WO2015039419A1 - Kit de réactif utilisé pour la prédiction ou le diagnostic précoce de troubles de l'hypertension gravidique - Google Patents

Kit de réactif utilisé pour la prédiction ou le diagnostic précoce de troubles de l'hypertension gravidique Download PDF

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WO2015039419A1
WO2015039419A1 PCT/CN2014/074071 CN2014074071W WO2015039419A1 WO 2015039419 A1 WO2015039419 A1 WO 2015039419A1 CN 2014074071 W CN2014074071 W CN 2014074071W WO 2015039419 A1 WO2015039419 A1 WO 2015039419A1
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eclampsia
pregnancy
weeks
smet
plasma
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PCT/CN2014/074071
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王雁玲
刘明
李玉侠
吉蕾
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中国科学院动物研究所
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/64—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving ketones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/723—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/321—Arterial hypertension
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to a test kit, and more particularly to a kit for predicting or early diagnosis of a pregnancy-induced hypertension disease.
  • Preeclampsia is a hypertensive disorder of pregnancy characterized by hypertension and proteinuria. The incidence of this disease is 2-7% of the pregnant population and is one of the leading causes of maternal and child mortality. Typical clinical features of pre-eclampsia can be detected by blood pressure and urine examination of hypertension and proteinuria after 20 weeks of gestation. However, before these clinical features emerged, pregnant women and their fetuses have suffered from pathological damage. Therefore, a specific early diagnosis before the onset of clinical features of pre-eclampsia allows mothers and babies to be treated in a timely and effective manner, thereby reducing the risk to mothers and babies.
  • preeclampsia The current routine examinations are not an early diagnosis of preeclampsia, and there is a clinical lack of one that can be effective before the onset of pre-eclampsia symptoms in the first trimester (6-13 weeks of gestation) or the middle (14-26 weeks of gestation).
  • the etiology of pre-eclampsia is still unclear, but it is generally believed that the placement of the placenta in the mother's uterus is one of the main causes of the disease. Therefore, predictors of preeclampsia, such as P1GF and sFlt-1, which are widely considered to have good prospects, are mostly hormones, factors or corresponding antagonists that have important functions in placental development.
  • angiogenic factors such as VEGF, P1GF
  • anti-angiogenic factors such as sFlt-1, sEndoglin
  • c-Met is a receptor tyrosine kinase located on the cell membrane.
  • the ligand for c-Met is hepatocyte growth factor (HGF), which combines to promote cell proliferation, migration and invasion.
  • HGF hepatocyte growth factor
  • the HGF/c-Met system not only acts on the development and formation of the placenta, but also filters the kidneys normally. The maintenance of function plays an important role.
  • Soluble Met (sMet) is an extracellular fragment of mature c-Met that is cleaved by proteases on the cell membrane.
  • SerpinF2 is a natural inhibitor of plasmin in the fibrinolytic system, and its high expression inhibits fibrinolysis and promotes thrombus accumulation. There is evidence that higher fibrin deposits are present in the preeclamptic placenta, a phenomenon that may reduce the supply of placental blood flow and thus correlate with preeclampsia.
  • Testosterone (To) and estradiol (E2) are the predominant forms of androgen and estrogen in the human body. In non-pregnant women, estrogen and androgen are mainly synthesized by the ovaries. During pregnancy, due to the inhibition of ovarian function, estrogen and androgen are mainly synthesized by the placenta.
  • the present invention first provides a marker for predicting or early diagnosis of a pregnancy-induced hypertension disease, the marker being selected from at least one of sMet, SerpinF2, testosterone, and estradiol.
  • the marker is sMet, and at least one of SerpinF2, testosterone or estradiol.
  • the markers are sMet, SerpinF2, testosterone and estradiol.
  • the marker is applied to the determination of related markers in plasma, serum, urine or placenta of pregnant women after 6 weeks of gestation.
  • the marker is applied to the determination of related markers in plasma, serum, urine or placenta of pregnant women after 14 weeks of gestation.
  • the marker is used for the determination of Serpin F2 protein levels and/or gene levels in the placenta.
  • the present invention also provides a kit for predicting or early diagnosis of a pregnancy-induced hypertension disease, the kit comprising an antibody that specifically binds to sMet or an agent that detects sMet.
  • the aforementioned kit also includes an antibody that specifically binds to SerpinF2 or an agent that detects SerpinF2.
  • the aforementioned kit also includes an antibody that specifically binds to testosterone or an agent that detects testosterone.
  • the aforementioned kit further includes an antibody that specifically binds to estradiol or a reagent that detects estradiol.
  • the antibody involved in the present invention may be a monoclonal antibody or a polyclonal antibody, and may also be a labeled antibody.
  • the present invention provides methods and applications for predicting the predisposition to preeclampsia or preeclampsia prior to the onset of pre-eclampsia symptoms.
  • the change in plasma sMet with gestational time in normal pregnancy and different pregnancy conditions is shown in Figure 1. After 14 weeks of gestation, plasma sMet levels were significantly lower in patients with severe pre-eclampsia or slower high-grade pre-eclampsia (similar to pre-eclampsia symptoms, but hypertensive symptoms were present before pregnancy).
  • the sMet includes a polypeptide molecule obtained by transcription and translation of a Met gene (GenBank ID: 4233) or an amino acid sequence having the same antigenicity formed by adding one or several amino acids, a coding gene thereof, and specific binding to the polypeptide. antibody.
  • the change in SerpinF2 in plasma with gestational time in preeclampsia and normal pregnancy is shown in Figure 2A. After 6 weeks of gestation, plasma SerpinF2 levels were significantly higher in patients with pre-eclampsia than in normal ones.
  • the increase in SerpinF2 protein and nucleic acid expression in the preeclamptic placenta is shown in Figure 2B.
  • the SerpinF2 includes a polypeptide molecule obtained by transcription and translation of a SerpinF2 gene (GenBank ID: 5345) or an amino acid sequence having the same antigenicity formed by adding one or several amino acids, a coding gene thereof, and specific binding to the polypeptide. antibody.
  • the increase in plasma testosterone (To) in preeclampsia cases in early pregnancy (11-14 weeks), mid-term (21-26 weeks) is shown in Figure 3A and Figure 3B, and estradiol (E2) in early pregnancy ( The reduction in the 11-14 weeks) and the medium (21-26 weeks) is shown in Figures 4A and 4B.
  • the To and E2 include the corresponding steroid hormone molecule, as well as an antibody that specifically binds to the molecule.
  • the determination of sMet, SerpinF2, testosterone, and estradiol in human plasma, serum or urine can be carried out using conventional immunoassay methods such as ELISA, RIA and the like. Other methods, such as colorimetric or chromatographic (including gas phase, liquid phase) methods, can also be used for the determination of the above molecules.
  • SerpinF2 can be assayed in human placenta using conventional molecular biology methods such as Western blotting, RT-PCR, RT-real time qPCR, and the like. Some other methods, such as ELISA, can also be used to determine the above molecules.
  • Two or more predictive indicators can be combined to predict pre-eclampsia using a formula.
  • Typical (not all) formulas are mathematical formulas and formulas. Depending on the formula used, a significantly higher or lower than the reference value is diagnosed as pre-eclampsia.
  • sMet can be used alone to diagnose pre-eclampsia or pre-eclampsia morbidity, or it can be combined with SerpinF2 or To/E2 to obtain more accurate pre-eclampsia diagnosis.
  • the various diagnostic methods described herein can also be used in conjunction with other methods to obtain more accurate pre-eclampsia or eclampsia diagnostic results.
  • levels can be independently performed at one or more time points after 6 weeks of pregnancy.
  • the test may be diagnosed as having a pre-eclampsia or having a pre-eclampsia predisposition if the overall result of the relevant level test changes compared to the control level.
  • the invention also provides a basis for the production of a diagnostic kit. If the diagnostic kit includes an antibody to sMet, or SerpinF2, or To, or ⁇ 2, whether the antibody or polypeptide of sMet, or SerpinF2, or To, or ⁇ 2 is labeled, whether sMet, or SerpinF2, or To, or ⁇ 2 Whether the antibody or polypeptide is bound by a substrate, as long as the antibody in the presence of sMet, or SerpinF2, or To, or ⁇ 2 binds to the related polypeptide and is determined by the relevant label, a diagnostic prediction can be made for the preeclampsia. Conventional ELISA detects the corresponding molecule or compound by binding the antibody-substrate to each other and can be used as a kit.
  • the present invention is the first to propose that the sMet level in plasma is significantly lower than that in the group of people with severe pre-eclampsia or predisposition to severe pre-eclampsia. Often in the crowd and several other gestational diseases.
  • the level of SerpinF2 or To/E2 in the plasma of pregnant women with preeclampsia or preeclampsia is significantly higher than that of the normal population.
  • Figure 1 shows changes in plasma sMet levels with gestational age; among them, 0: severe pre-eclampsia; 1: normal control; 2: preterm birth; 3: gestational diabetes; 4: neonatal underweight; 5: mild eclampsia Pre-stage; 6: Pregnancy-induced hypertension; 7: Chronic hypertension; 8: Mildly slow and complicated with pre-eclampsia; 9: Severely high and complicated with pre-eclampsia.
  • Figure 2 shows changes in SerpinF2 in pre-eclampsia patients and normal pregnancies; wherein, Figure A shows changes in plasma PAP (SerpinF2-plasmin complex) levels during pre-eclampsia and normal pregnancy during pregnancy, * indicates Compared with the control group, p ⁇ 0.05; ** indicates ⁇ .01 compared with the control group; B shows the expression of SerpinF2 mRNA in the placenta of preeclampsia patients and normal pregnant women (error bars indicate standard deviation); C map shows SerpinF2 Expression of protein in placenta of preeclampsia patients and normal pregnant women. * indicates p ⁇ 0.05 compared with the control group.
  • Figure A shows changes in plasma PAP (SerpinF2-plasmin complex) levels during pre-eclampsia and normal pregnancy during pregnancy, * indicates Compared with the control group, p ⁇ 0.05; ** indicates ⁇ .01 compared with the control group; B shows the expression of SerpinF2 mRNA in the placenta of pree
  • Figure 3 shows the levels of testosterone (To) in normal pregnancy and preeclampsia in the early, middle and late stages of pregnancy; wherein, Figure A shows the level of testosterone in plasma during early pregnancy (11-14 weeks); B shows the second trimester (21) -26 weeks) levels of testosterone in plasma; Panel C shows levels of testosterone in plasma during the third trimester (27-34 weeks). * indicates p ⁇ 0.05 compared with the control group; ** indicates p ⁇ 0.01 compared with the control group.
  • Figure 4 shows plasma estradiol (E2) in normal pregnancy and preeclampsia in early, middle and late pregnancy. The level of the period; wherein, Figure A shows the level of plasma estradiol in the first trimester (11-14 weeks); B shows the level of estradiol in the plasma in the second trimester (21-26 weeks); 27-34 weeks) levels of estradiol in plasma. * indicates p ⁇ 0.05 compared with the control group; ** indicates p ⁇ 0.01 compared with the control group.
  • Figure 5 is a line graph of sMet levels as a function of gestational age in each case;
  • Figure A shows the difference between normal controls, preterm birth, gestational diabetes, and neonatal underweight and severe pre-eclampsia cases; The difference between mild pre-eclampsia, pregnancy-induced hypertension, chronic hypertension, slow-high with mild pre-eclampsia, and slow-high with severe pre-eclampsia and severe pre-eclampsia.
  • 0 severe pre-eclampsia
  • 1 normal control
  • 2 premature delivery
  • 3 gestational diabetes
  • 4 neonatal underweight
  • 5 mild pre-eclampsia
  • 6 gestational hypertension
  • 7 chronic hypertension
  • 9 Slow high with severe pre-eclampsia.
  • Figure 6 shows the best cut-off point for 15-26 weeks of gestation and its resolution for severe pre-eclampsia; wherein Figure A shows the location of the best cut-off point in the overall sMet level between 15-20 and 21-26 weeks of gestation. B is the best cut-off point (284.5 ng/mL) for severe preeclampsia at 15-20 weeks of gestation; C is the best cut-off point at 21-26 weeks of gestation (295.6 ng/mL) Specific resolution of severe pre-eclampsia.
  • Figure 7 is a graph showing the effect of plasma sMet levels on severe pre-eclampsia in the cohort; where A plots the mean level of plasma sMet in normal cases and severe pre-eclampsia cases in the cohort, and error bars indicate mean standard error (SEM) B is a scatter plot showing the overlap of plasma sMet levels in severe pre-eclampsia cases with plasma sMet levels in normal cases; C is the ROC curve and its AUC value for distinguishing severe pre-eclampsia cases and control cases; The figure shows the optimal cut-off point (284.5 ng/mL) at 15-20 weeks of gestation in the training cohort for the specific resolution of severe pre-eclampsia in the validation cohort.
  • SEM mean standard error
  • Figure 8 shows the ratio of testosterone to estradiol in plasma (To/E2) during normal pregnancy and preeclampsia at early, middle and late stages of pregnancy; wherein, Figure A shows To/E2 in plasma during early pregnancy (11-14 weeks) Level B; Figure shows the level of To/E2 in plasma during the second trimester (21-26 weeks); Shows the level of To/E2 in plasma in the third trimester (27-34 weeks). * indicates p ⁇ 0.05 compared with the control group; ** indicates p ⁇ 0.01 compared with the control group.
  • Figure 9 shows the effect of the ratio of testosterone to estradiol in plasma (To/E2) on severe pre-eclampsia and normal pregnancy, which is expressed as the ROC curve and its AUC value; wherein, Figure A shows early pregnancy (11-14 weeks) B shows the second trimester (21-26 weeks); C shows the third trimester (27-34 weeks).
  • the experiment can be divided into two parts: training-validation.
  • the training training cohort included 3,336 Chinese Han pregnant women. Plasma levels of sMet were measured in these pregnant women at 6-10, 11-14, 15-20, 21-26, 27-34 weeks, and >34 weeks of gestation.
  • the validation study cohort included 62 Chinese Han women with normal pregnancy and 62 Chinese Han women with preeclampsia. The levels of plasma sMet in these pregnant women at 14-20 weeks of gestation were determined.
  • the prospective cohort involved in the training institute was from the prenatal care group of Peking University Third Hospital from June 2009 to December 2010.
  • the population included 3,336 pregnant women, of whom 194 had multiple pregnancies, 5 were younger than 18 or older, 23 were delivered before 28 weeks of gestation, and 784 had other diseases unrelated to hypertension. All of the above cases were excluded from the study. Of the remaining 2330 cases, 1911 were normal pregnancy, 78 were severe pre-eclampsia, 77 were mild pre-eclampsia, 102 were premature, and 21 were gestational sugar. Urine disease, 11 cases of fetal growth restriction, 32 cases of pregnancy-induced hypertension, 41 cases of chronic hypertension, 57 cases of slow high with pre-eclampsia.
  • Pregnant women in this population collected 2 mL of peripheral blood and obtained plasma at 6-10, 11-14, 15-20, 21-26, 27-34 weeks, and >34 weeks of gestation.
  • the harvested plasma was stored at -80 °C for 12 hours after blood collection.
  • the prospective cohort involved in the validation study was from the pregnant women who underwent Down's screening at the Beijing First Hospital and Beijing Miyun Hospital from July 2009 to September 2011.
  • the population consisted of 2,455 pregnant women, and blood samples from all pregnant women were obtained at 14-20 weeks of gestation.
  • the harvested plasma was stored at -80 °C for 12 hours after blood collection.
  • 62 normal pregnancy controls and 62 severe preeclampsia cases were randomly selected for sMet testing.
  • pregnancy syndrome is defined by the definition of Williams Obstetrics (23 edition) and the International Association of Pregnancy and Hypertension. Cases with kidney disease or fetal chromosomal abnormalities are outside the scope of this study. Normal pregnancy cases are defined as cases of physical health before pregnancy, no obvious symptoms during pregnancy, delivery after 37 weeks of gestation, and normal weight of newborns. Severe pre-eclampsia refers to a history of chronic hypertension, but systolic blood pressure > 160 mmHg or diastolic blood pressure > llO mmHg in two independent examinations after 20 weeks of gestation, or accompanied by significant proteinuria (>2 g/24h or The independent test strips were determined to be 3+) at intervals of more than 4 hours.
  • Mild pre-eclampsia refers to systolic blood pressure between 140-160 mmHg or diastolic blood pressure between 90-110 mmHg in two independent examinations after 20 weeks of gestation, accompanied by 300 mg-2 g/24h or 2
  • the secondary test strips were greater than 4 hours and the independent test strips were determined to be 2+ proteinuria.
  • Early and late preeclampsia is defined primarily by whether the clinical symptoms of pre-eclampsia occur before the 34th week of gestation.
  • Pregnancy-induced hypertension is defined as a blood pressure that occurs after 20 weeks of gestation above 140/90 mmHg, but is not accompanied by proteinuria.
  • Chronic hypertension refers to hypertension that occurs before pregnancy and lasts until pregnancy.
  • Chronic high pre-eclampsia refers to chronic hypertension and pre-eclampsia comorbidities, and can be further divided into severe and mild groups according to whether proteinuria is >2 g/24h.
  • Preterm birth is defined as a delivery that is earlier than 37 weeks.
  • Fetal growth restriction is defined as the neonatal birth weight being less than the 10th percentile of the corresponding week.
  • Gestational diabetes is defined as a condition in which blood glucose is normal during pregnancy and hyperglycemia during pregnancy, but is not accompanied by high blood pressure. All cases of preterm birth and fetal growth restriction are not accompanied by hypertension or diabetes. 3. Measurement of sMet7] in plasma
  • the level of sMet in plasma was measured by ELISA and all operations were performed according to the instructions of the ELI S A test reagent produced by R&D. The surveyor did not know in advance the specifics of the sample being tested. All samples were duplicated and the sample concentration was calculated based on the absorbance of the sample and standard at a wavelength of 450 nm. The ELISA assay was repeated a total of 3 times and the final result was the average of 3 experiments.
  • sMet Personnel performing mathematical statistics do not participate in the measurement process of sMet.
  • the Mann-Whitney U test was used to analyze differences in sMet levels between normal pregnancy and the following cases, such as preterm birth, gestational diabetes, fetal growth restriction, gestational hypertension, chronic hypertension, slow high with mild pre-eclampsia, slow High combined with severe pre-eclampsia, mild pre-eclampsia, and severe pre-eclampsia. All tests were two-tailed and PO.05 considered the difference to be significant.
  • ROC analysis was used to test the predictive value of plasma sMet levels for pre-eclampsia.
  • the ROC curve takes each measured sMet level as a cut-off point, and continuously calculates the corresponding 1-specificity and sensitivity, and plots the plot.
  • the optimal cut-off point is defined as the value of the sum of specificity and sensitivity. All analyses were done using the R package.
  • the group of patients with hypertension including pregnancy-induced hypertension, chronic hypertension, slow-high with mild pre-eclampsia, slow-high with severe pre-eclampsia, mild pre-eclampsia, and severe pre-eclampsia, Systolic and diastolic blood pressures were significantly increased compared to controls.
  • Urine protein was only detected in patients with pre-eclampsia, including slow-high with mild pre-eclampsia, slow-high with severe pre-eclampsia, mild pre-eclampsia, and severe pre-eclampsia.
  • the pregnancy time in the normal pregnancy group was significantly longer than in the case of comorbidities. Except for cases of gestational diabetes and cases of pregnancy-induced hypertension, the birth weight of newborns with comorbidities was lower than that of normal pregnancy. Glucose tolerance did not change significantly between groups.
  • the samples from the training experiments were from 120 randomly selected normal pregnancies in the cohort of Clause 2 above, as well as all preterm birth, gestational diabetes, fetal growth restriction, gestational hypertension, chronic hypertension, slow high with mild pre-eclampsia, Slow high combined with severe pre-eclampsia, mild pre-eclampsia, and severe pre-eclampsia.
  • Plasma was measured by sMet7] in each case at 6-10, 11-14, 15-20, 21-26, 27-34, and >34 weeks of gestation. The results are shown in Figure 1. In normal pregnancy, the average plasma sMet level is as high as 566 ng/mL at 6-10 weeks of gestation.
  • Plasma sMet levels gradually decrease, and decrease from 15-20 weeks of gestation. To 343 ng/mL; however, at 21-26 weeks of gestation, plasma sMet7] had a distinct peak, reaching 460 ng/mL; it gradually decreased during the subsequent gestation period and decreased to 280 ng/mL before delivery.
  • Plasma sMet levels in preterm, gestational diabetes, fetal growth restriction, gestational hypertension, chronic hypertension, chronic hypertonic mild preeclampsia, and mild preeclampsia are similar to normal pregnancy (Figure 1).
  • Plasma sMet levels in patients with severe pre-eclampsia and slow-high and severe pre-eclampsia continued to decrease from 6-10 weeks of gestation until delivery, and there was no similar normal pregnancy group at 21-26 weeks of gestation. The peak. Plasma sMet levels in patients with severe pre-eclampsia and slow-high and severe pre-eclampsia were significantly lower than those in normal pregnancy and other individual cases from 11-14 weeks of gestation until delivery.
  • the present invention summarizes a line graph of sMet levels as a function of gestational age in each case, as shown in FIG.
  • the sMet level showed a gradual decrease with increasing pregnancy time, and compared with normal pregnancy, premature delivery at 20-27 weeks of gestation.
  • the present invention further utilizes ROC analysis to detect sMet's ability to predict and diagnose severe pre-eclampsia.
  • ROC analysis to detect sMet's ability to predict and diagnose severe pre-eclampsia.
  • Table 3 it can be seen from the AUC value that after 15 weeks of gestation, plasma sMet levels can effectively distinguish between severe pre-eclampsia and slow-high with severe pre-eclampsia and various other pregnancy conditions, including normal pregnancy and preterm birth, pregnancy. Diabetes, fetal growth restriction, gestational hypertension, chronic hypertension, mild pre-eclampsia, slow high and mild pre-eclampsia.
  • a ROC analysis between the two groups was performed to determine the optimal cut-off point for sMet levels at 29-26 weeks of gestation was 295.6 ng/ml, at which time the sensitivity, specificity, and positive predictive value of "severe pre-eclampsia" were predicted.
  • the negative predictive value and AUC were 1.00, 0.995, 0.980, 1.00, and 0.998, respectively.
  • the best cut-off point for sMet levels at 28-20 weeks of gestation was 284.5 ng/mL, at which time the sensitivity of "severe pre-eclampsia” was predicted.
  • the specificity, positive predictive value, negative predictive value, and AUC were 0.948, 0.935, 0.798, 0.988, and 0.980, respectively.
  • Verification Experiment 1 detects the predictive potential of plasma sMet levels in severe preeclampsia in an independent cohort
  • the present invention selected a validation cohort independent of the training cohort at Peking University First Hospital and Beijing Miyun Hospital, and plasma samples of the cases were collected during the 14-20 weeks of gestation.
  • this cohort 62 cases of normal pregnancy and 62 cases of severe pre-eclampsia were randomly selected according to case-control analysis, and plasma sMet levels were measured.
  • the mean plasma sMet level in severe pre-eclampsia cases was 215.74 ng/mL, which was significantly lower than 320.87 ng/mL in normal pregnancy (Fig. 7A); the scatter plot showed plasma sMet levels in severe preeclampsia cases.
  • Fig. 7B There was a small overlap between the normal pregnancies (Fig. 7B); ROC analysis showed that the AUC values for the severe preeclampsia and normal pregnancy groups reached 0.986 (Fig. 7C); the best cutoff point summed up in the applied training study When 284.5 ng/mL was segmented, there were 11 (8.87%) false positive cases and 1 (0.81%) false negative cases (Fig. 7D), and the predicted accuracy was 0.903.
  • the above results correspond well to the results obtained in the training queue at 15-20 weeks.
  • This study used a prospective nested case-control study that was approved by the Institute of Zoology of the Chinese Academy of Sciences and the Ethics Committee of the First Hospital of Peking University.
  • the subjects were pregnant women who underwent antenatal care at the First Hospital of Peking University from November 2005 to December 2007.
  • the peripheral blood of pregnant women was collected at different stages of pregnancy. After delivery, the subjects were divided into normal pregnancy groups according to the pregnancy outcome. And severe pre-eclampsia group.
  • Pre-pregnancy indicators are normal, no pre-eclampsia clinical indicators such as hypertension, proteinuria during pregnancy Signs and other pregnancies; severe preeclampsia is defined according to the International Society for Pregnancy and Hypertension: no pre-pregnancy, no history of proteinuria, systolic blood pressure > 160 mmHg and/or diastolic blood pressure 110 > mmHg after 20 weeks of gestation, With 24-hour proteinuria > 2g or qualitatively above ++.
  • SerpinF2 in plasma usually binds to plasmin Plasmin to form complex PAP, and SerpinF2, which binds to Plasmin, is a functional component, and thus the concentration of PAP complex reflects the concentration of SerpinF2.
  • the PAP concentration was measured using a human PAP ELISA kit (purchased from Wuhan EIAab Science Co., Ltd.). The specific steps are as described in the kit. The microplate was measured at a wavelength of 450 nm.
  • ROC analysis of plasma PAP concentration determination of plasma PAP level by calculating the AUC, accuracy, sensitivity, and cut-off value of each stage of pregnancy
  • Predictive value of preeclampsia (Table 4). The results indicate that plasma PAP levels are ideal predictors of severe preeclampsia and have good predictive value at all stages of pregnancy, especially at 6-14 weeks and 15-18 weeks of pregnancy, with a higher AUC value of 0.937. With 0.804, the optimal cutoff values were 0.117 g/mL and 1.04 g/mL, respectively.
  • the prospective cohort involved in the study was from the prenatal care population of the Third Hospital of Beijing University from June 2009 to December 2010.
  • the population included 3,336 pregnant women, of whom 194 were multiple pregnancies, 5 were younger than 18 years of age or older, 23 were delivered before 28 weeks of gestation, and 784 had other diseases unrelated to hypertension. All of the above cases were excluded from the study. Of the remaining 2330 cases, 1911 were normal pregnancy, 78 were severe pre-eclampsia, and the rest were other pregnancy complications.
  • 2 mL of peripheral blood was collected and blood plasma was obtained at 6-10, 11-14, 15-20, 21-26, 27-34 weeks, and >34 weeks of gestation. The collected plasma was stored at -80 °C within 12 hours after the blood was taken.
  • the ROC curve was plotted against plasma To/E2, and the predictive value of plasma To/E2 levels for pre-eclampsia was determined by calculating the AUC values at various stages of pregnancy (Fig. 9).
  • the results indicate that plasma To/E2 levels are of value in predicting severe pre-eclampsia, with AUC values of 0.749 and 0.779 at 21-26 weeks and 27-34 weeks of gestation, respectively ( Figure 9B, 9C), which can be used as predictors or diagnostics for severe neutrons.
  • a powerful auxiliary indicator for pre-eclampsia is of value in predicting severe pre-eclampsia.
  • kits for predicting or early diagnosis of a pregnancy-induced hypertension disease is provided for the determination of a marker in plasma, serum, urine or placenta of a pregnant woman after 6 weeks of gestation, the marker being selected from the group consisting of sMet At least one of SerpinF2, testosterone, and estradiol.
  • Table 1 (training population, including normal pregnancy, premature delivery, gestational diabetes, fetal growth restriction, gestational hypertension, chronic hypertension, slow high with mild pre-eclampsia, slow high with severe pre-eclampsia, mild eclampsia And the basic characteristics of severe pre-eclampsia cases ⁇ )
  • A The value in the table indicates the mean standard deviation; the P value only indicates a value less than 0.05.
  • ⁇ : BMI is the weight (kg) divided by the height (m) squared.
  • ⁇ Patients with gestational diabetes do not include 50g glucose tolerance data.
  • Urinary protein can only be detected in patients with mild high and mild pre-eclampsia, slow high with severe pre-eclampsia, mild pre-eclampsia, and severe pre-eclampsia.
  • Table 2 Validation population (including basic characteristics of normal pregnancy and severe pre-eclampsia cases A )
  • Urine protein (g/24h) ⁇ - 3.2 ⁇ 4.0
  • Neonatal weight 3316 ⁇ 486 2770 ⁇ 857 (P ⁇ 0.001)
  • ⁇ The values in the table indicate the mean ⁇ standard deviation; the P value is only marked with a value less than 0.05.
  • ⁇ : BMI is the weight (kg) divided by the height (m) squared.
  • ⁇ The value in parentheses in the table indicates a 95% confidence interval. Some AUC values of less than 0.5 at 6-10 weeks or 11-15 weeks are due to the fact that the average sMet level for this group is less than the severe pre-eclampsia group at this time.

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Abstract

La présente invention concerne un kit de réactif pour la prédiction ou le diagnostic précoce de troubles de l'hypertension gravidique. Le kit de réactif est utilisé pour la mesure d'un marqueur dans le plasma, le sérum, l'urine ou le placenta d'une femme à six semaines de grossesse, le marqueur étant au moins un marqueur choisi parmi sMet, SerpinF2, la testostérone et l'œstradiol.
PCT/CN2014/074071 2013-09-23 2014-03-26 Kit de réactif utilisé pour la prédiction ou le diagnostic précoce de troubles de l'hypertension gravidique WO2015039419A1 (fr)

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CN103513042A (zh) * 2013-09-23 2014-01-15 中国科学院动物研究所 用于预测或早期诊断妊娠高血压疾病的试剂盒
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WO2021073518A1 (fr) * 2019-10-17 2021-04-22 中国科学院动物研究所 Utilisation d'un inhibiteur d'un facteur inhibiteur de la plasmine pour prévenir ou traiter la prééclampsie ou l'éclampsie
CN117677715A (zh) * 2021-07-28 2024-03-08 北京沙东生物技术有限公司 一种骨髓瘤生物标志物serpinf2及其应用
CN116953255A (zh) * 2023-07-27 2023-10-27 山东大学 血清中总IgM和/或总IgG在子痫前期预测或诊断中的应用

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