KR102473036B1 - Novel ectopic pregnancy test devices by using beta core fragment hCG as diagnostic marker - Google Patents

Abstract

The immune device of the present invention includes: 1) a sample area accommodating a test sample to be analyzed; 2) a conjugate region linked to the sample region and containing a probe-label conjugated anti-I-hCG antibody and a probe-label conjugated anti-βcf hCG antibody; and 3) a signal detection linked to the conjugate region including a first detection line to which the anti-I-hCG antibody is immobilized, a second detection line to which the anti-βcf hCG antibody is immobilized, and a control line connected to the sample region. area; and an ectopic pregnancy if the signal intensity of βcf hCG / I-hCG is lower than that of a normal pregnancy, including an absorption region located downstream of the signal detection region, which absorbs the test sample for which the signal detection reaction has been completed. This makes it possible to accurately and conveniently diagnose ectopic pregnancy.

Description

Novel ectopic pregnancy test devices using beta core fragment hCG as a marker {Novel ectopic pregnancy test devices by using beta core fragment hCG as diagnostic marker}

The present invention relates to an immunization device for diagnosing ectopic pregnancy, and a method for diagnosing ectopic pregnancy using the same.

Ectopic pregnancy accounts for 9% of all deaths of pregnant women during the first trimester, increasing the mortality rate of pregnant women, and the prevalence of ectopic pregnancy is gradually increasing worldwide. Between 1976 and 1993, the incidence of ectopic pregnancies in Northern Europe increased from 11.2 to 18.8 per 1,000 pregnancies. The incidence of ectopic pregnancy in Korea is estimated to be 1 in 20 to 26 pregnancies, which is relatively higher than in Western countries.

In the past, there were difficulties in early detection by diagnosing ectopic pregnancy based on clinical findings such as vaginal bleeding and lower abdominal pain. It has become possible to detect extra-pregnancy. However, despite these advances, hCG measurement and vaginal ultrasonography still lack accuracy and cost-effectiveness, limiting their effectiveness in diagnosing ectopic pregnancy in primary care centers.

In addition, serum β-hCG measurement is performed when the diagnostic results are ambiguous. In the case of normal pregnancy, β-hCG hormone is detected in the blood from 7 days after ovulation, and the blood concentration continues to increase until 6 weeks of pregnancy, but ectopic In case of pregnancy, ectopic pregnancy is diagnosed when it takes more than 3 days for the blood concentration of β-hCG to double, based on the fact that the increase in β-hCG concentration is remarkably slow or not increased. Repeated quantification of hCG levels must be performed, which may require additional time and cost.

Serum progesterone level has been proposed as a serum marker for ectopic pregnancy, but some studies have shown that serum progesterone level is not effective in the differential diagnosis of normal pregnancy, ectopic pregnancy and spontaneous abortion and has a poor clinical picture. It is desirable to diagnose ectopic pregnancy quickly and accurately, but it is difficult, and delay in diagnosis may result in fallopian tube rupture, intra-abdominal bleeding, blood transfusion, and emergency open surgery. Early diagnosis and intervention by medical or surgical treatment of ectopic pregnancy can minimize mortality. In particular, initially minimally invasive laparoscopic surgery may promote preservation of the fallopian tubes and increase future fertility.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone produced during pregnancy. hCG is produced in the trophoblastic membrane of the placenta and continues to produce progesterone in the early stages of pregnancy to maintain implantation until the 10th week of pregnancy when the placental function is completed. Intact hCG (I-hCG), an active form in the body, has a molecular weight of about 37 kDa and consists of 244 amino acids. I-hCG largely consists of two subunits, alpha (α) and beta (β). The alpha-subunit contains 92 amino acids and the beta-subunit contains 145 amino acids. I-hCG is generally present at various stages during pregnancy and is the main structure present in the body, and in the early stages of pregnancy, this active structure exists in the form of polysaccharide hCG (hyperglycosylatedhCG; H-hCG) at a high rate. In addition to the above active forms, various dissociation and degradation products of hCG are found in blood and urine, and these include fragmented hCG (nicked hCG; N-hCG), free beta hCG (free β-hCG), free alpha hCG (free α-hCG), It exists as free β-core fragment hCG (free β-core fragment hCG).

Beta core fragment hCG (βcf hCG) is a fragment of free β-hCG, which is one distinct degradation product of hCG, and has a molecular weight of about 14 kDa. βcf hCG begins to appear in the urine at a concentration of about 0.01 pmol/mL, which is lower than that of I-hCG, in the urine from 2 weeks after the last menstrual period (LMP), and gradually increases as the gestational age progresses, and after 5 weeks, I -Appears in a concentration that is superior to that of hCG. In addition, βcf hCG is present only in urine, unlike other metabolites (J ClinEndocrinol Metab, 76, 1993, 704-10).

The concentration of I-hCG is highest between 9 and 12 weeks in serum and urine, and the concentration in urine of pregnant women is 150,000mIU/mL at 10 weeks, and high concentrations of 900,000mIU/mL have been detected depending on individual differences. do.

In the case of ectopic pregnancy, the present inventors found that the concentration of βcf hCG is very low compared to normal pregnancy, and when comparing the change in the concentration ratio of βcf hCG and I-hCG in urine between normal pregnancy and ectopic pregnancy, more accurately and efficiently The present invention was completed by confirming that diagnosis of ectopic pregnancy is possible. The present invention measures the βcf hCG / I-hCG level of a sample using a monoclonal antibody that specifically recognizes the dissociation of hCG and the structural change of the degradation product, resulting in a high βcf hCG / I-hCG level compared to normal pregnancy. Provides ectopic pregnancy diagnosis results with high level of accuracy. Therefore, unlike the case of diagnosis by ultrasound examination, it is not dependent on the technician's experience, the time required for diagnosis of ectopic pregnancy is relatively short compared to existing diagnostic methods, and it is cost-effective and time-efficient and accurate because it does not require repeated tests. An ectopic pregnancy test kit is provided.

Registration Publication No. 10-0403871

The present inventors found that a lower level of βcf hCG / I-hCG compared to βcf hCG / I-hCG in normal pregnancy was obtained through an immune system that added a second detection line for detecting βcf hCG in addition to the first detection line for detecting I-hCG. by inventing an immune device capable of diagnosing ectopic pregnancy, enabling efficient and accurate diagnosis of ectopic pregnancy.

In order to achieve the above object, the present invention provides an ectopic pregnancy diagnostic kit containing antibodies specifically recognizing I-hCG and βcf hCG.

In addition, the present invention

1) a sample region accommodating a test sample to be analyzed;

2) a conjugate region linked to the sample region and containing an anti-I-hCG antibody conjugated to a probe and an anti-βcf hCG antibody conjugated to a probe;

3) A signal detection region linked to the conjugate region, including a first detection line to which the anti-I-hCG antibody is immobilized, a second detection line to which the anti-βcf hCG antibody is immobilized, and a control line connected to the sample region ; and

4) Provided is an immunological device for diagnosing ectopic pregnancy including a moisture absorption region located downstream of the signal detection region, which absorbs the test sample after the signal detection reaction has been completed.

According to another aspect of the present invention,

1) applying the sample to the immunization device so that I-hCG and βcf hCG in the sample react with the probe-labeled anti-I-hCG antibody and the anti-βcf hCG antibody;

2) As a step of confirming the reaction of I-hCG and βcf hCG in the sample by the first and second detection lines, I-hCG and βcf hCG are signals generated by the presence of the sandwich complex in each detection line detected by; and

3) If the signal strength of the second detection line for the first detection line is lower than the signal strength of the second detection line for the first detection line of a normal pregnancy, ectopic pregnancy including the step of determining that the pregnancy is ectopic Provides a method for providing information necessary for diagnosis.

An immunochromatography strip according to an embodiment of the present invention includes a detection line for detecting βcf hCG in addition to a detection line for detecting I-hCG, and diagnoses ectopic pregnancy by the concentration ratio of βcf hCG / I-hCG, thereby efficiently diagnosing ectopic pregnancy. However, it was possible to diagnose ectopic pregnancy with high accuracy.

1 is a schematic diagram showing an embodiment of an immunochromatography strip manufactured according to the present invention.
2 is a model diagram showing a specific example of applying the immunochromatography strip according to the present invention to a kit.
3 is a diagram showing changes in concentrations of intact hCG and βcf hCG in a normal pregnancy group (Intact hCG: Circle spot, βcf hCG: Diamond spot).
4 is a diagram showing changes in concentrations of intact hCG and βcf hCG in the ectopic pregnancy patient group (Intact hCG: Circle spot, βcf hCG: Diamond spot).
5 shows the concentration ratio of βcf hCG / Intact hCG (normal pregnancy: Circle spot, ectopic pregnancy: Diamond spot) (Fig. 5a) and the concentration of βcf hCG / Intact hCG measured in normal pregnancy and ectopic pregnancy patient groups by gestational week It is a diagram showing the distribution of ratios (EP: Ectopic pregnancy, NP: Normal pregnancy) (FIG. 5b).
6 shows the concentration ratio of βcf hCG / Intact hCG (normal pregnancy: Circle spot, ectopic pregnancy: Diamond spot) measured in normal pregnancy and ectopic pregnancy patient groups by day (FIG. 6a) and βcf hCG / Intact hCG It is a diagram showing the distribution of the concentration ratio (EP: Ectopic pregnancy, NP: Normal pregnancy) (FIG. 6b).

Advantages and features of the present invention, and methods for achieving them, will become clear with reference to the embodiments described below in detail in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in a variety of different forms, only the present embodiments make the disclosure of the present invention complete, and those skilled in the art in the art to which the present invention belongs It is provided to fully inform the person of the scope of the invention, and the invention is only defined by the scope of the claims. With reference to the accompanying drawings below, specific details for the practice of the present invention will be described in detail. Like reference numbers refer to like elements, regardless of drawing, and "and/or" includes each and every combination of one or more of the recited items.

Terms used in this specification are for describing embodiments and are not intended to limit the present invention. In this specification, singular forms also include plural forms unless specifically stated otherwise in a phrase. As used herein, "comprises" and/or "comprising" does not exclude the presence or addition of one or more other elements other than the recited elements.

Unless otherwise defined, all terms (including technical and scientific terms) used in this specification may be used in a meaning commonly understood by those of ordinary skill in the art to which the present invention belongs. In addition, terms defined in commonly used dictionaries are not interpreted ideally or excessively unless explicitly specifically defined.

Hereinafter, an immunological device for diagnosing ectopic pregnancy according to embodiments of the present invention and a diagnostic method using the same will be described.

An immunological device for diagnosing ectopic pregnancy according to an embodiment of the present invention,

1) a sample area accommodating a test sample to be analyzed;

2) a conjugate region linked to the sample region and in which a probe-labeled anti-I-hCG antibody and a probe-labeled anti-βcf hCG antibody are bound;

3) A signal detection region linked to the conjugate region, including a first detection line to which the anti-I-hCG antibody is immobilized, a second detection line to which the anti-βcf hCG antibody is immobilized, and a control line connected to the sample region ; and

4) An immunization device for diagnosing ectopic pregnancy is provided, which includes a moisture absorption region located downstream of the signal detection region, which absorbs the test sample for which the signal detection reaction has been completed.

The anti-I-hCG antibody is an antibody that specifically recognizes I-hCG to which two subunits of α and β are bound, and the βcf hCG antibody is a fragment of free β-hCG, beta core fragment hCG (βcf hCG). It is an antibody that specifically recognizes. On the other hand, the anti-α-hCG antibody recognizes I-hCG and free α-hCG, and recognizes the α part of hCG-like hormones LH, FSH, and TSH, and when used for ectopic pregnancy diagnosis, anti-I-hCG antibody It is less sensitive and has a risk of cross-reactivity.

The immune device consisting of two detection lines of the present invention expands the accuracy of diagnosis of ectopic pregnancy by qualitatively, semi-quantitatively, and quantitatively analyzing the detection line and its signal strength for detecting I-hCG and βcf hCG in a sample and comparing them with normal pregnancy. It has features that improve efficiency.

3) When the signal of the second detection line is absent or insignificant compared to the first detection line of the signal detection area, it can be determined as an ectopic pregnancy through qualitative or semi-quantitative analysis with the naked eye.

In one embodiment of the present invention, standard signal data of the signal strength of the second detection line with respect to the first detection line of normal pregnancy may be additionally provided to the device. If the signal strength of the second detection line with respect to the first detection line is low compared to standard signal data completed in advance for a normal pregnancy, it can be determined as an ectopic pregnancy.

The sample area preferably accommodates a liquid sample such as blood or urine of a woman of childbearing age, but any sample expected to contain I-hCG and/or βcf hCG may be used.

The sample region may further have a filtering function in order to further improve the selectivity for the analyte or to minimize the effect of an interference substance that may be included in the sample. If necessary, an auxiliary region containing a substance capable of increasing the reaction between the analyte and the conjugate or eliminating the influence of the interfering substance may be further provided upstream of the sample region.

The control line of the conjugate region means a portion that generates a constant signal regardless of the concentration of I-hCG or βcf hCG in the test sample. The control line may be formed by immobilizing a ligand that does not bind to I-hCG or βcf hCG but binds to the third conjugate that moves along the detection area by the mobile phase together with the test sample. It is to immobilize a ligand capable of emitting a constant signal regardless of the presence or absence of I-hCG and βcf hCG in the specimen. Therefore, even if the first conjugate and / or the second conjugate passes the first detection line and / or the second detection line because it does not bind to the target sample, the third conjugate binds to the sample and can be captured on the control line. . For example, when the target sample does not exist in the sample, the color or the like is expressed in the control line even though the color or the like is not expressed in the detection line. As such, a response in the control line means that the liquid sample is properly passing through the sensor. The third conjugate may be mouse IgG or chicken IgY. As the control line, anti-rabbit IgG, anti-chicken IgY, streptavidin, bovine serum albumin, goat anti-mouse IgG or goat-anti-chicken IgY may be used.

The probe material of the conjugate region is gold nanoparticles, silver nanoparticles, quantum dot nanoparticles, carbon nanoparticles, latex beads/fluorescent nanoparticles, cellulose nanoparticles, magnetic nanoparticles, silica nanoparticles, polymer beads ( polymer beads), fluorescent substances, luminescent substances, dye beads, and at least one probe substance selected from the group consisting of proteins may be used. According to one embodiment of the present invention, the probe material is colloidal gold nanoparticles, but is not limited thereto.

According to one embodiment of the present invention, the signal detection region of 3) is the first detection line to which the anti-I-hCG antibody is immobilized, and the second detection line to which the anti-βcf-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 of 3) is the second detection line to which the anti-βcf hCG antibody is immobilized, and the first detection line to which the anti-I-hCG antibody is immobilized downstream of the second detection line. and a control line downstream of the first detection line.

The signal detection region is a medium in which a mobile phase and a sample are developed, and the mobile phase and the sample to be tested may move by capillarity of a porous membrane in the signal detection region. The signal detection area is a well plate made of nitrocellulose, cellulose, polyethylene, polyethersulfone, polystyrene, polycarbonate, polymethyl methacrylate, nylon, PVDF, polyvinyl resin or polystyrene resin. ) and one selected from the group consisting of glass slide glass may be used. According to one embodiment of the present invention, the signal detection region uses nitrocellulose, but is not limited thereto. According to one embodiment of the present invention, a nitrocellulose membrane having pores of 5 to 15 μm may be used, but is not limited thereto.

The moisture absorption region may include a porous support and an absorbent dispersed in the cavity of the porous support or adsorbed or coated on the fiber yarns of the porous support, and may further include a porous film layer on the upper surface of the porous support. Not limited to this. The absorbent may be selected from the group consisting of calcium chloride, magnesium chloride, diatomaceous earth, bentonite, dolomite, gypsum, silica gel, and mixtures thereof, but is not limited thereto.

In the present invention, the concentration measurement range of the target substance in the sample through the signal strength of the first detection line and the second detection line is 5mIU/mL or more for I-hCG and 0.01 pmol/mL or more for βcf hCG.

In the present invention, the anti-I-hCG antibody first conjugate present in the conjugate region binds to I-hCG in a liquid specimen such as blood or urine of a woman of childbearing age and moves toward the membrane (I-hCG )-conjugate to form the first complex. When the anti-βcf hCG antibody second conjugate present in the conjugate region migrates to the liquid sample and the signal detection region, it binds to βcf hCG in the sample to form a βcf hCG-conjugate second complex. The first complex reacts with the anti-I-hCG antibody immobilized on the first detection line during development, and the second complex reacts with the anti-βcf hCG antibody immobilized on the second detection line during development.

In addition, the immunization device of the present invention may include a solid support at the bottom.

The solid support may be formed of a material selected from the group consisting of nitrocellulose, nylon, PVDF, glass, and plastic. Since the strip is manufactured by attaching the strip to the solid support, durability of the strip can be increased, and handling and storage can be facilitated. In addition, it is possible to facilitate mounting of additional external devices.

As the plastic material that can be used as the solid support, a polypropylene film, a polyester film, a polycarbonate film, an acrylic film, etc. may be used, but is not limited thereto.

As another aspect, the present invention is a kit in which the immune device is additionally fixed in a device, wherein the lower device is provided with a guide and a strip support, and the upper device includes a sample inlet; and a result confirmation window at positions corresponding to the first detection line, the second detection line, and the control line.

The upper and lower devices may be manufactured using a common plastic material, and for example, materials such as polycarbonate and acrylonitrile butadiene styrene (ABS) may be used, but are not limited thereto.

According to one embodiment of the present invention,

1) In the present invention, a sample region accommodating a test sample to be analyzed;

2) a conjugate region linked to the sample region and containing an anti-I-hCG antibody conjugated to a probe and an anti-βcf hCG antibody conjugated to a probe;

3) A signal detection region linked to the conjugate region, including a first detection line to which the anti-I-hCG antibody is immobilized, a second detection line to which the anti-βcf hCG antibody is immobilized, and a control line connected to the sample region ;

4) a moisture absorption region located downstream of the signal detection region, which absorbs the test sample for which the signal detection reaction has been completed; and

5) An immunological device for diagnosing ectopic pregnancy including a quantitative analysis device is provided.

According to one embodiment of the present invention, the quantitative analysis device of 5) may evaluate the signal strength of the first detection line and the second detection line. The quantitative assay device includes a reader coupled with a software program capable of evaluating the intensity, color, luminescence and/or fluorescence of the labeled analyte complexes accumulated in the first and second detection lines. The reader can compare intensity, color, luminescence, and/or fluorescence against a pre-programmed threshold, and based on the comparison, provide a specific digital output if intensity, color, etc. are above a threshold, and if intensity, color, etc. If it is below the threshold, another digital output can be provided. According to one embodiment of the present invention, the quantitative analysis device uses a reflectance reader equipped with analysis software based on a CCD camera or laser light source, and diagnoses ectopic pregnancy with high accuracy.

In one embodiment of the present invention, the quantitative analysis device may further include a display providing information related to ectopic pregnancy.

In one embodiment of the present invention, the quantitative analysis device is programmed with a threshold value as a measurement ratio of the signal strength of the second detection line to the first detection line of normal pregnancy.

In one embodiment of the present invention, when the signal intensity of the second detection line with respect to the first detection line is lower than the signal intensity of the second detection line with respect to the first detection line of normal pregnancy, which is a threshold value, for example, 0.001 to 0.9, 0.001 to 0.8, 0.001 to 0.7, 0.001 to 0.6, 0.001 to 0.5, 0.001 to 0.4, 0.001 to 0.3, or 0.001 to 0.2 times, it can be determined as an ectopic pregnancy. The ratio may be applied differently depending on the number of weeks of LMP.

In one embodiment of the present invention, the signal intensity of the second detection line with respect to the first detection line is 0.001 in LMP (last menstrual period) 2 weeks compared to the signal intensity of the second detection line with respect to the first detection line of normal pregnancy. to 0.2 times, 0.001 to 0.4 times at 3 weeks of LMP, 0.001 to 0.5 times at 4 or more weeks of LMP, or a combination thereof, it can be determined as an ectopic pregnancy. Another means for evaluating test results in the present invention may also be included in the present invention. In addition, the ratio of βcf hCG / I-hCG signal intensity for distinguishing normal pregnancy from ectopic pregnancy may vary depending on the type of probe material, the type of signal detection region, the sensitivity of the quantitative analysis device, etc. It can be understood by those skilled in the art.

In the embodiment of the present invention, it was confirmed that the measured value of βcf hCG / I-hCG, which is the signal strength of the second detection line relative to the first detection line, was significantly lower in the ectopic pregnancy patient group than in the normal pregnancy group. From 2 weeks, it was confirmed that normal pregnancy and ectopic pregnancy were clearly distinguished.

According to another aspect of the present invention,

1) applying the sample to the immunization device so that I-hCG and βcf hCG in the sample react with the probe-labeled anti-I-hCG antibody and the anti-βcf hCG antibody;

2) As a step of confirming the reaction of I-hCG and βcf hCG in the sample by the first and second detection lines, I-hCG and βcf hCG are signals generated by the presence of the sandwich complex in each detection line detected by; and

3) A method for providing information necessary for diagnosing an ectopic pregnancy, including the step of determining that the pregnancy is ectopic if there is no signal strength of the second detection line relative to the first detection line or is insignificant.

According to one embodiment of the present invention, 1) applying the sample to the immunization device so that I-hCG and βcf hCG in the sample react with the probe-labeled anti-I-hCG antibody and the anti-βcf hCG antibody;

2) As a step of confirming the reaction of I-hCG and βcf hCG in the sample by the first and second detection lines, I-hCG and βcf hCG are signals generated by the presence of the sandwich complex in each detection line detected by; and

3) If the signal strength of the second detection line for the first detection line is lower than the signal strength of the second detection line for the first detection line of a normal pregnancy, ectopic pregnancy including the step of determining that the pregnancy is ectopic Provides a method for providing information necessary for diagnosis.

In one embodiment of the present invention, when the signal strength of the second detection line for the first detection line of 3) is lower than the signal strength of the second detection line for the first detection line of normal pregnancy, the first detection It may be a step of comparing the signal strength of the second detection line for the line with standard signal data for the signal strength of the second detection line for the first detection line of the normal pregnancy. Compared to the standard signal data completed in advance for normal pregnancy, when the signal intensity of the second detection line for the first detection line is lower than the signal intensity of the second detection line for the first detection line for normal pregnancy, the uterus It can be diagnosed as an extra-pregnancy.

In one embodiment of the present invention, the signal strength of the second detection line relative to the first detection line may be analyzed using a quantitative analysis device. The quantitative analysis device may evaluate signal intensities of the first detection line and the second detection line. The quantitative assay device includes a reader coupled with a software program capable of evaluating the intensity, color, luminescence and/or fluorescence of the labeled analyte complexes accumulated in the first and second detection lines. The reader can compare intensity, color, luminescence, and/or fluorescence against a pre-programmed threshold, and based on the comparison, provide a specific digital output if intensity, color, etc. are above a threshold, and if intensity, color, etc. If it is below the threshold, another digital output can be provided. The threshold value is provided based on the measured value of βcf hCG / I-hCG in normal pregnancy or the measured value of the signal strength of the second detection line relative to the first detection line in normal pregnancy, and the measurement of βcf hCG / I-hCG If the value is low compared to a normal pregnancy, for example, a ratio of 0.001 to 0.9, 0.001 to 0.8, 0.001 to 0.7, 0.001 to 0.6, 0.001 to 0.5, 0.001 to 0.4, 0.001 to 0.3 or 0.001 to 0.2 ectopic It can be determined that you are pregnant. For example, the signal intensity of the second detection line for the first detection line is 0.001 to 0.2 times at 2 weeks of LMP (last menstrual period) compared to the signal intensity of the second detection line for the first detection line of normal pregnancy. 0.001 to 0.4 fold at 3 weeks of LMP, 0.001 to 0.5 fold at 4 weeks or more of LMP, or a combination thereof can be determined as an ectopic pregnancy.

In one embodiment of the present invention, 3) the signal strength of the second detection line with respect to the first detection line is 0.001 to 0.9, 0.001 to 0.8 compared to the signal strength of the second detection line with respect to the first detection line of normal pregnancy. , 0.001 to 0.7, 0.001 to 0.6, 0.001 to 0.5, 0.001 to 0.4, 0.001 to 0.3, or 0.001 to 0.2, it is determined that the pregnancy is ectopic.

In one embodiment of the present invention, the signal intensity of the second detection line with respect to the first detection line is 0.001 in LMP (last menstrual period) 2 weeks compared to the signal intensity of the second detection line with respect to the first detection line of normal pregnancy. to 0.2 times, 0.001 to 0.4 times at 3 weeks of LMP, 0.001 to 0.5 times at 4 or more weeks of LMP, or a combination thereof, it can be determined as an ectopic pregnancy.

According to another aspect of the present invention, a method for diagnosing ectopic pregnancy using the immunochromatographic strip is provided.

1) applying the test sample to the immunization device so that I-hCG and βcf hCG in the sample react with the probe-labeled anti-I-hCG antibody and the probe-labeled anti-βcf hCG antibody, respectively;

2) As a step of confirming the reaction of I-hCG and βcf hCG in the sample by the first and second detection lines, I-hCG and βcf hCG are signals generated by the presence of the sandwich complex in each detection line detected by; and

3) When the signal intensity of the second detection line for the first detection line is lower than the signal intensity of the second detection line for the first detection line of normal pregnancy, for example, 0.001 to 0.9, 0.001 to 0.8, 0.001 to 0.7, 0.001 to 0.6, 0.001 to 0.5, 0.001 to 0.4, 0.001 to 0.3, or 0.001 to 0.2, a method for diagnosing ectopic pregnancy is provided.

In one embodiment of the present invention, the signal intensity of the second detection line with respect to the first detection line is 0.001 in LMP (last menstrual period) 2 weeks compared to the signal intensity of the second detection line with respect to the first detection line of normal pregnancy. to 0.2 fold, 0.001 to 0.4 fold over 3 weeks of LMP, 0.001 to 0.5 fold over 4 weeks of LMP, or a combination thereof, a method for diagnosing ectopic pregnancy is provided.

embodiment

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example 1: Preparation of immunochromatography strips

A. Fabrication of membrane with first inspection line, second inspection line and control line

Three assay ligands were dispensed onto a nitrocellulose membrane. Anti-I-hCG monoclonal antibody (Monoclonal anti-hCG) was used as the I-hCG antibody of the first detection line, and anti-βcf hCG monoclonal antibody (Monoclonal anti-βcf hCG) was used as the ligand of the control line as the βcf hCG antibody of the second detection line. was dried after dispensing goat anti-mouse immunoglobulin (Goat anti-mouse IgG).

B. Fabrication of conjugate pad

A first conjugate solution in which colloidal gold nanoparticles and anti-I-hCG antibody (Monoclonal anti-hCG) were conjugated was prepared, and colloidal gold nanoparticles and anti-βcf hCG antibody (Monoclonal anti-βcf hCG ) A second conjugate solution was prepared. In addition, a third conjugate solution in which colloidal gold nanoparticles and mouse immunoglobulin (Mouse IgG) are combined was prepared. The first conjugate solution, the second conjugate solution, and the third conjugate solution were dispensed onto the pretreated conjugate pad, completely dried, and then cut into appropriate sizes.

C. Preparation of sample pad

The sample pad was sufficiently soaked in the sample pad pretreatment solution containing buffer and preservative, dried completely, and then cut into appropriate sizes.

D. Manufacture of absorbance pad

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

E. Fabrication of immunochromatographic strips

Assemble the membrane, conjugate pad, sample pad, and moisture absorption pad prepared through each of the above processes according to the structure shown in Figure 1.

That is, the sample pad is attached so as to overlap one end of the conjugate pad, one end of the detection pad is attached so as to overlap the other end of the conjugate pad, and the other end of the detection pad and one end of the moisture absorption pad are attached. attached so that they overlap each other.

In FIG. 1, the meaning of each reference numeral is as follows.

1: Sample pad

2: Conjugate pad

3: Nitrocellulose membrane

4: Absorbent Pad

5: first detection line immobilized with I-hCG antibody

6: βcf hCG antibody immobilized second detection line

7: control line immobilized with goat anti-mouse immunoglobulin

F. Quantitative analysis device

It was used to quantify the color development or contrast sensitivity of the detection line using a reflection reader equipped with analysis software based on a CCD camera or laser light source.

G. Device Assembly

After inserting the prepared immunochromatography strip for ectopic pregnancy diagnosis into the strip fixing position of the lower plastic device, insert it into the upper device having the sample inlet and the window for checking the results, and then assemble.

Example 2 Measurement of Intact hCG and βcf hCG Concentration Changes in Normal Pregnancy Group and Ectopic Pregnancy Patient Group

1) Measurement of change in concentration of intact hCG and βcf hCG in normal pregnancy group

The changes in the concentrations of intact hCG and βcf hCG in 11 normal pregnant women were measured and shown in FIG. 3 . In the normal pregnancy group, intact hCG and βcf hCG gradually increased according to the number of weeks after 2 weeks of LMP, and it was confirmed that intact hCG was detected first. βcf hCG was weakly detected in the 2nd week of LMP, but it was confirmed that it continuously increased and exceeded the detection amount of Intact hCG after 4 weeks.

2) Measurement of change in concentration of intact hCG and βcf hCG in the ectopic pregnancy patient group

Intact hCG and βcf hCG concentration changes in the ectopic pregnancy patient group were measured and shown in FIG. 4 . In the case of ectopic pregnancy patients, intact hCG was detected, but the concentration did not show an increasing pattern according to the gestational age, and βcf hCG similarly showed no correlation according to the number of weeks from the point of initial weak detection.

As a result, the concentrations of intact hCG and βcf hCG increased between 2 and 8 weeks of LMP in the case of normal pregnancy, while the concentrations of ectopic pregnancy increased slightly until 5 weeks and then decreased. confirmed as a result.

Example 3: Intact hCG / βcf hCG measurement of normal pregnancy group and ectopic pregnancy patient group

In order to use the results of Example 2 for diagnosing the ectopic pregnancy patient group, the concentrations of βcf hCG / Intact hCG in the normal pregnancy group and the ectopic pregnancy patient group were measured and compared.

1) Measurement of βcf hCG / Intact hCG concentration in normal and ectopic pregnancy patients by gestational week

Distribution of the concentration ratio of βcf hCG / Intact hCG (Fig. 5a) and the concentration ratio of βcf hCG / Intact hCG (Fig. 5b) based on the detected concentrations of intact hCG and βcf hCG measured in normal pregnancy and ectopic pregnancy patient groups by gestational age showed As a result, in the case of ectopic pregnancy, it was confirmed that the concentration ratio and the distribution of the concentration ratio were lower than those of normal pregnancy, and the P-value value was 1.66 x 10 ^-7 in the T test result, indicating a significant difference, βcf It was confirmed that hCG / Intact hCG concentration values could be used to distinguish between normal and ectopic pregnancy patients.

2) Measurement of concentration of βcf hCG / Intact hCG in normal pregnancy and ectopic pregnancy patient groups by pregnancy day

Concentration ratio of βcf hCG / Intact hCG (Fig. 6a) and βcf hCG / Intact hCG based on detected concentrations of intact hCG and βcf hCG measured in normal pregnancy and ectopic pregnancy patient groups by day during LMP 2-3 weeks The distribution of the concentration ratio of (FIG. 6b) was shown. As a result, in the case of ectopic pregnancy, it was confirmed that the concentration ratio and distribution of the concentration ratio were lower than those of normal pregnancy, and the P-value value was 5. 82 x 10 ^-10 in the T test result, indicating a very significant difference. , it was confirmed that the βcf hCG / Intact hCG concentration values could be used to distinguish between normal and ectopic pregnancy patients.

3) Experimental results

During the period of 2 to 8 weeks of LMP, the βcf hCG / Intact hCG concentration ratio gradually approached 1 in the case of normal pregnancy (Median: 1.092), and the βcf hCG / Intact hCG concentration ratio approached 0.2 in the case of ectopic pregnancy (Median: 0.186 ), the βcf hCG / Intact hCG concentration ratio between normal pregnancy and ectopic pregnancy showed a difference as the gestational age progressed.

In addition, the concentration ratio between normal pregnancy and ectopic pregnancy was 5.82 x 10 ^-10 in the day results between 2 and 3 weeks of LMP and 1.66 x 10 ^-7 (0.001>p-value) in the results by week, showing a significant difference. As a result, it was confirmed that the βcf hCG / Intact hCG concentration value could be used to distinguish between normal pregnancy and ectopic pregnancy patient groups, and began to show significant differences from 2 weeks or more of LMP, and the device of the present invention showed a significant difference from 2 weeks or more of LMP It has been proven that it is a device that can distinguish normal pregnancy from ectopic pregnancy.

Example 4: Results of normal pregnancy and ectopic pregnancy analysis

An experiment was conducted to confirm the accuracy of the diagnosis of ectopic pregnancy by the immunodiagnostic device according to the present invention. There was no significant difference between the average age and estimated date after LMP between the normal pregnancy and ectopic pregnancy test groups, and it was confirmed that the immunodiagnostic device according to the present invention accurately diagnosed all ectopic pregnancy patient groups as a result of the blind experiment.

Claims (9)

1) a sample area accommodating a test sample to be analyzed;
2) a conjugate region linked to the sample region and containing a probe-label conjugated anti-I-hCG antibody and a probe-label conjugated anti-βcf hCG antibody;
3) A signal detection region linked to the conjugate region, including a first detection line to which the anti-I-hCG antibody is immobilized, a second detection line to which the anti-βcf hCG antibody is immobilized, and a control line connected to the sample region ;
4) an absorption region located downstream of the signal detection region, which absorbs the test sample for which the signal detection reaction has been completed; and
5) If the signal strength of the second detection line with respect to the first detection line is lower than the signal strength of the second detection line with respect to the first detection line of a normal pregnancy, the uterus including a quantitative analysis device that determines that the pregnancy is ectopic. Immunotherapy device for diagnosis of pregnancy. delete delete According to claim 1,
1) The signal intensity of the second detection line relative to the first detection line is 0.001 to 0.2 times at 2 weeks of LMP (last menstrual period) compared to the signal intensity of the second detection line relative to the first detection line of normal pregnancy, LMP 3 An immunological device for diagnosing ectopic pregnancy, characterized in that it is judged as an ectopic pregnancy in the case of 0.001 to 0.4 times per week, 0.001 to 0.5 times over 4 weeks of LMP, or a combination thereof. The probe material of claim 1, wherein the probe material of 2) is gold nanoparticles, silver nanoparticles, quantum dot nanoparticles, carbon nanoparticles, latex beads/fluorescent nanoparticles, cellulose nanoparticles, or magnetic nanoparticles. An immune device for diagnosing ectopic pregnancy, characterized in that it is at least one selected from the group consisting of silica nanoparticles, polymer beads, fluorescent substances, luminescent substances, pigment beads, and proteins. The uterus according to claim 1, wherein the signal detection region of 3) is any one selected from the group consisting of nitrocellulose, cellulose, polyethylene, polyethersulfone, polystyrene, polycarbonate, polymethyl methacrylate, and nylon. Immunotherapy device for diagnosis of pregnancy. The immunological device for diagnosing ectopic pregnancy according to claim 1, wherein the absorption area of 4) includes an absorbent dispersed in the pores of the porous support or adsorbed or coated on the fibers of the porous support. 1) Applying the test sample to the immune device according to any one of claims 1, 4 to 7, the I-hCG and βcf hCG in the sample are probe-labeled anti-I-hCG antibody and probe- reacting with the anti-βcf hCG antibody conjugated with the label;
2) As a step of confirming the reaction of I-hCG and βcf hCG in the sample by the first and second detection lines, I-hCG and βcf hCG are signals generated by the presence of the sandwich complex in each detection line detected by; and
3) The signal intensity of the second detection line relative to the first detection line is 0.001 to 0.2 times at 2 weeks of LMP (last menstrual period) compared to the signal intensity of the second detection line relative to the first detection line of normal pregnancy, LMP 3 0.001 to 0.4 times per week, 0.001 to 0.5 times over 4 weeks of LMP, or a combination thereof, a method for providing information necessary for ectopic pregnancy diagnosis comprising the step of determining that the pregnancy is ectopic. The method according to claim 8, wherein 3) the signal strength of the second detection line with respect to the first detection line of normal pregnancy is programmed as a threshold value.

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