops on ultrasound, but the fetus is not visible. In cases of aborted ovum, the placenta may develop for a period without an embryo, but ultimately, it results in a spontaneous abortion, with abdominal pain and bleeding, followed by the expulsion of the conceptus from the uterus.

[0023] In addition, the present invention provides an immunoassay device for determining pregnancy or abnormal pregnancy, comprising: i) a sample region for receiving a test sample to be analyzed; ii) a conjugate region linked to the sample region and including an anti-intact hCG / βcf hCG multi-binding anti-hCG monoclonal antibody conjugated to a probe material; iii) a signal detection region linked to the conjugate region, the signal detection region including a first detection line connected to the sample region and having an anti-βcf hCG antibody specifically binding only to βcf hCG immobilized thereon, a second detection line having an anti-intact hCG antibody specifically binding only to intact hCG immobilized thereon, and a control line; and iv) an absorption region located downstream of the signal detection region for absorbing a test sample for which a signal detection reaction has been completed.

[0024] The above i) specimen area is preferably designed to accommodate a liquid sample such as pregnant woman's urine, but any sample expected to contain intact hCG and / or βcf hCG may be used.

[0025] The above-mentioned sample region may additionally have a filtering function to further enhance 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 increases the reaction between the analyte and the conjugate or eliminates the influence of interfering substances may be additionally provided upstream of the sample region.

[0026] The probe material of the conjugate region of the above ii) may be at least one 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, pigment beads, and proteins, but is not limited thereto.

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

[0028] The signal detection region of the above iii) is a medium in which the mobile phase and the sample are developed, and the mobile phase and the sample to be tested can move by the capillary phenomenon of the porous membrane of the signal detection region. The signal detection region may be any one selected from the group consisting of a well plate synthesized with nitrocellulose, cellulose, polyethylene, polyethersulfone, polystyrene, polycarbonate, polymethyl methacrylate, nylon, PVDF, polyvinyl resin or 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 to which the second detection line is immobilized. 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 to which the first detection line is immobilized.

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

[0031] The present invention is an immune device composed of two detection lines, and has the characteristic of being able to accurately determine gestational weeks, normal pregnancy, and abnormal pregnancy including ectopic pregnancy by quantitatively or qualitatively analyzing the color development intensity of the detection lines that detect βcf hCG and intact hCG in a sample, and comparing the distribution ratio of βcf hCG by digitizing it using the following formula derived in the present invention.

[0032]

[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 to 60% or more at 5 to 6 weeks of LMP, it can be determined as a normal pregnancy, if it is 5 to 15% or less, it can be determined as an ectopic pregnancy, and if it is maintained at 20 to 30% or less until 6 weeks and 6 days, it can be determined as a dead ovum.

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

[0035] The above iv) absorption region may include an absorbent dispersed in the pores of the porous support or adsorbed or coated on the fibers of the porous support, and may additionally include a porous film layer on the upper surface of the porous support, but is not limited thereto.

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

[0037] The immunoassay device of the present invention may include a solid support at the bottom. The solid support may be formed of any material selected from the group consisting of nitrocellulose, nylon, PVDF, glass, and plastic, but is not limited thereto. Since the device is manufactured by attaching a pad, membrane, etc. to the solid support, the durability of the strip can be increased and handling and storage can be facilitated. In addition, the device can facilitate the attachment of additional external devices.

[0038] Plastic materials that can be used as the above 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 analysis device. The analysis device may evaluate the color intensity of the first detection line and the second detection line. The analysis device may include a reader coupled with a software program capable of evaluating the intensity, color, luminescence, and / or fluorescence of the analyte complex formed in the first detection line and the second detection line. The reader may compare the intensity, color, luminescence, and / or fluorescence with a preprogrammed threshold value and provide a digital output based on the comparison. According to one embodiment of the present invention, the analysis device used a reflectance reader equipped with analysis software based on a CCD camera or a laser light source, and was capable of determining normal pregnancy and abnormal pregnancy, including ectopic pregnancy, with high accuracy. The analysis device may further include a display that provides information related to abnormal pregnancy, including ectopic pregnancy.

[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 chromogenic intensity of βcf hCG to the chromogenic intensity of βcf hCG and Intact hCG increases by 50 to 60% or more at 5 to 6 weeks of LMP, the gestational sac and fetus can be observed in a normal pregnancy, whereas if it is 5 to 15% or less, the gestational sac cannot be observed in an 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., the measured value of βcf hCG / (βcf hCG + Intact hCG), was compared, and it was confirmed that it was significantly lower in the case of abnormal pregnancy, including ectopic pregnancy, compared to normal pregnancy.

[0042] Additionally, the present invention provides a kit characterized in that the immune device is additionally fixed within the device.

[0043] The above kit is a kit in which a guide and a strip support are provided in the lower device, a sample input port is provided in the upper device, and a result confirmation window is provided at positions corresponding to the first detection line, the second detection line, and the control line.

[0044] The upper and lower devices can be manufactured using conventional plastic materials, for example, polycarbonate, acrylonitrile butadiene styrene (ABS), etc., but are not limited thereto.

[0045] In addition, the present invention provides a method for interpreting information necessary for determining pregnancy or abnormal pregnancy, comprising the following steps: i) a step of measuring the amount or concentration of βcf hCG and Intact hCG in a test sample to be analyzed; and ii) a step of determining pregnancy or abnormal pregnancy by 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 multivalent anti-hCG monoclonal antibody that commonly recognizes βcf hCG and Intact hCG.

[0046] The above amount or concentration can be measured by the color intensity proportional thereto.

[0047] If the ratio of the amount or concentration of βcf hCG to the amount or concentration of the above βcf hCG and Intact hCG is greater than 50% at 5 weeks and 6 days of LMP, it is judged to be a normal pregnancy, and if it is less than 30% at 5 weeks and 6 days, it can be judged to be an abnormal pregnancy such as an ectopic pregnancy or a dead ovum, but is not limited thereto.

[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 above-described immunological device so that Intact hCG and βcf hCG in the sample react with a multi-conjugated anti-hCG monoclonal antibody conjugated as a probe substance; ii) confirming the reaction of βcf hCG and Intact hCG in the sample by 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 in each detection line; and iii) determining that a normal pregnancy is present if the ratio of the color intensity of the first detection line to the first detection line and 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 to 60% or more at LMP 5 to 6 weeks, that is, LMP 5 weeks and 6 days, and determining that an ectopic pregnancy or a dead ovum is present if the ratio is 30% or less.

[0049] The above color development intensity is the color development intensity according to the concentration of βcf hCG and Intact hCG, and in a specific embodiment of the present invention, it was confirmed that the concentration and the color development intensity are proportional.

[0050] In addition, the present invention provides a method for determining pregnancy or abnormal pregnancy using the immunochromatography strip.

[0051] The method for determining the pregnancy or abnormal pregnancy comprises the steps of: i) applying a sample to the immunological device so that Intact hCG and βcf hCG present in the sample react with a multi-conjugated anti-hCG monoclonal antibody conjugated as a probe substance; ii) confirming the reaction of βcf hCG and Intact hCG in the sample by 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 in each detection line; and iii) determining a normal pregnancy if the ratio of the color intensity of the first detection line to the first detection line and 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 an abnormal pregnancy if it is 30% or less at 5 weeks and 6 days.

[0052] The present invention provides a novel immunodiagnostic device that measures the beta core fragment hCG (βcf hCG) distribution ratio to determine normal and abnormal pregnancy. An increase in the βcf hCG distribution ratio thus confirmed indicates that the fetus is implanting and growing normally, which will have the effect of relieving anxiety in early pregnant women. In addition, if the βcf hCG distribution ratio drops significantly, prompt action and treatment in conjunction with a hospital can be expected, and it will be helpful in providing pregnant women with information on maintaining a normal pregnancy.

[0053] Figure 1 is a schematic diagram showing the reaction principle due to the concentration ratio of intact hCG and βcf hCG in a sample of the present invention and an example of an implementation of an immunochromatography strip.

[0054] Figure 2 is a diagram showing the correlation between the color intensity according to the amount or concentration of intact hCG and βcf hCG.

[0055] Figure 3 is a diagram showing changes in the distribution of Intact hCG and βcf hCG during normal pregnancy.

[0056] Figure 4 is a diagram showing changes in the distribution of Intact hCG and βcf hCG in ectopic pregnancy.

[0057] Figure 5 is a graph comparing the color intensity ratio of βcf hCG / (Intact hCG + βcf hCG) in the normal pregnancy group and the ectopic pregnancy group by gestational week.

[0058] Figure 6 is a diagram showing the results of a clinical evaluation using the diagnostic device of the present invention on a normal pregnancy specimen.

[0059] Figure 7 is a diagram showing the results of a clinical evaluation using the diagnostic device of the present invention on a normal pregnancy specimen.

[0060] Figure 8 is a diagram showing the results of a clinical evaluation using the diagnostic device of the present invention for an ectopic pregnancy specimen.

[0061] Figure 9 is a diagram showing the results of a clinical evaluation using the diagnostic device of the present invention for an ectopic pregnancy specimen.

[0062] Figure 10 is a diagram showing the color development intensity correlation results according to each concentration ratio when Intact hCG alone, βcf hCG alone, Intact hCG and βcf hCG mixed positive samples were prepared and reacted at different concentrations using a kit manufactured according to the present invention.

[0063] Hereinafter, the present invention will be described in more detail 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 includes modifications of technical concepts equivalent thereto.

[0064] Example 1. Preparation of immunochromatography strips

[0065] 1-1. Fabrication of a membrane with a first detection line, a second detection line, and a control line

[0066] Three different antibodies were dispensed onto a nitrocellulose membrane. The first detection line was an anti-βcf hCG monoclonal antibody as a βcf hCG antibody, the second detection line was an anti-intact hCG monoclonal antibody as an intact hCG antibody, and the control line was goat anti-mouse IgG, which were dispensed and then dried.

[0067] 1-2. Fabrication of conjugate pads

[0068] A first conjugate solution was prepared, in which colloidal gold nanoparticles were conjugated with a multimeric anti-hCG monoclonal antibody that commonly recognizes intact hCG and βcf-hCG. Furthermore, a second conjugate solution was prepared, in which colloidal gold nanoparticles were conjugated with mouse immunoglobulin (Mouse IgG). The first and second conjugate solutions were dispensed onto pretreated conjugate pads, completely dried, and then cut into appropriate sizes for preparation.

[0069] 1-3. Sample pad production

[0070] The specimen pad was sufficiently soaked in a specimen pad pretreatment solution containing a buffer and preservative, completely dried, and then cut to an appropriate size.

[0071] 1-4. Manufacturing an absorption pad

[0072] The dried absorbent pad was prepared by cutting it into an appropriate size.

[0073] 1-5. Fabrication of immunochromatography strips

[0074] The membrane, conjugate pad, specimen pad, and absorbent pad prepared through each of the above processes are assembled as shown in Fig. 1.

[0075] That is, the sample pad was attached so as to overlap with one end of the conjugate pad, one end of the detection pad was attached so as to overlap with the other end of the conjugate pad, and the other end of the detection pad and one end of the absorbent pad were attached so as to overlap with each other.

[0076] In Fig. 1, the immunochromatography strip includes the following structure.

[0077] 1: Sample pad

[0078] 2: Conjugate pad

[0079] 3: Nitrocellulose membrane

[0080] 4: Absorption pad

[0081] 5: First detection line (Test line 1) with immobilized anti-βcf hCG monoclonal antibody

[0082] 6: Second detection line (Test line 2) with immobilized anti-intact hCG monoclonal antibody

[0083] 7: Control line with fixed goat anti-mouse immunoglobulin

[0084] 1-6. Analysis device

[0085] A reflectance reader equipped with analysis software based on a CCD camera or laser light source was used to quantify the color or brightness sensitivity of the detection line and digitize it as signal intensity.

[0086] 1-7. Device Assembly

[0087] The manufactured immunochromatographic strip for determining normal pregnancy, gestational weeks, and abnormal pregnancy, including ectopic pregnancy and dead ovum, is inserted into the strip fixing position of the plastic lower device, and then assembled by inserting it into the upper device with the sample input port and result confirmation window.

[0088] Example 2. Measurement of color intensity changes according to the concentration of intact hCG and βcf hCG in normal pregnancy groups and abnormal pregnancy groups including ectopic pregnancy.

[0089] Previously, diagnosis was made based on concentration ratios, but the pregnancy diagnosis device of the present invention determines the diagnosis by confirming the color intensity. The color intensity was measured equally in all color intensity measuring devices and quantified.

[0090] It was confirmed that the concentration and color intensity were proportional when measuring using the analysis device, which is a component of the pregnancy diagnosis device of the present invention (Fig. 2).

[0091] 2-1. Measurement of color intensity changes according to the concentration of intact hCG and βcf hCG in normal pregnancy groups

[0092] In a normal pregnancy group, changes in color intensity according to the concentration of intact hCG and βcf hCG were measured. In the normal pregnancy group, intact hCG begins to be detected from 4 days after 3 weeks of LMP and increases until 5 weeks. This allows for the detection of early pregnancy. βcf hCG begins to be detected from the late 4th week of LMP, increases rapidly from 5 weeks, and remains at high concentrations after 6 weeks (Fig. 3).

[0093] Additionally, when βcf hCG was detected at 5 weeks of LMP, the gestational sac (baby's uterus) could be confirmed by ultrasound examination. From 6 weeks of LMP, intact hCG decreased, while βcf hCG remained, a reversal phenomenon occurred, and the fetus could be confirmed by ultrasound examination.

[0094] 2-2. Measurement of color intensity changes according to the concentration of intact hCG and βcf hCG in abnormal pregnancy groups, including ectopic pregnancies.

[0095] As a result of measuring the change in the concentration of Intact hCG and βcf hCG in the ectopic pregnancy group, it was confirmed that in the ectopic pregnancy group, Intact hCG was detected at 5 weeks of LMP, but βcf hCG was not detected at 5 weeks of LMP or was detected at a very low concentration (Fig. 4).

[0096] As a result, in the case of normal pregnancy between 4 and 7 weeks of LMP, the concentration of intact hCG increases and then decreases, and the concentration of βcf hCG gradually increases and remains constant, whereas in the case of ectopic pregnancy, βcf hCG is not detected or is detected at a very low concentration, which was confirmed as a significant result that distinguishes normal pregnancy from ectopic pregnancy.

[0097] Meanwhile, in the case of aborted ovum, βcf hCG remains low without increasing to less than 30% from the 5th week and 6th day of LMP, and ultrasound observation confirms the gestational sac, but the fetus and heart sounds are not confirmed.

[0098] Example 3. Measurement of the intensity ratio of βcf hCG / (Intact hCG + βcf hCG) in normal pregnancy groups and abnormal pregnancy groups including ectopic pregnancy.

[0099] In order to use the results of Example 2 above to determine the gestational weeks, normal pregnancy group, and abnormal pregnancy group including ectopic pregnancy, the intensity ratio according to the concentration of βcf hCG / (Intact hCG + βcf hCG) by gestational weeks in the normal pregnancy group and abnormal pregnancy group was measured and compared.

[0100] 3-1. Verification of measurement principle and derivation of calculation formula for βcf hCG distribution ratio (%)

[0101] The pregnancy diagnosis device of the present invention comprises 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 a conjugate region among the components of the pregnancy diagnosis device, and the stationary phase corresponds to a signal detection region including a first detection line, a second detection line, and a control line.

[0102] The mobile phase is a dried state in which a novel antibody developed by the inventors of the present invention, a multi-conjugated anti-hCG monoclonal antibody that can commonly recognize Intact hCG and βcf hCG, is bound to an indicator that can express the concentration intensity of the sample, i.e., a gold particle, a nano bead, a fluorescent or phosphorescent indicator.

[0103] That is, the characteristics of the antibody bound to the marker are characterized by recognizing and reacting with both intact hCG and βcf hCG.

[0104] In the first detection line of the fixed phase, an anti-βcf hCG monoclonal antibody is fixed, so only βcf hCG is detected in the specimen sample, and in the second detection line, an anti-intact hCG monoclonal antibody is fixed, so only intact hCG is detected in the specimen sample.

[0105] Accordingly, depending on the concentration ratio of Intact hCG and βcf hCG included in the specimen sample, the conjugate primarily reacts and moves to the stationary phase, and each sample is separated and detected according to the characteristics of the antibody fixed to the stationary phase.

[0106] Meanwhile, the calculation formula for the βcf hCG distribution ratio (%) was derived through the color development intensity ratio of βcf hCG / (Intact hCG + βcf hCG), which enabled more clear measurement and analysis.

[0107] 3-2. Measurement of the color intensity ratio of βcf hCG / (Intact hCG + βcf hCG) in normal pregnancies and abnormal pregnancies including ectopic pregnancies by gestational week.

[0108] Based on the color intensity according to the detected concentration ratio of Intact hCG and βcf hCG measured in the normal pregnancy group and the ectopic pregnancy group by gestational week, the distribution ratio (%) of βcf hCG through the color intensity ratio of βcf hCG / (Intact hCG + βcf hCG) was confirmed using the following formula.

[0109]

[0110] As a result, as shown in Table 1 and Figures 5 to 9 below, in the case of a normal pregnancy in which a fertilized egg is normally implanted in the uterus and a baby grows, the distribution ratio of βcf hCG is approximately 10 to 20% on day 0 of LMP week 5, increases to 50% on day 6 of LMP week 5, and is maintained at 70% at week 6 of LMP, and 80% after week 8 of LMP. On the other hand, in the case of an ectopic pregnancy in which implantation occurs abnormally, the distribution ratio of βcf hCG is maintained at 10% or less on week 5 to 6 of LMP, and no increase is observed, and it is confirmed that a gestational sac (baby's sac) is not observed on ultrasound.

[0111]

[0112] ** The % for each week is based on the median.

[0113] In addition, in the case of aborted oocytes, it was confirmed that there was no increase from the 5th week and 6th day of LMP, and the distribution ratio of βcf hCG was maintained at 30% or less. Ultrasound observation confirmed the gestational sac, but the fetus and heart sounds were not confirmed.

[0114] Through this, the pregnancy diagnosis device of the present invention distinguishes between a normal pregnancy group and an abnormal pregnancy group including an ectopic pregnancy based on the intensity of color development according to the detected concentration of Intact hCG and βcf hCG, and determines the time when the gestational sac can be confirmed through the ratio of the intensity of color development of βcf hCG / (Intact hCG + βcf hCG) and the distribution ratio (%) of βcf hCG, and can determine more accurately and quickly with a single measurement.

[0115] Example 4. Kit performance (specificity) verification and color intensity correlation analysis

[0116] Based on the kit manufactured according to the present invention, the results of reactions by manufacturing and reacting positive samples of Intact hCG alone, βcf hCG alone, and Intact hCG and βcf hCG mixed at different concentrations are shown in Fig. 10.

[0117] As a result of reacting by diluting intact hCG in a negative standard sample to 100, 600, and 800 mIU / mL concentrations to prepare a positive sample, the first detection line (βcf hCG) did not develop color, while the color development intensity for each concentration was confirmed in the second detection line (Intact hCG).

[0118] As a result of reacting by diluting βcf hCG in a negative standard sample to 0.03, 0.5, and 5 pmol / mL concentrations to prepare a positive sample, the second detection line (Intact hCG) did not develop color, while the color development intensity according to concentration was confirmed in the first detection line (βcf hCG).

[0119] The above Intact hCG and βcf hCG were mixed and diluted in a negative standard sample at a concentration 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, and reacted to prepare a positive sample. As a result, both the first and second detection lines showed a color intensity corresponding to each concentration, and the color intensity was equivalent to the results of treatment with Intact hCG alone and βcf hCG alone for each concentration.

[0120] These results verify that the multi-conjugated anti-hCG monoclonal antibody used in the present invention specifically reacts to intact hCG and βcf hCG without 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, allowing for accurate distribution ratio measurement.

[0121] While the embodiments of the present invention have been described above, the present invention is not limited to the above embodiments, but can be manufactured in various different forms. Those skilled in the art will understand that the present invention can be implemented in other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood to be illustrative in all respects and not restrictive.

Claims

1. i) Specimen area for receiving the test sample to be analyzed; ii) a conjugate region comprising a multimeric anti-hCG monoclonal antibody that is linked to the above-mentioned sample region and commonly recognizes Intact hCG and βcf hCG conjugated to a probe substance; iii) a signal detection region capable of completely separating and detecting intact hCG and βcf hCG bound in reaction to the conjugate, characterized in that it includes 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; and iv) An immunoassay device for determining pregnancy or abnormal pregnancy, comprising an absorbent region located downstream of the signal detection region, which absorbs a test sample after a signal detection reaction has been completed.

2. In paragraph 1, An immune device, characterized in that the immune device further includes an analysis device.

3. In paragraph 1 or 2, An immune device characterized in that the above immune device determines pregnancy or abnormal pregnancy by the ratio of the color intensity of βcf hCG to the color intensity of βcf hCG and Intact hCG measured at the detection line derived by the following formula.

4. In paragraph 1, An immune device, characterized in that the probe material of the above ii) is at least one 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, pigments, and proteins.

5. In paragraph 1, An immune device, characterized in that the signal detection area of ​​the above iii) is any one selected from the group consisting of nitrocellulose, cellulose, polyethylene, polyethersulfone, and nylon.

6. In paragraph 1, An immune device, characterized in that the absorption region of the above iv) includes an absorbent dispersed in the pores of the porous support or adsorbed or coated on the fibers of the porous support.

7. In paragraph 1, An immune device characterized in that the above immune device can confirm the gestational week at which the gestational sac can be observed by the color development of the first detection line.

8. How to interpret the information needed to determine pregnancy or abnormal pregnancy, including the following steps: i) a step of measuring the amount or concentration of βcf hCG and Intact hCG in the test sample to be analyzed; and ii) a step of determining pregnancy or abnormal pregnancy by 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 multivalent anti-hCG monoclonal antibody that commonly recognizes βcf hCG and Intact hCG.

9. In paragraph 8, A method characterized in that the above amount or concentration is measured by a color development intensity proportional thereto.

10. In clause 8 or 9, A method characterized in that when the ratio of the color development intensity of βcf hCG to the color development intensity of the above βcf hCG and Intact hCG exceeds 50% at 5 weeks and 6 days of LMP, it is determined as a normal pregnancy, and when it is 30% or less, it is determined as an abnormal pregnancy including an ectopic pregnancy and a dead ovum.

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