Application of C1INH in preparation of product for prediction or auxiliary diagnosis of preeclampsia and product

Abstract

The invention discloses application of C1INH in preparation of a product for prediction or auxiliary diagnosis of preeclampsia and the product, belongs to the technical field of biological medicines, and aims to detect the content of C1INH in a blood sample on a protein level by taking the blood sample of a subject as a detection object. According to the invention, C1INH molecules in blood are taken as a detection index, prediction and auxiliary diagnosis of preeclampsia of pregnant women can be realized, high sensitivity and specificity are realized, rapid detection can be realized, and a new way is provided for early screening and diagnosis of preeclampsia in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application and products of C1INH in the preparation of products for predicting or assisting in the diagnosis of preeclampsia. Background Technology

[0002] Preeclampsia refers to the onset of high blood pressure and proteinuria after 20 weeks of gestation, accompanied by symptoms such as headache, blurred vision, nausea, vomiting, and upper abdominal discomfort. Eclampsia develops from preeclampsia into more severe symptoms, causing seizures or coma. The causes of both preeclampsia and eclampsia remain unclear, but they can lead to serious maternal and fetal complications, and there is no effective treatment other than termination of pregnancy. Therefore, the diagnosis and assessment of preeclampsia in pregnant women are crucial.

[0003] Because the symptoms of preeclampsia vary greatly in clinical diagnosis, it is often necessary to differentiate it from other diseases, including kidney disease, chronic hypertension, immune disorders, and liver disease. Furthermore, to diagnose preeclampsia, it is essential to assess the condition of both the pregnant woman and the fetus, including physical examination, liver and kidney function tests, routine blood and urine tests, coagulation parameters, and evaluation of the fetus's intrauterine condition. In some cases, further examinations such as head CT scans, electrocardiograms, and echocardiograms may be required. Therefore, the clinical diagnosis of preeclampsia currently relies on a combination of medical history and other functional examinations, with the diagnosis made by a physician based on the test results.

[0004] Currently, there are few reports on diagnostic biomarkers for preeclampsia. Fu Yao et al., in their study "The Role of Cathepsin B in the Pathogenesis of Preeclampsia and Eclampsia" (Chinese Journal of Maternal and Child Health, 2011, Issue 20), used enzyme-linked immunosorbent assay (ELISA) to detect serum cathepsin B (CB) levels in normal late-pregnancy women and patients with preeclampsia and eclampsia, and immunohistochemistry to determine CB expression in the placenta of 40 of these cases. The results showed that serum CB levels in patients with preeclampsia and eclampsia were not significantly different from those in the normal late-pregnancy group (P>0.05). However, placental CB expression in the PE group was significantly increased compared to the normal late-pregnancy group (P<0.05), and was positively correlated with the severity of the disease. Therefore, increased CB expression in the placenta of PE patients may be one of the pathogenic mechanisms of PE and eclampsia. In addition, Chinese patent CN112305220A also discloses the use of cathepsin Z (CTSZ) as a detection indicator for preeclampsia. Since the content of cathepsin Z in the peripheral blood of pregnant women with preeclampsia is significantly higher than that in healthy pregnant women, the prediction and diagnosis of preeclampsia can be quickly obtained by detecting the changes in the content of cathepsin Z in the blood samples of the subjects.

[0005] C1 inhibitor (C1INH) is a protein molecule in the blood whose main functions include inhibiting excessive activation of the complement system, regulating the coagulation and fibrinolytic systems, and playing an important role in the kallikrein-kinin system. It is also currently used as a protein biomarker for the diagnosis of gastric cancer. However, no research or reports have been found regarding C1INH in the prediction and auxiliary diagnosis of preeclampsia. Summary of the Invention

[0006] The purpose of this invention is to provide the application and products of C1INH in the preparation of products for predicting or assisting in the diagnosis of preeclampsia. Using C1INH molecules in the blood as a detection indicator, it can realize the prediction and assist in the diagnosis of preeclampsia in pregnant women. It has high sensitivity and specificity, and can achieve rapid detection, providing a new approach for the early screening and diagnosis of preeclampsia.

[0007] This invention is achieved through the following technical solution: the application of C1INH in the preparation of products for predicting or assisting in the diagnosis of preeclampsia, wherein the product uses a blood sample of a subject as the detection object and detects the content of C1INH in the blood sample at the protein level.

[0008] The subjects were pregnant women in the early or mid-stage of pregnancy, with the early stage of pregnancy referring to the period before the end of the 13th week of pregnancy and the mid-stage of pregnancy referring to the period from the 14th to the end of the 27th week of pregnancy.

[0009] The blood sample may include whole blood, serum, or plasma.

[0010] During the test, when the concentration of C1INH in the blood sample is higher than 43 μg / ml, it is predicted that the subject may develop preeclampsia after 20 weeks of gestation.

[0011] The detection methods include immunofluorescence assay, ELISA, biochip assay, and electrochemiluminescence assay.

[0012] Furthermore, the present invention also provides a product for predicting or assisting in the diagnosis of preeclampsia, containing a substance for detecting C1INH levels in a blood sample, the substance detecting the amount of C1INH in the subject's blood sample at the protein level.

[0013] The products include reagent kits, diagnostic reagents, or other medically feasible products.

[0014] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0015] (1) This invention uses C1INH molecules in the blood of the subject as a detection indicator. By measuring the C1INH level of pregnant women in the early or mid-pregnancy period, it can predict whether the pregnant woman is likely to develop preeclampsia after 20 weeks of gestation. This provides a new biological marker for the early screening and auxiliary diagnosis of preeclampsia and expands the development and research of subsequent blood test products or other diagnostic tools related to preeclampsia.

[0016] (2) The C1INH detected by this invention is a serine protease inhibitor. Its content can be detected by blood and the detection results can be obtained by known detection methods such as immunofluorescence analysis, ELISA, biochip method, electrochemiluminescence method, etc., which can achieve rapid detection with high sensitivity and high precision. Attached Figure Description

[0017] Figure 1 This is a standard curve of A450 value versus C1INH concentration involved in the detection of this invention.

[0018] Figure 2 This is a scatter plot of C1INH concentrations in samples from pregnant women in the early, middle, and late stages of pregnancy, as used in the detection process of this invention. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0020] Example 1: Preparation of a kit for predicting or assisting in the diagnosis of preeclampsia by detecting C1INH

[0021] Reagent preparation: Carbonate buffer at pH 9; Carbonate buffer at pH 9.6 with a concentration of 1 mol / L; Anhydrous methanol; Bovine serum albumin solution; Phosphate buffer at pH 7.2 with a concentration of 0.05 mol / L; Dimethylformamide; Biotinylate-N-hydroxysuccinimide; Avidin-horseradish peroxidase; 3',3',5,5'-Tetramethylbenzidine; C1INH protein standard; Sample diluent.

[0022] Instrument preparation: constant temperature chamber; magnetic stirrer; precision pipette; microplate reader capable of measuring absorbance at 450nm.

[0023] The specific preparation steps are as follows:

[0024] (1) Prepare an ELISA plate coated with mouse anti-human C1INH monoclonal antibody (purchased from Abcam, catalog number ab53365).

[0025] The mouse anti-human C1INH monoclonal antibody was diluted to a concentration of 10 μg / mL using carbonate buffer at pH 9. Anhydrous methanol was added to the antibody dilution, with the amount of anhydrous methanol limited to a concentration of 3 mL / 100 mL. Then, 100 μL of the methanol-containing mouse anti-human C1INH monoclonal antibody dilution was added to each well of a 96-well plate, and the plate was coated at 4°C for 12 hours. After the coating time was up, 120 μL of 2 g / 100 mL bovine serum albumin solution was added to each well of the multi-well plate, and the plate was blocked at 37°C for 1 hour. After the blocking reaction, the multi-well plate was washed with phosphate buffer at pH 7.2 and a concentration of 0.05 mol / L. Once the unreacted material on the multi-well plate was removed, the multi-well plate coated with mouse anti-human C1INH monoclonal antibody was obtained and stored at 4°C for later use.

[0026] (2) Preparation of biotin-labeled rabbit anti-human C1INH polyclonal antibody (purchased from: Abcam, catalog number ab244029) (Biotin-C1INH Ab) detection solution.

[0027] A 50 μg / μL solution of acyl-N-hydroxysuccinimide was prepared using N,N-dimethylformamide as solvent and biotinylate-N-hydroxysuccinimide as solute. A 24 mg / mL solution of rabbit anti-human C1INH polyclonal antibody was prepared using 1 mol / L carbonate buffer (pH 9.6) as solvent and rabbit anti-human C1INH polyclonal antibody as solute. The acyl-N-hydroxysuccinimide and rabbit anti-human C1INH polyclonal antibody solutions were mixed at a mass ratio of 1:7. The mixture was then reacted at room temperature for 4 hours with stirring (magnetic stirrer, 200 rpm) to obtain the biotin-labeled rabbit anti-human C1INH monoclonal antibody (Biotin-C1INH). After the reaction, the reaction solution containing biotin-labeled rabbit anti-human C1INH polyclonal antibody (Biotin-C1INH Ab) was placed into a dialysis bag and dialyzed at 4°C with 0.05 mol / L phosphate buffer at pH 9.2 for 12 hours, during which the dialysate was changed 3 times. Then, bovine serum albumin was added to the dialyzed reaction solution, with the amount of bovine serum albumin added limited to a concentration of 4 g / 100 mL.

[0028] The biotin-labeled rabbit anti-human C1INH polyclonal antibody (Biotin-C1INH Ab) prepared by the above process and the post-dialysis reaction solution of bovine serum albumin were mixed with glycerol at a volume ratio of 1:1 at room temperature to form the biotin-labeled rabbit anti-human C1INH polyclonal antibody (Biotin-C1INH Ab) detection solution, which was stored at -20℃.

[0029] (3) Each kit contains 12 mL of avidin-horseradish peroxidase, 12 mL of chromogenic substrate 3',3',5,5'-tetramethylbenzidine, 1 mL of C1INH protein standard (C1INH concentration is 600 μg / mL) and 30 mL of sample diluent. The sample diluent is prepared by adding bovine serum albumin to 0.02 mol / L tris(hydroxymethyl)aminomethane buffer (Tris buffer, TBS). The amount of bovine serum albumin added is limited to a concentration of 2 g / 100 mL. The volume ratio of avidin-horseradish peroxidase to biotin-labeled rabbit anti-human C1INH polyclonal antibody detection solution is 1:1.

[0030] Example 2: Detection of C1INH levels in the test sample

[0031] This embodiment describes the process of detecting C1INH levels in test samples using the preeclampsia prediction or auxiliary diagnostic kit prepared in Example 1.

[0032] The samples to be tested were taken from 80 outpatient and inpatient cases at West China Second Hospital of Sichuan University.

[0033] The inclusion criteria for the sample were: pregnant women who had registered for prenatal care at our hospital, and the researchers fully explained the project content to the participants. Enrollment was voluntary, and participants signed informed consent forms before being enrolled. 5-10 ml of peripheral venous blood and placental tissue discarded during delivery or cesarean section were collected from each participant. Preeclampsia patients were matched with suitable healthy pregnant controls at the same time. The control group included 38 participants, and the case group included 42 participants.

[0034] Specific testing methods:

[0035] 1. Sample to be tested

[0036] (1) Preparation of standard samples

[0037] The C1INH protein standard provided in the kit described in Example 1 was set as the standard sample with the highest C1INH concentration and named Standard Sample 1, with a C1INH concentration of 600 μg / mL. Then, the C1INH protein standard was diluted with the sample diluent provided in the kit described in Example 1 to prepare standard samples with C1INH concentrations of 600 μg / mL, 240 μg / mL, 96 μg / mL, 38.4 μg / mL, 15.36 μg / mL, 6.144 μg / mL, and 2.458 μg / mL, respectively, and named Standard Sample 1, Standard Sample 2, Standard Sample 3, Standard Sample 4, Standard Sample 5, Standard Sample 6, and Standard Sample 7, for a total of 7 standard samples.

[0038] (2) Sample

[0039] This study included female outpatients and inpatients at West China Second University Hospital of Sichuan University. Based on existing diagnostic methods, participants were divided into a case group and a healthy control group. Peripheral venous blood samples were collected from both groups, totaling 80 samples. Samples 1-38 were collected from the healthy control group, and samples 39-80 were collected from the case group. The healthy control group included 11 pregnant women in early pregnancy (before the end of the 13th week of gestation), designated as samples 1-11; 11 pregnant women in mid-pregnancy (14 to 27 weeks of gestation), designated as samples 12-22; and 16 pregnant women in late pregnancy (after 28 weeks of gestation), designated as samples 23-38. The case group included 18 pregnant women in early pregnancy (before the end of the 13th week of gestation), designated as samples 39-56; 18 pregnant women in mid-pregnancy (14 to 27 weeks of gestation), designated as samples 57-74; and 6 pregnant women in late pregnancy (after 28 weeks of gestation), designated as samples 75-80.

[0040] (3) Blank control

[0041] The sample diluent provided in the kit described in Example 1 is a blank control.

[0042] 2. Sample testing

[0043] (1) Before testing, place all reagents and samples at room temperature (18-25℃).

[0044] (2) Add the standard, blank control, and sample prepared in step 1 to the corresponding wells of a 96-well plate coated with mouse anti-human C1INH monoclonal antibody. Each standard, blank control, and sample should be replicated. The volume of sample added to each well is 100 μL. Cover the 96-well plate with a gel film and place it on a shaker to incubate at room temperature for 2.5 hours with shaking at a speed of 100 rpm.

[0045] (3) After the incubation time is up, aspirate the liquid from each well and set the cleaning program on the automatic plate washer. Use 400 μL / well washing buffer (0.02 mol / L phosphate buffer or 0.02 mol / L Tris buffer) to wash each well twice. Each time, the washing buffer should remain in the well for 10 to 15 seconds. After cleaning, flip the 96-well plate so that the well openings are facing down and gently tap the plate on absorbent filter paper to completely remove any remaining washing buffer from the wells.

[0046] (4) Add 100 μL of biotinylated antibody (Biotin-C1INH Ab) detection solution to each well of the 96-well plate, then cover the 96-well plate with a film and place the 96-well plate on a shaker and incubate at room temperature for 1.5 hours under shaking. After the incubation time is up, aspirate the liquid from each well and clean the 96-well plate according to the method in step (3).

[0047] (5) Add 100 μL of avidin-horseradish peroxidase to each well of the 96-well plate, then cover the 96-well plate with a film and place the 96-well plate on a shaker and incubate at room temperature for 1.5 hours under shaking. After the incubation time is up, aspirate the liquid from each well and clean the 96-well plate according to the method in step (3).

[0048] (6) Add 100 μL of 3',3',5,5'-tetramethylbenzidine to each well of the 96-well plate, and then incubate at room temperature in the dark for 10 minutes. After the incubation time is up, add 100 μL of stop solution to each well. The stop solution is an aqueous solution of sulfuric acid and sodium sulfite, with a sulfuric acid concentration of 2 mol / L and a sodium sulfite concentration of 0.1 mol / L.

[0049] (7) Place the 96-well plate in a microplate reader and measure the A450 value (optical density or absorbance) of each well. The results are shown in Tables 1 to 3 below (the A450 value in the table is the weighted average of two wells). The A450 value of the blank control is 0.066.

[0050] Table 1: A450 values ​​of standard samples

[0051] Standard sample 1 2 3 4 5 6 7 A450 value 1.098 0.941 0.694 0.473 0.262 0.152 0.102

[0052] Table 2: A450 values ​​of samples 1-38

[0053]

[0054]

[0055] Table 3: A450 values ​​of samples 39-80

[0056] Sample 39 40 41 42 43 44 45 A450 value 0.372 0.329 0.411 0.378 0.412 0.339 0.359 Sample 46 47 48 49 50 51 52 A450 value 0.456 0.386 0.434 0.558 0.554 0.314 0.414 Sample 53 54 55 56 57 58 59 A450 value 0.333 0.350 0.371 0.581 0.345 0.372 0.383 Sample 60 61 62 63 64 65 66 A450 value 0.398 0.299 0.322 0.338 0.317 0.364 0.381 Sample 67 68 69 70 71 72 73 A450 value 0.544 0.393 0.391 0.407 0.350 0.295 0.282 Sample 74 75 76 77 78 79 80 A450 value 0.496 0.190 0.199 0.663 0.163 0.156 0.191

[0057] (8) Based on the A450 value and C1INH concentration of each standard sample, construct an A450 value-C1INH concentration standard curve (see [reference]). Figure 1 The calculation formula is y = 0.0017x + 0.2012, where y is the A450 value of the sample and x is the C1INH content in the sample.

[0058] (9) Based on the measured A450 value of each sample, the concentration of C1INH in each sample is calculated using the formula y=((max / (x-min)-1)^(-1 / Hillslope))*EC50 (see Table 6). The calculation results are shown in Table 4 and Table 5 below.

[0059] Table 4: Calculation results for samples 1-38 (C1INH concentration unit: μg / mL)

[0060]

[0061]

[0062] Table 5: Calculation results for samples 39-80 (ClINH concentration unit: μg / mL)

[0063]

[0064] Table 6: Formulas and parameters for the four-parameter method

[0065]

[0066] The average C1INH concentrations for samples 1-11 from early pregnancy (Table 4) were calculated to be 44.56 ± 13.54 μg / mL; for samples 12-22 from mid-pregnancy (Table 4), the average C1INH concentration was 14.72 ± 6.42 μg / mL; and for samples 23-38 from late pregnancy (Table 5), the average C1INH concentration was 12.48 ± 5.74 μg / mL. The average C1INH concentrations for samples 39-56 from early pregnancy (Table 5) were calculated to be 66.25 ± 26.02 μg / mL; for samples 57-74 from mid-pregnancy (Table 5), the average C1INH concentration was 55.23 ± 18.95 μg / mL; and for samples 75-80 from late pregnancy (Table 5), the average C1INH concentration was 41.79 ± 63.17 μg / mL.

[0067] The calculation results show that the average C1INH concentration in samples 1-11 from the healthy control group of pregnant women in early pregnancy was significantly different from that in samples 39-56 from the case group of pregnant women in early pregnancy. The average C1INH concentration in samples 12-22 from the healthy control group of pregnant women in mid-pregnancy was significantly different from that in samples 57-74 from the case group of pregnant women in mid-pregnancy. However, there was no significant difference in the average C1INH concentration in samples 23-38 from the healthy control group of pregnant women in late pregnancy compared to that in samples 75-80 from the case group of pregnant women in late pregnancy.

[0068] The test results were analyzed using a t-test between group samples. See [link to t-test analysis]. Figure 2 The results showed that the average C1INH concentration in samples from pregnant women in the early and mid-pregnancy stages of the case group was significantly higher than that in the healthy control group, while the average C1INH concentration in samples from pregnant women in the late pregnancy stages of the case group was not different from that in the healthy control group. This demonstrates that the kit described in this invention can be used to predict the probability of preeclampsia in pregnant women in the early and mid-pregnancy stages.

[0069] For pregnant women in the first and second trimesters, the detection limit is set based on the ROC curve calculation results of the sample. Therefore, the detection limit for C1INH in the kit described in this invention is set to 43 μg / mL. That is, a C1INH concentration higher than 43 μg / mL in the test sample is considered positive, indicating the possibility of preeclampsia or a high probability of preeclampsia. A C1INH concentration lower than 43 μg / mL is considered negative, indicating that the woman does not have preeclampsia.

[0070] Example 3: Specificity and Sensitivity Test

[0071] This embodiment is a test of the specificity and sensitivity of the kit described in Example 1 for the prediction or auxiliary diagnosis of preeclampsia.

[0072] (1) Specificity

[0073] Source of test samples: The experimental subjects were women from the outpatient and inpatient departments of West China Second Hospital of Sichuan University. According to the existing diagnostic methods, the experimental subjects were divided into case group and control group. Anticoagulated blood samples were collected from both groups as test samples, totaling 58 samples. Among them, samples 1-22 were taken from the control group and samples 39-74 were taken from the case group (all pregnant women in the early or mid-pregnancy stage).

[0074] The inclusion criteria and detection methods for the samples to be tested are the same as in Example 2.

[0075] The test results are shown in Table 7 below.

[0076] Table 7: Detection results of the samples to be tested

[0077] True negative 16 False positive 6 True positive 30 False negative 6

[0078] Based on the ROC curve, the specificity of the kit described in this invention was calculated to be 72.73% for the test sample.

[0079] (2) Sensitivity

[0080] Source of test samples: The experimental subjects were women who were outpatients and inpatients at West China Second Hospital of Sichuan University. According to the existing diagnostic methods, the experimental subjects were divided into a case group and a control group. Plasma samples were collected from both groups as test samples, totaling 58 samples. Among them, samples 1-22 were taken from the healthy control group, and samples 39-74 were taken from the case group (all pregnant women in the early or mid-pregnancy stage).

[0081] The inclusion criteria and detection methods for the samples to be tested are the same as in Example 2.

[0082] Based on the ROC curve, the sensitivity of the kit described in this invention for the test sample was calculated to be 83.33%.

[0083] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. The application of C1INH in the preparation of products for predicting or assisting in the diagnosis of preeclampsia, characterized by: The product uses blood samples from test subjects as the detection target and detects the C1INH content in the blood samples at the protein level.

2. The application according to claim 1, characterized in that: The subjects were pregnant women in the first or second trimester of pregnancy.

3. The application according to claim 1, characterized in that: The blood sample may include whole blood, serum, or plasma.

4. The application according to claim 1, characterized in that: During the test, when the concentration of C1INH in the blood sample is higher than 43 μg / ml, it is predicted that the subject may develop preeclampsia after 20 weeks of gestation.

5. The application according to claim 1, characterized in that: The detection methods include immunofluorescence assay, ELISA, biochip assay, and electrochemiluminescence assay.

6. A product for predicting or assisting in the diagnosis of preeclampsia, characterized in that: It contains a substance that detects C1INH levels in blood samples, and this substance detects the amount of C1INH in the subject's blood sample at the protein level.

7. The product according to claim 7, characterized in that: The products include reagent kits, diagnostic reagents, or other medically feasible products.

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