Novel immunodiagnostic device for differentizing normal and abnormal generations by determining

Abstract

The present invention relates to an immune device for discriminating whether a pregnancy or an abnormal pregnancy occurs, and more particularly, to an immune device comprising a novel multi-binding anti-hCG monoclonal antibody capable of jointly recognizing a complete hCG and a beta cf hCG, comprising two detection lines and a quality control line capable of separately detecting the complete hCG and the beta cf hCG contained in a specimen, according to the present invention, it is possible to accurately and quickly distinguish the number of weeks of pregnancy, normal pregnancy, including extrauterine pregnancy, and abnormal pregnancy of a withered egg by numeralizing the ratio of the color development intensity of beta cf hCG (the distribution ratio of beta cf hCG) with respect to the color development intensity of complete hCG and beta cf hCG.

Description

Technical Field

[0001] The present invention relates to an immunological device capable of distinguishing normal pregnancy from abnormal pregnancy during pregnancy and an interpretation method using the same, and more specifically, to an immunological device capable of distinguishing normal pregnancy from abnormal pregnancy by measuring the distribution ratio of beta core fragment hCG (βcfhCG: Betacore framgment hCG) among various hCG secreted from pregnant women's urine and an interpretation method using the same. The core principle of the immunological device is to use a novel multi-binding anti-hCG monoclonal antibody that can only recognize and bind to intact hCG and βcfhCG that are diversely present in hCG proteins in pregnant women's urine as a mobile phase that binds to a marker, and to specifically separate intact hCG and βcfhCG from 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's chorionic villi and continues to produce progesterone in the early stages of pregnancy, thus maintaining implantation until the 10th week of pregnancy when the placenta completes its function.

[0003] The intact hCG (I-hCG: intact hCG) in the in vivo active form has a molecular weight of about 37 kDa and is composed of 244 amino acids. The intact hCG is roughly 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. The intact hCG is usually present in the blood during pregnancy and is the main structure with biological activity. In addition to the above-mentioned active forms, various dissociation and degradation products of hCG are also found in the blood and urine, which exist as nicked hCG (N-hCG: nicked hCG), free beta hCG (freeβ-hCG), free alpha hCG (freeα-hCG), β-core fragment hCG (β-corefragment hCG), etc.

[0004] β core fragment hCG (βcf hCG) is the hCG protein mainly present in the urine of pregnant women, with a molecular weight of about 14 kDa. Intact hCG is secreted predominantly in the 4th week of the last menstrual period (LMP), and βcf hCG gradually increases from the 5th week of LMP and is secreted at a concentration that is predominant over intact hCG on the 6th day of the 5th week or after the 6th week (John Walter Larsen et al., 2015).

[0005] Regarding the hCG protein distribution of pregnant women, intact hCG is present at a predominant concentration of 98% in the blood, but in the urine, as the number of weeks of pregnancy increases, βcf hCG increases to a maximum of 80% and exists at a concentration that is predominant over intact hCG. The existing pregnancy diagnostic kit using intact hCG as a marker determines whether pregnancy is present by qualitatively diagnosing intact hCG present in urine. However, as a result of testing 11,760 people who were judged to be pregnant using the existing pregnancy diagnostic kit, about 0.2% of 22 people were confirmed as false negatives, and the highest intact hCG blood concentration for them was measured to be 268,022.5 IU / mL, indicating that the existing urine pregnancy diagnostic kit has a false negative problem due to the hook effect. It was found that the root cause of this is caused by βcfhCG present in large quantities in urine (Richard T. Griffey et.al., 2013).

[0006] False negative results in urine pregnancy diagnosis can have serious consequences in emergency department (ED) situations. For example, even if a female patient is pregnant, if a false negative result is obtained when using urine with a general pregnancy diagnosis kit, a medical malpractice may occur when treating the female patient without regard for the fetus.

[0007] In addition, extrauterine pregnancy accounts for 9% of all deaths in pregnant women within the first trimester, increases the mortality rate of pregnant women, and the prevalence of extrauterine pregnancy is gradually increasing throughout the world.

[0008] Under such circumstances, the inventors of the present invention conducted resolute research to solve the fundamental hook effect and develop a method capable of distinguishing normal pregnancy from abnormal pregnancy. As a result, they developed a novel multi-binding anti-hCG monoclonal antibody capable of jointly recognizing intact hCG and βcf hCG, solved the hook effect by separately and specifically separating and detecting intact hCG and βcfhCG in urine samples, and confirmed that by quantifying the ratio of the color intensity of βcfhCG to the color intensity of intact hCG and βcfhCG (distribution ratio of βcfhCG), abnormal pregnancy including gestational age, normal pregnancy, extrauterine pregnancy and atrophied ovum can be distinguished accurately and quickly, thereby completing the present invention. Summary of the invention

[0009] Technical issues

[0010] The object of the present invention is to provide an immunological device and an interpretation method capable of maintaining normal pregnancy and distinguishing abnormal pregnancy in the early stages of pregnancy by specifically separating and detecting βcfhCG and intact hCG contained in pregnant woman's urine and accurately measuring the βcfhCG distribution ratio.

[0011] The immunological device of the present invention is an immunological device for distinguishing pregnancy or abnormal pregnancy, and comprises: i) a specimen area, which accommodates a sample to be analyzed; ii) a binding area, which is linked to the specimen area and comprises a multi-binding anti-hCG monoclonal antibody bound to a probe substance; iii) a signal detection area, which is connected to the binding area and comprises a first detection line fixed with an anti-βcf hCG antibody that can only specifically bind to βcfhCG, a second detection line fixed with an anti-intact hCG antibody that can only specifically bind to intact hCG, and a quality control line; and iv) a hygroscopic area, which absorbs the sample to be tested after the signal detection reaction is completed and is located downstream of the signal detection area.

[0012] The interpretation method of the present invention is a method for interpreting information required for distinguishing pregnancy or abnormal pregnancy, which comprises the following steps: i) a step of combining with multi-binding anti-hCG monoclonal antibodies that recognize βcfhCG and intact (Intact) hCG in a sample to be analyzed to reflect the distribution ratio of βcfhCG and intact (Intact) hCG; ii) a step of measuring the intensity of color development of βcf hCG and intact (Intact) hCG; and iii) a step of distinguishing pregnancy or abnormal pregnancy using the intensity distribution ratio of βcfhCG color development.

[0013] Technical Solution

[0014] The advantages and features of the present invention and the methods for achieving the same will become clear with reference to the embodiments described in detail below in conjunction with the accompanying drawings. However, the present invention can be implemented in various forms different from each other and is not limited to the embodiments disclosed below. The embodiments are provided only to complete the disclosure of the present invention and to fully inform the ordinary technicians in the technical field to which the present invention belongs. The present invention is only defined by the scope of the claims.

[0015] The terms used in this specification are used to illustrate the embodiments and are not intended to limit the present invention. In this specification, unless specifically mentioned in a sentence, the singular includes the plural. "Includes (comprises)" and / or "comprising (comprising)" used in the specification do not exclude the existence or addition of one or more other constituent elements other than the mentioned constituent elements. Throughout the specification, the same reference numerals refer to the same constituent elements, and "and / or" includes each of the mentioned constituent elements and all combinations of more than one. Although "first", "second" and the like are used to illustrate various constituent elements, these constituent elements are obviously not limited to these terms. These terms are only used to distinguish one constituent element from another constituent element. Therefore, the first constituent element mentioned below is within the technical idea of ​​the present invention, and it is obvious that it can also be a second constituent element.

[0016] If there is no other definition, all terms (including technical and scientific terms) used in this specification can be used as the meaning commonly understood by those of ordinary skill in the art to which the present invention belongs. In addition, unless clearly defined otherwise, the terms defined in the commonly used dictionaries should not be interpreted ideally or excessively.

[0017] The present invention provides an immunological device and an interpretation method capable of maintaining normal pregnancy and distinguishing abnormal pregnancy in the early stage of pregnancy by specifically separating and detecting βcfhCG and intact hCG contained in the urine of pregnant women using a multi-binding anti-hCG monoclonal antibody that recognizes both intact hCG and βcfhCG and accurately determining the distribution ratio of βcfhCG.

[0018] The multi-binding anti-hCG monoclonal antibody that recognizes intact hCG and βcfhCG in common utilized in the present invention has a characteristic of accurately reflecting the distribution ratio of intact hCG and βcfhCG according to the gestational weeks, and thus can be usefully used in more accurately and effectively distinguishing normal pregnancy from abnormal pregnancy based on the interpretation of its distribution ratio.

[0019] The purpose of the present invention is to accurately distinguish normal pregnancy from abnormal pregnancy. This is difficult to achieve by simply measuring the concentration of intact hCG and βcfhCG using antibodies to each of intact hCG and βcfhCG, but can be achieved by measuring the ratio of the reaction with intact hCG and βcf hCG present in the sample. In the case of using antibodies to each of intact hCG and βcf hCG, the concentration of intact hCG and βcf hCG that can be recognized by the antibodies is limited, but it is difficult to accurately grasp the concentration and ratio of intact hCG and βcf hCG. In addition, in the case of using the monoclonal antibody of the present invention that recognizes intact hCG and βcfhCG together, even if the values ​​of intact hCG and βcf hCG that can be recognized are limited, the concentration and concentration ratio of intact hCG and βcfhCG can be predicted more accurately due to the measurement of the ratio of the reaction with intact hCG and βcfhCG present in the sample.

[0020] The present invention uses a monoclonal antibody that recognizes both intact hCG and βcfhCG, rather than using monoclonal antibodies corresponding to intact hCG and βcf hCG separately, because the content of all intact hCG and βcfhCG in the sample can be confirmed in proportion.

[0021] By using the multi-binding anti-hCG monoclonal antibody of the present invention, it is possible to accurately determine normal pregnancy or abnormal pregnancy by deriving an accurate distribution ratio.

[0022] The abnormal pregnancy of the present invention may be an extrauterine pregnancy or a blighted ovum, but is not limited thereto.

[0023] The ectopic pregnancy refers to a pregnancy in which the fertilized egg implants in a location other than the normal implantation location inside the uterus. The implantation location of an ectopic pregnancy may be an abnormal location in the uterus or other location outside the uterus. Most of them are tubal pregnancies that occur in the fallopian tube, and there are also rare ovarian, peritoneal, and cervical pregnancies. Ectopic pregnancy is one of the most common emergency diseases, accounting for about 1% to 2% of total pregnancies.

[0024] The blighted ovum refers to a situation where the placenta tissue is well developed but no embryo bud can be seen under ultrasound. In the case of a blighted ovum, the placenta can grow for a period of time without an embryo bud, but eventually ends with a spontaneous abortion that causes lower abdominal pain and bleeding and then discharges the material conceived in the uterus.

[0025] In addition, the present invention provides an immunological device for distinguishing pregnancy or abnormal pregnancy, which comprises: i) a specimen area, which accommodates a sample to be analyzed; ii) a binding area, which is linked to the specimen area and comprises a multi-binding anti-hCG monoclonal antibody against intact hCG / βcf hCG bound to a probe substance; iii) a signal detection area, which is connected to the specimen area, comprises a first detection line to which an anti-βcf hCG antibody that specifically binds only to βcf hCG is fixed, a second detection line to which an anti-intact hCG antibody that specifically binds only to intact hCG is fixed, and a quality control line, and is linked to the binding area; and iv) a hygroscopic area, which absorbs the sample to be tested after the signal detection reaction is completed, and is located downstream of the signal detection area.

[0026] Preferably, the specimen area i) contains a liquid sample such as pregnant woman's urine, but any sample can be used as long as it is expected to contain intact hCG and / or βcfhCG.

[0027] The sample area may also have a filtering function to further improve the selectivity for the analyte or minimize the influence of interfering substances that may be contained in the sample. If necessary, an auxiliary area containing substances that can increase the reaction between the analyte and the conjugate or eliminate the influence of interfering substances may be provided upstream of the sample area.

[0028] The probe substance in the binding area of ​​ii) can 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, silicon nanoparticles, polymer beads, fluorescent substances, luminescent substances, pigment beads and proteins, but is not limited thereto.

[0029] In a specific implementation example of the present invention, the probe material may be colloidal gold nanoparticles, but is not limited thereto.

[0030] The signal detection area of ​​iii) is a medium for developing a mobile phase and a sample, and the mobile phase and the sample to be detected can move by the capillary phenomenon of the porous membrane of the signal detection area. The signal detection area can be one selected from the group consisting of a hole plate synthesized by nitrocellulose, cellulose, polyethylene, polyethersulfone, polystyrene, polycarbonate, polymethyl methacrylate, nylon, polyvinylidene fluoride (PVDF), polyethylene resin or polystyrene resin, and a sliding glass composed of glass, but is not limited thereto. In a specific implementation example of the present invention, the signal detection area can use a nitrocellulose membrane with pores of 5μm to 15μm, but is not limited thereto.

[0031] According to a specific implementation example of the present invention, the signal detection area may include: a first detection line, on which anti-βcf hCG antibodies are fixed; a second detection line, downstream of the first detection line, on which anti-intact hCG antibodies are fixed; and a quality control line, downstream of the second detection line. According to another implementation example of the present invention, the signal detection area may include: a second detection line, on which anti-intact hCG antibodies are fixed; a first detection line, downstream of the second detection line, on which anti-βcf hCG antibodies are fixed; and a quality control line, downstream of the first detection line.

[0032] The anti-intact hCG antibody is an antibody that specifically recognizes the active intact hCG composed of two subunits, α and β, and the anti-βcfhCG antibody is an antibody that specifically recognizes the β core fragment hCG (βcfhCG).

[0033] The present invention is an immunological device consisting of two detection lines, characterized in that the color intensity of the detection lines of βcfhCG and intact (Intact) hCG in the test sample is quantitatively or qualitatively analyzed and detected, and the distribution ratio of βcfhCG is numerically converted and compared by the following formula derived in the present invention, so that the gestational age, normal pregnancy and abnormal pregnancy including ectopic pregnancy can be accurately distinguished.

[0034]

[0035] In a specific implementation example of the present invention, when the ratio of the color intensity of βcfhCG relative to the color intensity of βcfhCG and intact (Intact) hCG according to the above formula increases by 50% to 60% or more from the 5th week to the 6th week of LMP, it can be identified as a normal pregnancy; when it is below 5% to 15%, it can be identified as an ectopic pregnancy; when it remains below 20% to 30% until the 6th day of the 6th week, it can be identified as a blighted egg.

[0036] The control line refers to a portion that emits a predetermined signal regardless of the concentration of intact hCG or βcf hCG in the sample to be tested.

[0037] The hygroscopic region iv) may include an absorbent dispersed in the pores of the porous support or adsorbed or coated on the fiber filaments of the porous support. The upper surface of the porous support may further include a porous film layer, but is not limited thereto.

[0038] In the present invention, when the multi-binding anti-hCG monoclonal antibody conjugate present in the binding region reacts with a sample such as urine of a pregnant woman, the reaction proceeds according to the concentration ratio of intact hCG and βcf hCG present in the sample, and the reacted conjugate moves to the membrane side. The first detection line on the membrane where the anti-βcf hCG monoclonal antibody is immobilized forms a sandwich-shaped complex only for βcf hCG in hCG reacting with the conjugate, and the second detection line where the anti-intact hCG monoclonal antibody is immobilized forms a sandwich-shaped complex only for intact hCG in hCG reacting with the conjugate.

[0039] The immunological device of the present invention may include a solid support platform at the bottom. The solid support platform may be formed of a material selected from the group consisting of nitrocellulose, nylon, polyvinylidene fluoride (PVDF), glass and plastic, but is not limited thereto. Since it is manufactured by attaching a pad, a film, etc. to the solid support platform, the durability of the test strip can be improved, and it can be easily handled and kept. In addition, additional external equipment can be easily installed.

[0040] As the plastic material that can be used as the solid support, polypropylene film, polyester film, polycarbonate film, acrylic film, etc. can be used, but it is not limited thereto.

[0041] The immune device of the present invention may further include an analysis device. The analysis device can evaluate the color intensity of the first detection line and the second detection line. The analysis device may include a reader combined 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 can compare the intensity, color, luminescence and / or fluorescence with a pre-programmed threshold value, and a digital output can be provided based on the comparison. According to one embodiment of the present invention, the analysis device uses a reflectance reader equipped with an analysis software based on a CCD camera or a laser light source, and can distinguish normal pregnancy and abnormal pregnancy including ectopic pregnancy with high accuracy. The analysis device may also include a display that provides information related to abnormal pregnancy including ectopic pregnancy.

[0042] The immunological device of the present invention can confirm the gestational week at which the gestational sac and the embryonic bud can be observed. In a specific embodiment of the present invention, if the ratio of the color intensity of βcfhCG to the color intensity of βcfhCG and intact (Intact) hCG increases by 50% to 60% or more from the 5th to 6th week of LMP, it is a normal pregnancy and the gestational sac and the embryonic bud can be observed. On the contrary, if it is less than 5% to 15%, it is an extrauterine pregnancy and the gestational sac can be observed.

[0043] In another specific embodiment of the present invention, as a result of comparing the ratio of the color intensity of βcfhCG to the color intensity of βcfhCG and intact (Intact) hCG (i.e., the measured value of βcfhCG / (βcfhCG+intact (Intact) hCG)), it was confirmed that the incidence of abnormal pregnancy including extrauterine pregnancy was significantly lower than that of normal pregnancy.

[0044] In addition, the present invention provides a kit, characterized in that the immunological device is further fixed in a device.

[0045] The reagent kit is equipped with a guide and a test strip support in the lower device, and equipped with a sample input port and a result confirmation window located at positions corresponding to the first test line, the second test line and the quality control line in the upper device.

[0046] The upper and lower devices may be made of conventional plastic materials, such as polycarbonate, acrylonitrile-styrene-butadiene styrene (ABS), etc., but are not limited thereto.

[0047] Furthermore, the present invention provides a method for interpreting information required for distinguishing pregnancy or abnormal pregnancy, comprising the steps of: i) a step of determining the amount or concentration of βcfhCG and intact (Intact) hCG in a test sample to be analyzed; and ii) a step of determining pregnancy or abnormal pregnancy using the ratio of the amount or concentration of βcf hCG relative to the amount or concentration of βcfhCG and intact (Intact) hCG, wherein the amount or concentration is determined using a multi-binding anti-hCG monoclonal antibody that recognizes both βcfhCG and intact (Intact) hCG.

[0048] The amount or concentration can be determined using the intensity of the color development which is proportional thereto.

[0049] When the ratio of the amount or concentration of βcfhCG to the amount or concentration of βcfhCG and intact (Intact) hCG exceeds 50% on the 6th day of the 5th week of LMP, it can be determined as a normal pregnancy. When it is 30% or less on the 6th day of the 5th week of LMP, it can be determined as an abnormal pregnancy such as an extrauterine pregnancy or a blighted egg, but the present invention is not limited thereto.

[0050] Specifically, a method for interpreting information required for distinguishing pregnancy or abnormal pregnancy is provided, comprising the following steps: i) a step of reacting intact hCG and βcfhCG in the sample with a multi-binding anti-hCG monoclonal antibody conjugated with a probe substance by applying the sample to the immunoassay device; ii) a step of confirming the reaction of βcfhCG and intact hCG in the sample by means of a first detection line and a second detection line, wherein βcf hCG and intact hCG are detected by the color intensity generated by a sandwich complex present in each detection line; and iii) a step of determining that the pregnancy is normal when 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 from the 5th to 6th week of LMP (i.e., on the 6th day of the 5th week of LMP), and that the pregnancy is ectopic pregnancy or blighted ovum when the ratio is less than 30%.

[0051] The color intensity is the color intensity according to the concentration of βcf hCG and intact (Intact) hCG. In a specific implementation example of the present invention, it was confirmed that the concentration is proportional to the color intensity.

[0052] In addition, the present invention provides a method for distinguishing pregnancy or abnormal pregnancy using the immunochromatographic test strip.

[0053] The method for distinguishing pregnancy from abnormal pregnancy comprises the following steps: i) a step of reacting intact hCG and βcfhCG present in the sample with a multi-binding anti-hCG monoclonal antibody conjugated with a probe substance by applying the sample to the immunological device; ii) a step of confirming the reaction of βcfhCG and intact hCG in the sample by means of a first detection line and a second detection line, wherein βcf hCG and intact hCG are detected by the color intensity generated by a sandwich complex present in each detection line; and iii) a step of determining that the pregnancy is normal when 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% on the 6th day of the 5th week of LMP, and determining that the pregnancy is abnormal when the ratio is 30% or less on the 6th day of the 5th week of LMP. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 This is a schematic diagram showing the reaction principle of the concentration ratio of intact hCG and βcfhCG in a sample caused by the present invention and an implementation example of an immunochromatographic test strip.

[0055] Figure 2 The graph shows the correlation between the color intensity (Intensity) of different amounts or concentrations of intact (Intact) hCG and βcf hCG.

[0056] Figure 3 It is a graph showing the changes in the distribution degree of intact (Intact) hCG and βcfhCG during normal pregnancy.

[0057] Figure 4 It is a graph showing the changes in the distribution degree of intact (Intact) hCG and βcfhCG during extrauterine pregnancy.

[0058] Figure 5 This is a graph showing the comparison of the color intensity ratio of βcf hCG / (intact hCG+βcfhCG) in the normal pregnancy group and the extrauterine pregnancy group at different gestational weeks.

[0059] Figure 6 The figure shows the results of clinical evaluation of normal pregnancy specimens using the diagnostic apparatus of the present invention.

[0060] Figure 7 The diagram shows the results of clinical evaluation of normal pregnancy specimens using the diagnostic apparatus of the present invention.

[0061] Figure 8The diagram shows the results of clinical evaluation of an extrauterine pregnancy specimen using the diagnostic apparatus of the present invention.

[0062] Fig. 9 The diagram shows the results of clinical evaluation of an extrauterine pregnancy specimen using the diagnostic apparatus of the present invention.

[0063] Fig.10 This is a graph showing the results of the correlation between color intensity at various concentration ratios when positive samples of intact (Intact) hCG alone, βcfhCG alone, and a mixture of intact (Intact) hCG and βcfhCG were prepared at different concentrations in the kit prepared according to the present invention and reacted. DETAILED DESCRIPTION

[0064] Hereinafter, the content of the present invention will be described in more detail by the following embodiments and experimental examples. However, the scope of the claims of the present invention is not limited to the following embodiments and experimental examples, but also includes the deformation of the technical concept equivalent thereto.

[0065] Example 1: Preparation of immunochromatographic test strips

[0066] 1-1. Manufacturing of a membrane having a first detection line, a second detection line and a quality control line

[0067] Three different antibodies were separated onto nitrocellulose membranes. The first test line was separated into anti-βcf hCG monoclonal antibody (Anti-βcf hCG monoclonal antibody) as βcf hCG antibody, the second test line was separated into anti-intact hCG monoclonal antibody (Anti-Intact hCG monoclonal antibody) as intact hCG antibody, and the quality control line was separated into goat anti-mouse immunoglobulin (Goat anti-mouse IgG), and then dried.

[0068] 1-2. Conjugate pad manufacturing

[0069] A first conjugate solution in which colloidal gold nanoparticles are conjugated with a multi-binding anti-hCG monoclonal antibody that recognizes both intact hCG and βcf hCG is prepared. In addition, a second conjugate solution in which colloidal gold nanoparticles are conjugated with mouse immunoglobulin (Mouse IgG) is prepared. The first conjugate solution and the second conjugate solution are separated into pre-treated conjugate pads, and after being completely dried, they are cut into appropriate sizes for use.

[0070] 1-3. Sample pad manufacturing

[0071] The specimen pad is fully soaked in a specimen pad pretreatment solution containing a buffer and a preservative, and after being completely dried, it is cut into appropriate sizes for use.

[0072] 1-4. Absorbance pad manufacturing

[0073] The dry absorbent pad is cut into appropriate sizes for later use.

[0074] 1-5. Manufacturing of immunochromatographic test strips

[0075] The membrane, conjugate pad, specimen pad and moisture absorbent pad prepared in the above steps are placed in the Figure 1 Assemble the structure shown.

[0076] That is, the specimen pad is attached to overlap one end of the conjugate pad, one end of the detection pad is attached to overlap the other end of the conjugate pad, and the other end of the detection pad and one end of the moisture absorbent pad are attached to overlap each other.

[0077] exist Figure 1 In the present invention, the immunochromatographic test strip comprises the following structure.

[0078] 1: Sample pad

[0079] 2: Conjugate pad

[0080] 3: Nitrocellulose membrane (Membrane)

[0081] 4. Absorption pad

[0082] 5: Test line 1 with fixed anti-βcfhCG monoclonal antibody

[0083] 6: Test line 2 with anti-intact hCG monoclonal antibody

[0084] 7: Control line with immobilized goat anti-mouse immunoglobulin

[0085] 1-6. Analytical device

[0086] The color development or light-dark sensitivity of the detection line is quantified using a reflectance reader equipped with analysis software based on a CCD camera or a laser light source, and can be used as a numerical signal intensity.

[0087] 1-7. Equipment assembly

[0088] The manufactured immunochromatographic test strip for distinguishing normal pregnancy, gestational age, and abnormal pregnancy including ectopic pregnancy and blighted ovum is placed in the test strip fixing position of the plastic lower device, and then inserted into the upper device with a specimen input port and a result confirmation window for assembly.

[0089] Example 2: Determination of the color intensity change of intact hCG and βcf hCG concentrations in the normal pregnancy group and the abnormal pregnancy group including extrauterine pregnancy

[0090] In the prior art, diagnosis is performed based on the concentration ratio, but the pregnancy diagnosis device of the present invention performs identification by confirming the color intensity. The color intensity is measured in the same way by all instruments that measure the color intensity and is digitized.

[0091] The analysis device used as the composition of the pregnancy diagnosis device of the present invention was used for measurement, and it was confirmed that the concentration was proportional to the color intensity ( Figure 2 ).

[0092] 2-1. Determination of the color intensity change of intact hCG and βcf hCG concentrations in the normal pregnancy group

[0093] In the normal pregnancy group, the amount of change in color intensity according to the concentration of intact hCG and βcfhCG was measured. In the normal pregnancy group, intact hCG was detected from the 4th day after the 3rd week of LMP and increased to the 5th week. Thus, it was possible to check whether it was early pregnancy. βcf hCG was detected from the late 4th week of LMP, increased sharply from the 5th week, and maintained a high concentration from the 6th week onwards ( Figure 3 ).

[0094] In addition, if βcfhCG is detected in the 5th week of LMP, the ultrasound examination results can confirm the fetal sac (baby room). From the 6th week of LMP, the intact (Intact) hCG decreases, the βcf hCG is maintained and reversed, and the ultrasound examination results can confirm the fetal bud.

[0095] 2-2. Determination of the color intensity change of intact hCG and βcf hCG concentrations in the abnormal pregnancy group including extrauterine pregnancy

[0096] The results of measuring the concentration changes of intact hCG and βcf hCG in the extrauterine pregnancy group showed that in the case of the extrauterine pregnancy group, intact hCG was detected at the 5th week of LMP, but βcf hCG was not detected at the 5th week of LMP or was detected at a very low concentration ( Figure 4 ).

[0097] As a result, between the 4th and 7th weeks of LMP, in the case of normal pregnancy, the concentration of intact (Intact) hCG first increased and then decreased, and the concentration of βcf hCG gradually increased and remained. In contrast, in the case of ectopic pregnancy, βcfhCG was not detected or was detected at a very low concentration. This was confirmed to be a clear result for distinguishing normal pregnancy from ectopic pregnancy.

[0098] In the case of blighted ovum, βcfhCG was maintained at a low level of less than 30% from the 6th day of the 5th week of LMP without increasing, and although the gestational sac was confirmed by ultrasound observation, the embryo bud and heartbeat were not confirmed.

[0099] Example 3: Determination of the color intensity ratio of βcf hCG / (intact hCG+βcfhCG) in the normal pregnancy group and the abnormal pregnancy group including extrauterine pregnancy

[0100] In order to use the results of Example 2 to distinguish the gestational weeks, the normal pregnancy group, and the abnormal pregnancy group including extrauterine pregnancy, the color intensity ratios of βcf hCG / (intact hCG+βcfhCG) according to the concentration at different gestational weeks in the normal pregnancy group and the abnormal pregnancy group were measured and compared.

[0101] 3-1. Verification of the assay principle and derivation of the calculation formula for the βcf hCG distribution ratio (%)

[0102] The pregnancy diagnostic device of the present invention is composed of a "mobile phase" that reacts with a sample and moves toward a stationary phase, and a "stationary phase" that can separate and detect intact hCG and βcfhCG in the sample. In the present invention, the mobile phase is equivalent to the binding area in the composition of the pregnancy diagnostic device, and the stationary phase is equivalent to the signal detection area including the first detection line, the second detection line and the quality control line.

[0103] The mobile phase is in a dry state in which a multi-binding anti-hCG monoclonal antibody capable of jointly recognizing intact hCG and βcfhCG, which is a novel antibody developed by the inventors of the present invention, is bound to an indicator capable of expressing the concentration intensity of the sample (i.e., gold particles, nano beads, fluorescence or phosphorescence).

[0104] That is, the property of the antibody binding to the marker is characterized by reacting by jointly recognizing intact hCG and βcf hCG.

[0105] Anti-βcf hCG monoclonal antibodies are immobilized on the first detection line of the stationary phase, thereby detecting only βcf hCG in the test sample, and anti-intact hCG monoclonal antibodies are immobilized on the second detection line, thereby detecting only intact hCG in the test sample.

[0106] Thus, according to the concentration ratio of intact hCG and βcf hCG contained in the test sample, the hCG first reacts with the conjugate and migrates to the stationary phase, and various samples are separated and detected according to the characteristics of the antibody fixed on the stationary phase.

[0107] In addition, by deriving a calculation formula for the βcfhCG distribution ratio (%) from the color intensity ratio of βcfhCG / (intact hCG+βcfhCG), more specific measurement and analysis can be performed.

[0108] 3-2. Determination of the color intensity ratio of βcfhCG / (intact hCG+βcf hCG) in normal pregnancy groups and abnormal pregnancy groups including extrauterine pregnancy at different gestational weeks

[0109] Based on the color intensity of the detected concentration ratio of intact hCG and βcf hCG measured in the normal pregnancy group and the extrauterine pregnancy group at different gestational weeks, the distribution ratio (%) of βcfhCG using the color intensity ratio of βcfhCG / (intact hCG+βcfhCG) was confirmed by the following formula.

[0110]

[0111] The results are shown in Table 1 and Figures 5 to 9As shown, in the case of a normal pregnancy (Normal pregnancy) in which the fertilized egg implants normally in the uterus and a baby grows, it is confirmed that the distribution ratio of βcfhCG is about 10% to 20% on the 0th day of the 5th week of LMP, increases to 50% on the 6th day of the 5th week of LMP, is 70% in the 6th week of LMP, and continues to maintain a ratio of 80% after the 8th week of LMP. On the contrary, in the case of an ectopic pregnancy (Ectopic pregnancy) in which the implantation is abnormal, it is confirmed that the distribution ratio of βcfhCG is maintained at less than 10% from the 5th week to the 6th week of LMP, and the phenomenon of increase is not observed, and the fetal sac (baby room) is not observed under ultrasound.

[0112] [Table 1]

[0113] Comparison of intensity ratios of normal pregnancy and extrauterine pregnancy at different weeks

[0114] unit(%)

[0115] 3 weeks 4 weeks 5 weeks 6 weeks 7 weeks 8 weeks Extrauterine pregnancy 0 0 1.11 5.49 13.88 - Normal pregnancy 4.98 4.23 31.07 70.02 81.11 84.27

[0116] **The % according to each week number is expressed on a median basis.

[0117] In the case of blighted ovum, it was confirmed that the distribution ratio of βcfhCG did not increase from the 5th week to the 6th day of LMP, and remained below 30%. The result of ultrasound observation showed that although the gestational sac was confirmed, the embryo bud and heartbeat were not confirmed.

[0118] Thus, the pregnancy diagnostic apparatus of the present invention distinguishes a normal pregnancy group from an abnormal pregnancy group including an extrauterine pregnancy by the color intensity ratio of βcf hCG / (intact hCG+βcfhCG) and the distribution ratio (%) of βcfhCG based on the color intensity of the detected concentrations of intact hCG and βcf hCG, and confirms the period when the gestational sac can be confirmed, thereby enabling more accurate and rapid identification through a single measurement.

[0119] Example 4. Test kit performance (specificity) verification and color intensity correlation analysis

[0120] The results of the reaction of intact hCG alone, βcfhCG alone, and mixed positive samples of intact hCG and βcfhCG prepared at different concentrations based on the kit prepared according to the present invention are shown in FIG. Fig.10 .

[0121] Intact hCG was diluted to a negative standard sample at concentrations of 100mIU / mL, 600mIU / mL, and 800mIU / mL to prepare a positive sample and reacted. As a result, no color was developed on the first test line (βcfhCG), and the color intensity of the second test line (intact hCG) was confirmed at different concentrations.

[0122] When βcfhCG was diluted to a negative standard sample at concentrations of 0.03 pmol / mL, 0.5 pmol / mL, and 5 pmol / mL to prepare a positive sample and reacted, the second test line (intact hCG) did not develop color, and the color intensity of the first test line (βcfhCG) was confirmed at different concentrations.

[0123] The intact (Intact) hCG and βcfhCG were mixed and diluted with a negative standard specimen at concentrations of 100mIU / mL (intact (Intact) hCG) + 0.03pmol / mL (βcfhCG), 800mIU / mL (intact (Intact) hCG) + 0.5pmol / mL (βcfhCG), and 600mIU / mL (intact (Intact) hCG) + 5pmol / mL (βcfhCG) to prepare a positive specimen and reacted. The first detection line and the second detection line both showed color intensity corresponding to each concentration, and the color intensity of different concentrations was equivalent to the results of single treatment of intact (Intact) hCG and single treatment of βcfhCG.

[0124] This result confirmed that the multi-binding anti-hCG monoclonal antibody used in the present invention reacts specifically with intact hCG and βcf hCG respectively without causing interference, and the color intensity increases in a positive correlation with the concentration of intact hCG and βcf hCG mixed in the sample, suggesting that the distribution ratio can be accurately measured.

[0125] The embodiments of the present invention are described above with reference to the accompanying drawings, but those skilled in the art should understand that the present invention can be implemented in other specific forms without changing its technical ideas or essential features. Therefore, the embodiments described above are illustrative in all aspects and should be understood as non-restrictive.

Claims

1. An immunological device for distinguishing pregnancy or abnormal pregnancy, comprising: i) Sample area, which contains samples to be analyzed; ii) a binding region, linked to the sample region and comprising a multi-binding anti-hCG monoclonal antibody that recognizes both intact hCG and βcfhCG and is conjugated to the probe substance; iii) a signal detection area, which is an area capable of completely separating and detecting intact hCG and βcf hCG bound by the reaction with the conjugate, characterized by comprising a first detection line immobilized with a monoclonal antibody that can only form a complex specifically with βcf hCG, a second detection line immobilized with a monoclonal antibody that can only form a complex specifically with intact hCG, and a quality control line; and iv) a moisture absorption area, which absorbs the sample to be tested after the signal detection reaction is completed and is located downstream of the signal detection area.

2. The immunological device according to claim 1, characterized in that: The immunological device also includes an analytical device.

3. The immunological device according to claim 1 or 2, characterized in that: The immunological device discriminates pregnancy or abnormal pregnancy using the ratio of the color intensity of βcf hCG relative to the color intensities of βcf hCG and intact hCG measured at the test line derived from the following formula.

4. The immunological device according to claim 1, characterized in that: The probe substance ii) is selected from one or more of the group consisting of gold nanoparticles, silver nanoparticles, quantum dot nanoparticles, carbon nanoparticles, latex beads / fluorescent nanoparticles, cellulose nanoparticles, magnetic nanoparticles, silicon nanoparticles, polymer beads, fluorescent substances, luminescent substances, pigments and proteins.

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

6. The immunological device according to claim 1, characterized in that: The hygroscopic region iv) comprises an absorbent, which is dispersed in the pores of the porous support or adsorbed or coated on the fiber filaments of the porous support.

7. The immunological device according to claim 1, characterized in that: The immunoassay device utilizes the color development of the first detection line to confirm the gestational age at which the fetal sac can be observed.

8. A method for interpreting information required to identify pregnancy or abnormal pregnancy, comprising the following steps: i) a step of determining the amount or concentration of βcf hCG and intact hCG in a test sample to be analyzed; as well as ii) a step of determining pregnancy or abnormal pregnancy using the ratio of the amount or concentration of βcf hCG relative to the amount or concentration of βcf hCG and intact hCG, wherein said amount or said concentration is determined using said multi-binding anti-hCG monoclonal antibody that recognizes both βcf hCG and intact hCG.

9. The method according to claim 8, characterized in that The amount or concentration is determined by the intensity of the color development which is directly proportional thereto.

10. The method according to claim 8 or 9, characterized in that: If the ratio of the color intensity of βcf hCG to the color intensity of βcf hCG and intact hCG exceeds 50% on the 6th day of the 5th week of LMP, it is judged as a normal pregnancy. If it is less than 30%, it is judged as an abnormal pregnancy including extrauterine pregnancy and blighted ovum.

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