Pre-eclampsia marker and application and kit thereof

Abstract

The invention discloses a preeclampsia marker as well as application and a kit thereof. Comprising phosphatase and tension protein (PTEN), and the marker can be used for preparing a serological detection preeclampsia diagnostic reagent or diagnostic tool to perform early screening on preeclampsia risk population and fill the blank of current preeclampsia serological diagnosis. The invention belongs to the technical field of medicine. Furthermore, the invention also provides an ELISA kit for detecting preeclampsia, the sampling means is non-invasive and non-invasive, high-precision endoscope equipment is not needed, the kit can be applied to primary medical institutions, the early diagnosis of preeclampsia and the adoption of prevention means for intervention are facilitated, and the kit has good application value.

Description

Technical Field

[0001] This invention belongs to the field of medical technology. More specifically, it relates to a preeclampsia biomarker, its application, and a reagent kit. Background Technology

[0002] Preeclampsia (PE) is a condition characterized by hypertension and proteinuria that occurs after 20 weeks of gestation. Postpartum symptoms usually resolve spontaneously. Globally, the incidence of PE is 2%–8%. Early-onset PE, in particular, is a significant cause of adverse pregnancy outcomes for both mother and baby. Eclampsia is a serious complication leading to maternal and perinatal mortality. Once PE occurs, clinical treatment primarily involves expectant management measures such as antihypertensives, anticonvulsants, and sedation. The goal is to prolong gestation as much as possible while ensuring the mother's safety and improving perinatal survival. Termination of pregnancy is the only effective way to cure PE.

[0003] Although the etiology of preeclampsia is unclear, it has certain high-risk clinical factors. It typically develops after 20 weeks of gestation and progresses in two stages: ① Genetic, immune, nutritional, or environmental factors cause placental trophoblasts to invade and replace the smooth muscle cells of the uterine spiral arteries, leading to shallow placental implantation, increased resistance in the uterine spiral arteries, and reduced uteroplacental blood flow, resulting in placental ischemia and hypoxia; ② Abnormal expression of certain cytokines and vasoactive substances related to placental trophoblasts and vascular endothelial cells. Vasoactive substances cause spasm of small blood vessels throughout the body, which further aggravates vascular endothelial damage, causing endothelial cell swelling, increased vascular permeability, extravasation of body fluids and proteins, and a decrease in anticoagulant and vasodilator factors. This leads to the synthesis of procoagulant factors and activation of the coagulation system at the damaged site, resulting in platelet aggregation, thrombosis, vasoconstriction, and proteinuria. Regarding its pathophysiological mechanisms, obstetricians both domestically and internationally have conducted extensive clinical research on the population characteristics, pathophysiological features, and placental biomarkers of pregnant women with preeclampsia, in order to find effective and easily clinically applicable early predictive factors or screening methods. This aims to achieve early assessment of the risk of preeclampsia in pregnant women and prediction of adverse pregnancy outcomes, thereby enabling the implementation of effective intervention measures. This is an ideal solution to block the disease and reduce maternal and infant harm.

[0004] The phosphatase and tensin homology deleted on chromosome 10 (PTEN) gene is the only tumor suppressor gene discovered to date with dual phosphatase activities of both lipid phosphatase and protein phosphatase. Studies have shown that PTEN is expressed in various tissues, and its protein loss of function is involved in the development and progression of various tumor types. As a novel tumor suppressor gene, PTEN has attracted attention due to its unique pathway of action and alterations during the progression of various human tumors. However, current research is mostly limited to PTEN mutational inactivation in tumors, with limited research on the role and value of PTEN in non-tumor diseases. Trophoblastic cells infiltrate the maternal decidua, superficial myometrium, and corresponding uterine spiral arteries with behavior similar to malignant tumor cells, and their biological behaviors are similar. As a tumor suppressor gene, PTEN may play a regulatory role in trophoblastic cell invasiveness. The PTEN gene also plays an important role in the pathogenesis of hypertension by regulating PI3K / Akt signaling.

[0005] Specific expression of PTEN in placental tissue of preeclampsia and normal pregnant women has been reported. Furthermore, in our animal experiments, PTEN inhibitors significantly improved the disease activity index and histopathological condition of a mouse model of preeclampsia induced by L-NAME, PS / PC (phosphatidylserine / phosphatidylcholine) clusters, and STBM (syncytiotrophoblastic microvilli), suggesting that abnormal PTEN expression in PE models may trigger abnormal autoimmune responses. Therefore, detecting serum PTEN levels can not only non-invasively assess the occurrence, development, and prognosis of PE, enabling timely diagnosis and treatment even in the early stages with mild symptoms, but also facilitate early screening and intervention in at-risk populations, nipping the disease in the bud.

[0006] Currently, there are no serological early screening and diagnostic reagents for PE in clinical practice, and traditional predictive methods for PE diagnosis, such as the turning test and mean arterial pressure during pregnancy, have low sensitivity. On the one hand, these methods require highly experienced personnel, resulting in a high rate of misdiagnosis and missed opportunities for early detection and treatment. On the other hand, many primary care hospitals in China cannot afford the purchase and maintenance of such equipment, so local residents need to travel to higher-level hospitals with better facilities for diagnosis. Furthermore, the above testing methods can only be used for patients with certain symptoms and cannot be used for early screening of at-risk populations. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a preeclampsia biomarker, its application, and a reagent kit, thus solving the aforementioned problems.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a serological marker for the diagnosis of preeclampsia, wherein the marker is phosphatase and tensin (PTEN).

[0009] Preferably, the present invention provides the application of the above-mentioned biomarkers in the preparation of diagnostic reagents or tools for detecting preeclampsia.

[0010] Preferably, the present invention provides the use of the above-mentioned biomarkers in the preparation of antibodies for detecting phosphatases and tensin in human serum.

[0011] The present invention provides the application of the detection reagents for the above-mentioned markers in the preparation of diagnostic reagents or tools for detecting preeclampsia, or in the preparation of antibodies for detecting phosphatase and tensin in human serum.

[0012] Preferably, the present invention provides a kit for diagnosing preeclampsia, which contains the aforementioned phosphatase and tensin or their detection reagents.

[0013] Specifically, the present invention provides an ELISA kit for diagnosing preeclampsia, the kit containing the above-mentioned biomarkers phosphatase and tensin, an ELISA plate, a coating buffer, a blocking solution, and an ELISA-labeled antibody.

[0014] Preferably, as an alternative implementation, the phosphatase and tensin antibody can be obtained through a monoclonal antibody preparation reaction.

[0015] Preferably, as an optional implementation, the coating buffer contains the following components: 1.0–2.0 g / L Na₂CO₃ and 3–5 g / L NaHCO₃; the blocking solution contains the following components: 3–6 g / L bovine serum albumin, 2–5 g / L casein, 5–10 g / L NaCl, 0.2–0.4 g / L KH₂PO₄, 1–3 g / L Na₂HPO₄·12H₂O, and 0.2–0.5 g / L KCl.

[0016] Preferably, as an optional implementation, the enzyme-labeled antibody is a 0.2–1 μg / mL HRP-labeled anti-Human IgG antibody; the chromogenic solution includes chromogenic agent A and chromogenic agent B; chromogenic agent A includes the following components: 22–30 g / L sodium acetate, 3–5 g / L citric acid, and 0.1–0.5 mL / L 30% hydrogen peroxide; each liter of chromogenic agent B includes the following components: 200–500 mg / L tetramethylbenzidine, 3–5 mL / L dimethyl sulfoxide, and 1–2 g / L citric acid monohydrate.

[0017] Preferably, as an alternative implementation, the ELISA kit further includes a diluent, a washing solution, and a stop solution.

[0018] Preferably, the diluent comprises the following components: 6–8 g / L NaCl, 0.1–0.3 g / L KH₂PO₄, 1–5 g / L Na₂HPO₄·12H₂O, and 0.1–0.5 g / L KCl;

[0019] Preferably, the washing solution comprises the following components: 6-8 g / L NaCl, 0.2-0.4 g / L KH2PO4, 3-5 g / L Na2HPO4·12H2O, 0.2-0.4 g / L KCl, and 0.3-0.5 mL / L Tween 20;

[0020] Preferably, the terminating solution is a 1.5–2.5 mol / L H2SO4 solution.

[0021] Preferably, as an optional implementation, the method of using the ELISA kit includes the following steps:

[0022] S1. Coating with phosphatase and tensin (PTEN) antibodies: Dilute phosphatase and tensin (PTEN) with antibody coating buffer to 100 μg / ml, add 100 μL to each well of a 96-well microplate, and incubate overnight at 4°C. The next day, discard the liquid in the wells, and fill each well with washing buffer three times. After the first wash, let stand for 2 minutes and then discard the buffer; after the next two washes, let stand for 1 minute each. Invert the microplate onto filter paper and blot away any remaining liquid.

[0023] S2. Sealing: Fill each well with sealing solution (about 300 μL), cover or seal the plate with sealing film, incubate in a 37°C incubator for 60 min, pour out the liquid in the well, and wash 3 times as above.

[0024] S3. Sample addition: Add 100 μL each of the serum to be tested and the blank control (diluent) to the wells of the plate, seal the plate with sealing film, incubate at 37℃ for 60 min, and wash 5 times as above;

[0025] S4. Add enzyme-labeled antibody: Add 100 μL of HRP-labeled antibody to each well, seal the plate and incubate at 37°C for 1 h, wash 5 times as above, pat dry on filter paper and remove residual liquid;

[0026] S5. Color development and termination of reaction: Mix color development solution A and color development solution B at a ratio of 1:1 in a dark environment, then add 100 μL to each well, incubate at 37°C for 20 min in the dark, and then immediately add 100 μL of 2 mol / L H2SO4 to each well to terminate the reaction. The solution immediately changes from blue to yellow.

[0027] S6. Detection: Use an ELISA reader to detect the absorbance at a wavelength of 450nm and record the data;

[0028] S7. Result determination: Calculate the positive / negative ratio (P / N) between the antibody to be tested and the blank control. When P / N is greater than 2, it is positive; when P / N is less than 2, it is negative. The positive result with the highest dilution factor is taken as the measured antibody titer.

[0029] Beneficial effects

[0030] This invention provides a preeclampsia biomarker, its application, and a reagent kit. Compared with existing technologies, it has the following advantages:

[0031] The present invention provides serological markers for the diagnosis of preeclampsia, namely phosphatase and tensin (PTEN), which are released into the bloodstream before the onset of disease symptoms. Therefore, detecting their corresponding antibodies can serve as a diagnostic basis for patients with subtle early symptoms, enabling early screening of individuals at risk for preeclampsia and filling a gap in current serological diagnosis of preeclampsia. Furthermore, the ELISA kit provided by this invention employs a non-invasive sampling method, significantly improving patient and at-risk population compliance. It also has low requirements for implementation location, equipment, and personnel, eliminating the need for sophisticated endoscopic equipment, thus providing convenient diagnosis and screening for residents in primary healthcare institutions. Attached Figure Description

[0032] Figure 1 The results of placental tissue analysis in rats with PE model induced by L-NAME, PS / PC (phosphatidylserine / phosphatidylcholine) microclusters, and STBM (syncytiotrophoblast microvilli) combined (n≥2); A is the HE staining result of kidney tissue sections; B is the WB detection result of PTEN monoclonal antibody. Blank, control group; L-NAME-induced PE control group; Model, combined PE model group;

[0033] Figure 2 Bar graph showing the changes in serum PTEN expression in rats with PE model induced by L-NAME, PS / PC (phosphatidylserine / phosphatidylcholine) microclusters, and STBM (syncytiotrophoblast microvilli). Blank: control group; Control: L-NAME-induced PE control group; Model: combined PE model group.

[0034] Figure 3 The changes in cell function of HTR8 / SVneo cells after PTEN gene expression was interfered with by shRNA and then re-transfected with a PTEN overexpression plasmid (rescue experiment); A shows the change in cell invasion ability in the transfected group compared to the shRNA group; B shows the change in cell migration ability; C shows the change in cell apoptosis rate.

[0035] Figure 4The effects of combined induction by L-NAME, PS / PC (phosphatidylserine / phosphatidylcholine) microclusters, and STBM (syncytiotrophoblast microvilli) on a rat model of PE (porcine encephalopathy). A is a comparison of placenta and fetuses (n≥6); B is a scatter plot of fetal weight; C is a scatter plot of placental weight. Blank, control group; L-NAME-induced PE control group; Model, combined induction PE model group. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0037] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0038] 1. Experimental Materials

[0039] (1) Reagents

[0040] PTEN shRNA was purchased from Santa Fe Biomedical, USA; PS / PC was purchased from MP Biomedicals, USA; skim milk powder was purchased from Guangzhou Suju Biotechnology Co., Ltd.; BCA protein concentration assay kit, RIPA lysis buffer, 5× protein loading buffer, PBS powder, TBS powder, and SDS-PAGE rapid gel preparation kit were purchased from Guangzhou Youbai Biotechnology Co., Ltd.; GST-tagged protein purification kit and immunoprecipitation kit (Protein A+G agarose gel method) were purchased from Nanjing ACE Biotechnology Co., Ltd.; Freund's complete adjuvant, Freund's incomplete adjuvant, β-actin mouse monoclonal antibody, Goat Anti-Mouse IgG Antibody (H+L), HRP Conjugated, and Goat Anti-Rabbit IgG were purchased. Antibody (H+L), HRP Conjugated were purchased from Suzhou Bio-Long Pharmaceutical Co., Ltd.; OCT was purchased from Scigen Pharmaceuticals, Inc. (USA); Bovine serum albumin (BSA) was purchased from BBI Life Sciences Co., Ltd.; Sodium citrate antigen retrieval solution was purchased from Wuhan Boster Biological Engineering Co., Ltd.; Mounting solution containing DAPI anti-fluorescence quencher was purchased from Beijing Solarbio Science & Technology Co., Ltd.; Anti-PTEN antibody was purchased from Abcam.

[0041] (2) Preparation of reagents and reagents

[0042] 3% DSS aqueous solution: Prepared at a ratio of 3 / 100 for DSS powder and RO water.

[0043] PBS buffer: Take a pack of PBS buffer salts according to the instructions, add distilled water to make up to 2 L, adjust the pH to 7.2 - 7.4 after complete dissolution.

[0044] 0.9% NaCl solution: Weigh 9 g of NaCl powder, add distilled water to make up to 1 L, and dissolve completely.

[0045] TBST solution: Take a pack of TBS powder, make up to 2 L with ultrapure water, dissolve completely, and add 2 mL of Tween 20.

[0046] 1× electrophoresis buffer: Weigh 3.02 g of Tris-base, 18.8 g of Glycine, and 1 g of SDS, add ultrapure water to make up to 1 L, and store at 4 °C for later use.

[0047] 1× transfer buffer: Weigh 3.3 g of Tris-base, 14.4 g of Glycine, and 0.1 g of SDS, add ultrapure water to make up to 800 mL and dissolve completely, then add methanol to make up to 1 L, and store at 4 °C for later use.

[0048] (3) Experimental instruments

[0049] BSA223S ten-thousandth electronic analytical balance (Sartorius Scientific Instruments Beijing Co., Ltd.), MULTISKAN MK3 multifunctional microplate reader (Thermo Fisher Scientific China Co., Ltd.), BX41 fluorescence microscope (OLYMPUS Corporation, Japan), HT-6000 chemiluminescence analyzer (Beijing Hongtao Jiye Technology Development Co., Ltd.), HT-Mini04 electrophoresis tank (Beijing Hongtao Jiye Technology Development Co., Ltd.), S-TRANS rapid multi-channel semi-dry transfer instrument (Nanjing ACE Co.), TGL18W high-speed refrigerated centrifuge (Hunan Yingtai Co.), MICROM STP 120 tissue dehydrator (Thermo Fisher Scientific China Co., Ltd.), HistoStar tissue embedding machine (Thermo Fisher Scientific China Co., Ltd.), HM340E semi-automatic rotary microtome (Thermo Fisher Scientific China Co., Ltd.), LSM720 laser scanning confocal microscope (Carl Zeiss AG, Germany)

[0050] (4) Experimental animals

[0051] SPF-grade SD rats, female, 222 ± 10 g, purchased from Guangdong Gewu Biotechnology Co., Ltd., license number: SCXK(Guangdong)2023 - 0059; raised in the animal house of Guangdong Gewu Biotechnology Co., Ltd., unit use license: SCXK(Guangdong)2023 - 0059. The experimental animals were housed separately in cages, adaptively raised for 5 days, and had free access to water and food during the experiment. The breeding environment temperature was 22 ± 2 °C, the relative humidity was 60%, and there was a 12-hour light and 12-hour dark cycle every day.

[0052] 2. Experimental Methods

[0053] 2.1 Clinical Sample Collection Methods for Preeclampsia (PE)

[0054] Based on the pathological analysis of eclampsia, 24 patients each from normal pregnancy, preeclampsia, and posteclampsia were collected according to the following criteria, and serum samples were collected.

[0055] ① Preeclampsia: Preeclampsia is a disease that occurs in pregnant women after 20 weeks of gestation, characterized by high blood pressure and proteinuria. The clinical symptoms of patients can be relieved spontaneously after delivery.

[0056] ② Late-onset preeclampsia: Pregnant women who develop the condition after 34 weeks of gestation have better maternal and infant outcomes after termination of pregnancy; however, pregnant women who develop the condition before 34 weeks of gestation, especially before 28 weeks of gestation, experience rapid disease progression and are difficult to treat. The expectant management window is generally a few days to two weeks, and continuing the pregnancy will endanger the health of the pregnant woman. At this time, obstetricians must terminate the pregnancy early at the expense of perinatal interests. Therefore, the prevention and treatment of early-onset preeclampsia is an important issue for obstetricians.

[0057] 2.2 ELISA was used to detect the levels of phosphatase and tensin in the serum of the above patients.

[0058] Peripheral blood (approximately 3-5 ml) was collected from patients with PE at the above-mentioned diseases. After standing at room temperature for 20 minutes, the blood was centrifuged at 3000 rpm, 4°C, for 10 minutes. The supernatant was collected, and phosphatase and tensin (PTEN) were detected according to the ELISA kit instructions.

[0059] 2.3 Western Blot (WB)

[0060] A portion of rat colon tissue was collected and weighed. RIPA lysis buffer and PMSF were added according to the instructions. The tissue was homogenized thoroughly, and total protein was extracted using standard methods. Protein concentration was determined by the BCA method. The protein was denatured, and the loading volume was determined based on the concentration.

[0061] The total protein content reached 20 μg. Proteins were separated by molecular weight using 10% separating gel and 5% stacking gel SDS-PAG electrophoresis (80V, 60 min). The protein was transferred to a PVDF membrane at 500 mA for 10 min, blocked with 5% skim milk powder on a shaker for 1 h, washed three times with TBST for 8 min each time, incubated with PTEN and β-actin antibodies overnight at 4°C, washed three times with TBST for 8 min each time, incubated with secondary antibody at room temperature for 2 h, washed three times with TBST, and developed using a chemiluminescence system to obtain bands. The grayscale values ​​were analyzed using ImageJ.

[0062] 2.5 Methods for establishing the PE rat animal model

[0063] Female SD rats were randomly divided into three groups: a normal group, a control group, and a model group, with 10 rats in each group. Starting from day 10 of gestation, a PE rat model was induced by intravenous injection of L-NAME, PS / PC (phosphatidylserine / phosphatidylcholine) microclusters, and STBM (syncytiotrophoblast microvilli). The injections were administered continuously for 10 days, and samples were collected on day 21 to detect various indicators.

[0064] 2.6 Evaluation of rat blood routine

[0065] Blood samples were taken from pregnant mice on days 10 and 20 to test their blood routine, urine routine, liver function, and kidney function.

[0066]

[0067] Example 1: Preeclampsia induced an increase in PTEN protein expression levels in the placenta of rats.

[0068] A PE rat model was induced using L-NAME, PS / PC (phosphatidylserine / phosphatidylcholine) microclusters, and STBM (syncytiotrophoblast microvilli). After sacrifice, kidney tissue was collected for protein extraction, and a portion of the intestine was harvested for pathological sectioning. HE staining and Western blotting were performed to detect PTEN protein expression levels. The HE staining results of the kidney tissue sections are shown below. Figure 1 As shown in Figure A, compared with the normal group, the model group rats showed significantly greater vacuolation of renal tubular epithelial cells in the kidney region. Placental tissue Western blot results are as follows... Figure 1 As shown in Figure B, compared with the normal group, the expression level of PTEN protein in the placenta of rats in the control and model groups was significantly increased. These results indicate that PTEN protein undergoes significant changes during PE disease, and PE also causes associated kidney damage.

[0069] Example 2: Serum PTEN expression levels were significantly increased in SD rats.

[0070] To determine the changes in PTEN expression levels in serum during PE disease, this invention utilizes ELISA to detect PTEN levels in rat serum. The results are as follows... Figure 2 As shown, compared with the normal group, the serum PTEN protein level in the model group was significantly increased (P<0.0001), indicating statistical significance. PTEN, as a tumor suppressor gene, may play a regulatory role in trophoblast invasion.

[0071] The PTEN gene also plays an important role in the pathogenesis of hypertension by regulating PI3K / Akt signaling.

[0072] Example 3: Function of the PTEN gene in chorionic trophoblast cells (HTR8 / SVneo)

[0073] This study aimed to improve conventional gene function research methods in the laboratory. After constructing a stable HTR8 / SVneo (PTEN shRNA) cell line, the cells were transfected with a PTEN overexpression plasmid to investigate the major role of the PTEN gene in HTR8 / SVneo cells. Results are as follows: Figure 3 As shown, compared with the shRNA group, the OE group showed a significant decrease in apoptosis rate, cell migration rate, and invasion ability, indicating that the PTEN gene can improve the severity and prognosis of PE disease.

[0074] Example 4: Correlation between PTEN and preeclampsia

[0075] The PTEN gene is the first tumor suppressor gene discovered to date with phosphatase activity. Its protein product is a dual-specificity phosphatase with both lipid phosphatase and protein phosphatase activities. PTEN mutations or deletions are associated with various tumors such as glioma, breast cancer, melanoma, prostate cancer, and pancreatic cancer, and its inactivation mutations are closely related to tumor occurrence and development. Studies have shown that the lower the PTEN protein expression rate, the higher the malignancy of the tumor, and it can be used as a marker for judging tumor invasion and malignancy level. PTEN also plays an important role in embryonic development. Mouse models by DiCristofano et al. and Podsypanina et al. have confirmed that PTEN plays an important role in animal growth and development, and embryos with PTEN inactivation die early. The etiology and pathogenesis of preeclampsia have always been an important topic in obstetric research. Currently, it is generally believed that shallow placental implantation and vascular endothelial cell damage are the main causes of preeclampsia. Figure 4 As shown, compared with the normal group, the fetal development in the control group and the model group was significantly poor, and the placental and fetal weights were also significantly lower. Figure B is a scatter plot of fetal weight, and Figure C is a scatter plot of placental weight.

[0076] Example 5

[0077] This embodiment provides a biomarker for diagnosing preeclampsia and an ELISA kit containing the biomarker, wherein the biomarker is phosphatase and tensin (PTEN). This kit can be used to detect phosphatase and tensin (PTEN) in human serum, providing a detection method for the early diagnosis of preeclampsia. Specifically, the kit includes antibodies against the biomarkers phosphatase and tensin (PTEN), coating buffer, enzyme-labeled antibody, chromogenic solution, blocking solution, diluent, washing solution, and stop solution.

[0078] The preparation method of phosphatase and tensin (PTEN) antibodies is as follows: Recombinant vectors for constructing phosphatase and tensin (PTEN) are transformed into *E. coli*, inducing the expression of phosphatase and tensin (PTEN). After separation, purification, and Western blotting, purified phosphatase and tensin (PTEN) are obtained. These are then injected into mice with Freund's adjuvant. After multiple immunizations, serum antibody titers are measured by ELISA. Spleen cells from immunized mice are then fused with *SP / 0* myeloma cells of the same strain. Monoclonal antibodies are selected, and the cells are expanded and cultured in vitro. The supernatant is collected, and the antibodies are purified to obtain monoclonal antibodies against phosphatase and tensin (PTEN).

[0079] Preparation of coating buffer: Weigh 1.5g Na2CO3 powder and 2g NaHCO3 powder, and dilute to 1L with ultrapure water to fully dissolve.

[0080] The enzyme-labeled antibody was 0.1 μg / mL HRP-labeled anti-Human IgG antibody.

[0081] Preparation of blocking solution: Weigh 4g bovine serum albumin, 7g NaCl, 0.1g KH2PO4, 2g Na2HPO4·12H2O, 0.1g KCl, and ddH2O, mix them, and dilute to 1L with ultrapure water to fully dissolve them.

[0082] The colorimetric solution includes colorimetric agent A and colorimetric agent B.

[0083] Preparation of colorimetric reagent A: Take 26g sodium acetate, 2g citric acid, and 0.4mL 30% hydrogen peroxide, and dilute to 1L with ultrapure water. Preparation of colorimetric reagent B: Weigh 600 mg tetramethylbenzidine, 32 mL dimethyl sulfoxide, and 8 g citric acid monohydrate, and dilute to 1 L with ultrapure water.

[0084] Preparation of diluent: Weigh 7g NaCl, 0.1g KH2PO4, 2g Na2HPO4·12H2O, and 0.1g KCl, and dilute to 1L with ultrapure water.

[0085] Preparation of washing solution: Weigh 7g NaCl, 0.1g KH2PO4, 2g Na2HPO4·12H2O, 0.1g KCl, and 0.4mL Tween 20, and dilute to 1L with ultrapure water.

[0086] The stop solution was a 2 mol / L H2SO4 solution.

[0087] This kit undergoes destructive testing to determine the concentration changes of each component after treatment at 37°C for 3, 7, 10, and 14 days. Simultaneously, the concentration changes of each component were measured at 4°C for 1 month, 6 months, and 12 months. This kit can be stored at 4°C for one year. The diagnostic accuracy of this kit for preeclampsia is over 80%.

[0088] Example 6

[0089] This embodiment provides a method for using the ELISA kit for diagnosing preeclampsia from Embodiment 5 above, specifically including the following steps:

[0090] S1. Coating with phosphatase and tensin (PTEN) antigens: Dilute phosphatase and tensin (PTEN) with antigen coating buffer to 100 μg / ml, add 100 μL to each well of a 96-well microplate, and incubate overnight at 4°C. The next day, discard the liquid from the wells, and fill each well with washing buffer three times. After the first wash, let it stand for 2 minutes and then discard it; after the next two washes, let it stand for 1 minute each. Invert the microplate onto filter paper and blot away any remaining liquid.

[0091] S2. Sealing: Fill each well with sealing solution (about 300 μL), cover or seal the plate with sealing film, incubate in a 37°C incubator for 60 min, pour out the liquid in the well, and wash 3 times as above.

[0092] S3. Sample addition: Add 100 μL each of the antibody to be tested and the blank control (diluent) to the wells of the plate, seal the plate with sealing film, incubate at 37℃ for 60 min, and wash 5 times as above;

[0093] S4. Add enzyme-labeled antibody: Add 100 μL of HRP-labeled antibody to each well, seal the plate and incubate at 37°C for 1 h, wash 5 times as above, pat dry on filter paper and remove residual liquid;

[0094] S5. Color development and termination of reaction: Mix color development solution A and color development solution B at a 1:1 ratio in a dark environment, then add 100 μL to each well, incubate at 37°C for 20 min in the dark, and then immediately add 100 μL of 2 mol / L H2SO4 to each well to terminate the reaction. The solution immediately changes from blue to yellow.

[0095] S6. Detection: Use an ELISA reader to detect the absorbance at a wavelength of 450nm and record the data;

[0096] S7. Result determination: Calculate the positive / negative ratio (P / N) between the antibody to be tested and the blank control. When P / N is greater than 2, it is positive; when P / N is less than 2, it is negative. The positive result with the highest dilution factor is taken as the measured antibody titer.

[0097] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A serological biomarker for diagnosing preeclampsia, characterized in that, The biomarkers are phosphatase and tensin (PTEN).

2. The use of the biomarker of claim 1 in the preparation of diagnostic reagents or tools for detecting preeclampsia, or in the preparation of antibodies for detecting antiphosphatase and tensin (PTEN) in human serum.

3. The use of the detection reagent for the marker of claim 1 in the preparation of diagnostic reagents or tools for detecting preeclampsia, or in the preparation of antibodies for detecting antiphosphatase and tensin (PTEN) in human serum.

4. A reagent kit for diagnosing preeclampsia, characterized in that, Contains the phosphatase and tensin (PTEN) as described in claim 1, or a detection reagent thereof.

5. An ELISA kit for diagnosing preeclampsia, characterized in that, The plate contains the phosphatase and tensin (PTEN) as described in claim 1, an ELISA plate, a coating buffer, a blocking solution, an enzyme-labeled antibody, and a colorimetric solution.

6. The ELISA kit according to claim 5, characterized in that, The coating buffer comprises the following components: 1.0–2.0 g / L Na₂CO₃, 3–5 g / L NaHCO₃; the blocking solution comprises the following components: 3–6 g / L bovine serum albumin, 2–5 g / L casein, 5–10 g / L NaCl, 0.2–0.4 g / L KH₂PO₄, 1–3 g / L Na₂HPO₄·12H₂O, and 0.2–0.5 g / L KCl.

7. The ELISA kit according to claim 5, characterized in that, The enzyme-labeled antibody is a 0.2–1 μg / mL HRP-labeled anti-Human IgG antibody.

8. The ELISA kit according to claim 5, characterized in that, The colorimetric solution includes colorimetric agent A and colorimetric agent B; colorimetric agent A includes the following components: 22-30 g / L sodium acetate, 3-5 g / L citric acid, and 0.1-0.5 mL / L 30% hydrogen peroxide; each liter of colorimetric agent B includes the following components: 200-500 mg / L tetramethylbenzidine, 3-5 mL / L dimethyl sulfoxide, and 1-2 g / L citric acid monohydrate.

9. The ELISA kit according to any one of claims 5 to 8, characterized in that, The kit also includes a diluent, a washing solution, and a stop solution.

10. The ELISA kit according to claim 9, characterized in that, The diluent comprises the following components: 6–8 g / L NaCl, 0.1–0.3 g / L KH₂PO₄, 1–5 g / L Na₂HPO₄·12H₂O, and 0.1–0.5 g / L KCl; the washing solution comprises the following components: 6–8 g / L NaCl, 0.2–0.4 g / L KH₂PO₄, 3–5 g / L Na₂HPO₄·12H₂O, 0.2–0.4 g / L KCl, and 0.3–0.5 mL / L Tween 20; the stop solution is a 1.5–2.5 mol / L H₂SO₄ solution.

Images