PLGF and sFlt-1 double-marker joint detection kit and application

Abstract

The embodiment of the invention provides a PLGF and sFlt-1 double-marker joint detection kit and application. The kit comprises an sFlt-1 labeled antibody, a PlGF labeled antibody, an antibody coated fluorescent magnetic bead working solution, an antibody calibrator, a sample diluent, a cleaning solution, an enzyme solution and an enzyme substrate solution. According to the application, specific capture antibodies of sFlt-1 and PlGF are respectively coupled to different fluorescence coded magnetic beads, so that the specific capture antibodies, an antigen and a detection antibody form a double-antibody sandwich structure, and the double-antibody sandwich structure is cleaned, uniformly mixed and synchronously imaged, so that synchronous and simultaneous testing of sFlt-1 and PlGF in the same sample is realized, and high-resolution quantitative analysis is carried out. Therefore, the application of the joint detection kit significantly simplifies the detection process, reduces the cost, improves the detection flux and efficiency, effectively improves the sensitivity and accuracy of preeclampsia risk assessment, and has important clinical popularization value.

Description

Technical Field

[0001] This application relates to the field of in vitro diagnostic testing, and in particular to a kit for the detection of PLGF and sFlt-1 dual markers and its application. Background Technology

[0002] Preeclampsia is a pregnancy-specific condition characterized by hypertension (systolic blood pressure ≥140 mmHg and / or diastolic blood pressure ≥90 mmHg) accompanied by proteinuria (≥0.3 g / 24 h) after 20 weeks of gestation. It affects approximately 4.6% of pregnant women worldwide, posing a serious threat to maternal and infant health. According to the World Health Organization, preeclampsia and its complications cause approximately 76,000 maternal deaths and 500,000 fetal deaths globally each year.

[0003] The core pathological change in preeclampsia involves dysplasia of the uteroplacental spiral arteries, which reduces uteroplacental blood flow in late pregnancy. Placental ischemia and hypoxia trigger systemic inflammatory responses and oxidative stress. High-risk factors include a history of preeclampsia, chronic hypertension, pregnancy complicated by medical conditions (systemic lupus erythematosus (SLE), antiphospholipid syndrome (APS), kidney disease, diabetes, etc.), and obesity (BMI ≥ 28.0 kg / m²). 2 Risk factors include: ) and pregnant women aged ≥40 years; medium-risk factors include being overweight (BMI 24.0~27.9 kg / m²). 2 Preeclampsia is a risk factor for multiple pregnancies, maternal age between 35 and 40 years, a history of adverse pregnancy outcomes, and a family history of preeclampsia. Low-risk factors include primiparity (or remarriage), pregnancy intervals >10 years, assisted reproductive technology pregnancy, history of gestational hypertension, sleep apnea syndrome, gestational diabetes mellitus, and urinary tract infections. Preeclampsia increases the risk of cerebrovascular accidents by 5 times, and due to insufficient uteroplacental perfusion, fetal growth restriction (incidence 10-25%), acute distress (15-20%), and even intrauterine fetal death (1-2%). In early-onset cases, the perinatal mortality rate is as high as 44% for those terminating pregnancy before 32 weeks, and the incidence of respiratory distress syndrome and intraventricular hemorrhage among surviving preterm infants is 33%.

[0004] Angiogenic factor (PlGF) promotes endothelial cell proliferation and migration, thereby promoting angiogenesis and vascular endothelial cell differentiation. In low-risk pregnant women with preeclampsia, the sensitivity of PlGF in early pregnancy for predicting early and late-onset preeclampsia was 55% and 33%, respectively (specificity criterion set at 90%). Therefore, PlGF has limited value as an independent predictor of preeclampsia. Soluble fms-like tyrosine kinase 1 (sFlt-1), as a receptor tyrosine kinase, regulates the biological activity of PlGF and inhibits angiogenesis in the placenta. The "Guidelines for the Diagnosis and Treatment of Hypertensive Disorders in Pregnancy (2020)" indicates that the sFlt-1 / PlGF ratio has clinical value for short-term prediction of preeclampsia. When the sFlt-1 / PlGF ratio is ≤38, the negative predictive value (excluding preeclampsia within 1 week) is 99.3%; when the sFlt-1 / PlGF ratio is >38, the positive predictive value (predicting preeclampsia within 4 weeks) is 36.7%. The "Guidelines for Prediction and Prevention of Preeclampsia (2025)" state that decreased PlGF levels, elevated sFlt-1 levels, or an increased sFlt-1 / PlGF ratio during pregnancy indicate an increased risk of preeclampsia. When medical resources are sufficient, it is recommended to use these guidelines in conjunction with maternal risk factors, maternal MAP (metastatic leukemia), and ultrasound parameters to comprehensively assess the risk of preeclampsia in pregnant women.

[0005] Because sFlt-1 is a soluble receptor that can bind to PlGF, current clinical kits for detecting these two biomarkers typically use separate detection methods, requiring two separate experiments to obtain relevant data. This method of separating and detecting a single biomarker is complex and inefficient. Its limitations become increasingly apparent, especially in high-throughput screening or when multiple indicators need to be comprehensively evaluated. Separate detection can easily lead to significant time differences between different experimental results, making it difficult to meet the clinical need for rapid and simultaneous diagnosis. Therefore, developing a new detection technology with high sensitivity, a wide detection range, and the ability to simultaneously quantify sFlt-1 and PlGF is of significant clinical importance. Summary of the Invention

[0006] This application aims to at least partially address one of the technical problems in the related art. This application proposes a dual-biomarker detection kit for PLGF and sFlt-1 and its application, realizing the synergistic detection of two key biomarkers, sFlt-1 and PLGF, in a digital single-molecule detection system platform. This kit has the advantages of high sensitivity, strong specificity, low sample requirement, simple operation, good repeatability, and combined detection.

[0007] According to an embodiment of the first aspect of this application, a PLGF and sFlt-1 dual-marker detection kit is provided, comprising: sFlt-1 labeled antibody, PLGF labeled antibody, antibody-coated fluorescent magnetic bead working solution, antibody calibrator, sample diluent, washing solution, enzyme solution and enzyme substrate solution.

[0008] In some embodiments, the antibody-coated fluorescent magnetic bead working solution includes PlGF antibody-coated fluorescent magnetic beads and sFlt-1 antibody-coated fluorescent magnetic beads, which are respectively coated with fluorescent dyes with different emission wavelengths.

[0009] In some embodiments, both the sFlt-1 labeled antibody and the PlGF labeled antibody are labeled with biotin; the molar ratio of the sFlt-1 labeled antibody and the PlGF labeled antibody to the biotin is 1:10-80.

[0010] In some embodiments, the enzyme solution contains 10-200 pM streptavidin-labeled β-galactosidase.

[0011] In some embodiments, the enzyme substrate solution contains 10-200 nM halogen-β-galactoside.

[0012] In some embodiments, the sample diluent contains 1-10% BSA, 1-10mM EDTA, 0.05-1% preservative, 50-500μg / mL blocking agent, and 0.1-1% surfactant.

[0013] In some embodiments, the antibody calibrators include PlGF calibrators and sFlt-1 calibrators; the cleaning solution includes cleaning solution A and cleaning solution B.

[0014] In some embodiments, the washing solution A comprises 10-100 mM PBS, 1-10% sucrose, 0.1-1% Tween 20, and 0.05-1% Proclin 300;

[0015] The washing solution B contains 10-100 mM PBS, 1-10% sucrose and 0.05-1% Proclin 300.

[0016] According to embodiments of the second aspect of this application, the use of the combined detection kit described in any of the above embodiments in the preparation of products for the identification and treatment of preeclampsia is proposed.

[0017] In some embodiments, the subjects are pregnant subjects, with a gestational age of 8 to 38 weeks, and the application is to detect the levels of sFlt-1 and PlGF in the subjects' blood.

[0018] The beneficial effects of this application are as follows:

[0019] (1) The kit of this application is used with the DiSMS platform and the Simoa platform. It has high sensitivity and good repeatability and accuracy of the test results. It solves the problem that in related technologies, due to the interaction between sFlt-1 and PlGF in blood samples, clinical tests usually need to detect the sFlt-1 and PlGF values ​​in blood samples separately. This is not only cumbersome to operate, but also easily affected by factors such as temperature, reagent batch and operator differences, which leads to fluctuations in the sFlt-1 / PlGF ratio and thus affects the accurate assessment of the risk of preeclampsia.

[0020] (2) This application conjugates specific capture antibodies for sFlt-1 and PlGF onto different fluorescently encoded magnetic beads, forming a double-antibody sandwich structure with the antigen and detection antibody. After washing and mixing, simultaneous imaging is performed, enabling parallel high-resolution quantitative analysis of sFlt-1 and PlGF in the same sample. This allows for simultaneous quantitative analysis of both sFlt-1 and PlGF markers, saving detection time and further improving the accuracy of early diagnosis of preeclampsia. Compared with previous related technologies, this application significantly simplifies the detection process, reduces costs, increases detection throughput and efficiency, and effectively improves the sensitivity and accuracy of preeclampsia risk assessment, possessing significant clinical application value.

[0021] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0022] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings, wherein:

[0023] Figure 1 This is a schematic diagram of the detection principle of the DiSMS platform according to an embodiment of this application;

[0024] Figure 2 Calibration curves for single-inspection and combined-inspection of sFlt-1;

[0025] Figure 3 The calibration curves for single-detection and combined-detection of PlGF are shown. Detailed Implementation

[0026] Embodiments of this application are described in detail below. Examples of these embodiments are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this application, and should not be construed as limiting this application. Rather, this application includes all variations, modifications, and equivalents falling within the spirit and scope of the appended claims.

[0027] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, it is expected that ranges of 60-110 and 80-120 are also included. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In this application, unless otherwise stated, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0-5" indicates that all real numbers between "0-5" have been listed in this article; "0-5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0028] In the description of the embodiments in this application, the term "and / or" is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone. Additionally, the character " / " in this document generally indicates that the preceding and following related objects have an "or" relationship.

[0029] According to an embodiment of the first aspect of this application, a PLGF and sFlt-1 dual-marker detection kit is provided, comprising: sFlt-1 labeled antibody, PLGF labeled antibody, antibody-coated fluorescent magnetic bead working solution, antibody calibrator, sample diluent, washing solution, enzyme solution and enzyme substrate solution.

[0030] Both the sFlt-1 labeled antibody and the PlGF labeled antibody were biotin-labeled; the molar ratio of sFlt-1 labeled antibody and PlGF labeled antibody to biotin was 1:10-80. The biotin-labeled detection antibody was prepared, for example, using the Thermo Fisher EZ-Link™ NHS-PEG4-Biotin Biotinylation Kit to biotin-label the sFlt-1 labeled antibody and the PlGF labeled antibody. In this embodiment, the binding of the detection antibody to the enzyme was introduced using the biotin-streptavidin system. In other words, both the sFlt-1 labeled antibody and the PlGF labeled antibody were biotin-labeled, the enzyme solution contained 10-200 pM streptavidin-labeled β-galactosidase, and the enzyme substrate solution contained 10-200 nM halogen-β-galactosidase. This embodiment utilized the efficient binding of streptavidin-labeled β-galactosidase (SBG) to both the biotin-labeled sFlt-1 detection antibody and the biotin-labeled PlGF detection antibody. This embodiment uses the classic double-antibody sandwich method, which is simple to operate and has high conjugation efficiency. It can simultaneously detect sFlt-1 and PlGF, assisting clinicians in predicting preeclampsia and adverse pregnancy outcomes, helping doctors identify and treat high-risk groups, thereby ensuring the safety of mothers and babies during pregnancy.

[0031] The antibody-coated fluorescent magnetic bead working solution in this application includes PlGF antibody-coated fluorescent magnetic beads and sFlt-1 antibody-coated fluorescent magnetic beads, which are respectively coated with fluorescent dyes with different emission wavelengths.

[0032] In this application, PlGF antibody-coated fluorescent magnetic beads and sFlt-1 antibody-coated fluorescent magnetic beads are respectively coated with anti-PlGF capture antibody and anti-sFlt-1 capture antibody onto two different fluorescently labeled magnetic beads, using fluorescent dyes with different emission wavelengths to distinguish the detection signals of different markers. The preparation methods for PlGF antibody-coated fluorescent magnetic beads and sFlt-1 antibody-coated fluorescent magnetic beads are the same, including: coupling the capture antibody with EDC / NHS activated fluorescently encoded magnetic beads at 2-8℃; after washing the coupling product, blocking with blocking solution; and washing again to obtain the capture antibody-coated magnetic beads.

[0033] For example, the preparation methods for PlGF antibody-coated fluorescent magnetic beads and sFlt-1 antibody-coated fluorescent magnetic beads include: liquid replacement of the capture antibody with magnetic bead conjugation buffer; activation of fluorescently encoded magnetic beads with different emission wavelengths using EDC / NHS, with a magnetic bead activation concentration of 1-10 mg / mL, activation conditions of 2-8℃, and activation time of 20-60 min, the activation reagents including EDC concentration of 0.1-1 mg / mL and NHS concentration of 0.1-1.0 mg / mL; overnight conjugation of the capture antibody and activated magnetic beads at 2-8℃, wherein the reaction concentration of the capture antibody is 0.1-1 mg / mL; then blocking at room temperature using blocking buffer, wherein the blocking buffer includes 10-50 mM PBS, 0.2-2% BSA, pH 6.8-8.0; washing the magnetic beads twice with magnetic bead preservation solution and storing them at 4℃ using magnetic bead preservation solution, wherein the magnetic bead preservation solution is 50-100 mM Tris-HCl, 1-10 mM... EDTA, 0.01-0.1% Tween 20, 0.2-2% BSA, pH 7.0-8.0.

[0034] In some embodiments, the sample diluent contains 1-10% BSA, 1-10mM EDTA, 0.05-1% preservative, 50-500μg / mL blocking agent, and 0.1-1% surfactant.

[0035] In some embodiments, the antibody calibrators include PlGF calibrators and sFlt-1 calibrators; the washing solutions include washing solution A and washing solution B. Washing solution A contains 10-100 mM PBS, 1-10% sucrose, 0.1-1% Tween 20, and 0.05-1% Proclin 300; washing solution B contains 10-100 mM PBS, 1-10% sucrose, and 0.05-1% Proclin 300.

[0036] According to embodiments of the second aspect of this application, the application of the combined detection kit in any of the above embodiments in the preparation of products for the identification and treatment of preeclampsia is described. The subjects are pregnant subjects with a gestational age of 8 to 38 weeks, and the application is used to detect the levels of sFlt-1 and PlGF in the subjects' blood. For example, the method of use on a DiSMS platform includes operating each component of the kit according to the instructions, placing the fluorescent substrate solution in the corresponding position on the instrument as instructed, and exporting the test results after the experiment is completed.

[0037] For example, the kit includes sFlt-1 antibody-coated fluorescent magnetic beads, PlGF antibody-coated fluorescent magnetic beads, sFlt-1 labeled antibody, PlGF labeled antibody, enzyme solution, fluorescent substrate solution, washing solution A, washing solution B, calibrators, sample diluents, and electronic files of the assay method. Instructions for using the kit on the DiSMS platform are provided as follows:

[0038] 1) All components in the kit should be equilibrated to room temperature beforehand, and the reagents in the kit should be mixed thoroughly;

[0039] 2) Add 60 μL of the serum sample to be tested to a 1.5 mL EP tube, add 180 μL of sample diluent, mix and set aside;

[0040] 3) In a 96-well polystyrene plate, add 100 μL of sFlt-1 calibrator or diluted test sample, 25 μL of sFlt-1 antibody-coated fluorescent magnetic beads, and 20 μL of sFlt-1 detection antibody to column 1. Place the plate on a shaker at 30°C and 700 rpm for 30 min.

[0041] 4) In a 96-well polystyrene plate, add 100 μL of PlGF calibrator or diluted test sample, 25 μL of PlGF antibody-coated fluorescent magnetic beads, and 20 μL of PlGF detection antibody to column 2. Place the plate on a shaker at 30°C and 700 rpm for 30 min.

[0042] 5) Magnetic suction for 1 min, discard the liquid in the well, add 180 μL of cleaning solution A to each well, shake at 700 rpm for 1 min, mix well, magnetic suction for 1 min, discard the liquid, and repeat the washing process three times.

[0043] 6) Add the magnetic beads corresponding to the same sample in column 2 to column 1 for mixing;

[0044] 7) Add 100 μL SBG solution to each well, place on a shaker at 30°C and 700 rpm for 10 min;

[0045] 8) Magnetic suction for 1 min, discard the liquid in the well, add 180 μL of cleaning solution A to each well, shake at 700 rpm for 1 min, mix well, magnetic suction for 1 min, discard the liquid, and repeat the washing process three times.

[0046] 9) Add 180 μL of cleaning solution B to each well and wash. Shake at 700 rpm for 1 min, mix well, and then magnetically remove the liquid for 1 min. Repeat three times.

[0047] 10) Set up the well plate; import the test method electronic file into the Di2000, set the run name and sample name, and then click run;

[0048] 11) Export the sample test report.

[0049] Because sFlt-1 and PlGF interact in blood samples, clinical testing typically requires separate measurements. However, separate detection of sFlt-1 and PlGF in blood samples using existing techniques is not only cumbersome but also susceptible to fluctuations in the sFlt-1 / PlGF ratio due to factors such as temperature, reagent batches, and operator variations, thus affecting the accurate assessment of preeclampsia risk. This application addresses this issue by coupling specific capture antibodies for sFlt-1 and PlGF onto different fluorescently encoded magnetic beads, forming a double-antibody sandwich structure with the antigen and detection antibody. After washing and mixing, the beads are simultaneously imaged, enabling simultaneous high-resolution quantitative analysis of sFlt-1 and PlGF in the same sample.

[0050] To facilitate a further understanding of this application, the solutions described below are further described in conjunction with embodiments. Those skilled in the art will understand that the examples described in this application are only a portion of the examples, and any other suitable specific examples are within the scope of this application.

[0051] Example 1

[0052] This embodiment provides a dual-marker assay kit for the auxiliary detection of preeclampsia, comprising sFlt-1 antibody-coated fluorescent magnetic beads, PlGF antibody-coated fluorescent magnetic beads, sFlt-1 labeled antibody, PlGF labeled antibody, enzyme solution, fluorescent substrate solution, washing solution A, washing solution B, sFlt-1 calibrator, PlGF calibrator, sample diluent, and electronic file of the assay method. The preparation method of the kit in this application is as follows:

[0053] Preparation of sFlt-1 capture antibody-coated magnetic beads: The sFlt-1 capture antibody was subjected to liquid replacement treatment using a 10KD ultrafiltration tube, and the anti-sFlt-1 capture antibody was collected; Cy5-labeled fluorescent magnetic beads were added to EDC / NHS to a final concentration of 0.5 mg / mL, mixed well, and activated at 4℃ for 30 min; the activated magnetic beads were washed twice, and after changing the buffer, the sFlt-1 capture antibody was added to the Cy5-labeled fluorescent magnetic beads, and the reaction was carried out at 4℃ and 1500 rpm for 12 h; the coupled magnetic beads were washed twice, and the uncoupled sites were blocked with magnetic bead blocking buffer; after washing again, the beads were stored in magnetic bead dilution buffer to obtain sFlt-1 antibody-coated fluorescent magnetic beads.

[0054] Preparation of PlGF capture antibody-coated magnetic beads: Anti-PlGF capture antibody was subjected to liquid replacement treatment using a 10KD ultrafiltration tube, and the anti-PlGF capture antibody after liquid replacement was collected; AF488-labeled fluorescent magnetic beads were added to EDC / NHS to a final concentration of 0.5 mg / mL, mixed well, and activated at 4℃ for 30 min; the activated magnetic beads were washed twice, and after liquid replacement, PlGF capture antibody was added to AF488-labeled fluorescent magnetic beads, and the reaction was carried out at 4℃ and 1500 rpm for 12 h; the coupled magnetic beads were washed twice, and the uncoupled sites were blocked with magnetic bead blocking buffer; after washing again, the beads were stored in magnetic bead dilution buffer to obtain sFlt-1 antibody-coated fluorescent magnetic beads.

[0055] Preparation method of sFlt-1 / PlGF biotinylated detection antibody: The sFlt-1 labeled antibody and PlGF labeled antibody were biotinylated using the Thermo Fisher EZ-Link™ NHS-PEG4-Biotin Biotinylation Kit, with a molar ratio of antibody to biotin of 1:60. Quanterix SBG concentrate was purchased and diluted to 50 pM using the accompanying diluent.

[0056] Example 2

[0057] This embodiment provides a method for using the kit from Example 1 on the DiSMS platform:

[0058] Step 1: Remove each component of the combined detection kit and allow it to equilibrate to room temperature.

[0059] Step 2: Add 60 μL of the serum sample to be tested to a 1.5 mL EP tube, add 180 μL of sample diluent, mix and set aside.

[0060] Step 3: In a 96-well polystyrene plate, add 100 μL of sFlt-1 calibrator or diluted test sample to column 1, 25 μL of sFlt-1 antibody-coated fluorescent magnetic beads, and 20 μL of sFlt-1 labeled antibody. Seal the plate with a sealing film and incubate at 37°C and 700 rpm for 30 min.

[0061] Step 4: In a 96-well polystyrene plate, add 100 μL of PlGF calibrator or diluted test sample, 25 μL of PlGF antibody-coated fluorescent magnetic beads, and 20 μL of PlGF-labeled antibody to column 2. Seal the plate with sealing film and incubate at 37°C and 700 rpm for 30 min.

[0062] Step 5: After incubation, place the magnetic beads on the magnetic plate for 1 minute, discard the liquid, add 180 μL of cleaning solution A, mix at 700 rpm for 1 minute, magnetically attach for 1 minute, discard the liquid, and repeat the washing process 3 times.

[0063] Step 6: Add the magnetic beads from column 2 corresponding to the same sample to column 1 for mixing.

[0064] Step 7: Add 100 μL of SBG solution to each well, seal with a sealing film, and incubate at 30°C and 700 rpm for 10 min. After incubation, wash three times each with washing solution A (180 μL) and washing solution B (180 μL).

[0065] Step 8: Let the 96-well plate stand at room temperature for 10 minutes. Transfer the 96-well plate to the Di2000 analyzer.

[0066] Step 9: Import the electronic file of the test method into the Di2000 (for first-time use), and place the fluorescent substrate solution in the corresponding position on the instrument according to the instructions.

[0067] Step 10: After setting the run name and sample name according to the 96-well plate sample content, click Run.

[0068] Step 11: Export the sample test report.

[0069] Example 3

[0070] The sFlt-1 and PlGF calibrators were calibrated on the device according to the usage method in Example 2. Serum samples from 10 healthy pregnant women at different gestational weeks (13-20 weeks, 21-28 weeks, and 29-36 weeks) and preeclampsia risk individuals were prepared. The concentrations of sFlt-1 and PlGF in the samples were calculated using their respective calibration curves. Figures 1-2 The results were compared between single and combined sFlt-1 tests, single and combined PlGF tests, and single and combined sFlt-1 / PlGF tests. Table 1 shows the sFlt-1 concentration in the calibration curves for single and combined sFlt-1 tests; Table 2 shows the PlGF concentration in the calibration curves for single and combined PlGF tests; Table 3 shows the sFlt-1 single and combined sFlt-1 test results for samples at different gestational weeks; Table 4 shows the PlGF single and combined sFlt-1 test results for samples at different gestational weeks; and Table 5 shows the sFlt-1 / PlGF single and combined sFlt-1 test results for samples at different gestational weeks.

[0071] Table 1 shows the sFlt-1 concentrations of samples in the sFlt-1 single-detection and combined-detection calibration curves.

[0072]

[0073] Table 2 shows the PlGF concentrations of samples in the calibration curves for single and combined PlGF detection.

[0074]

[0075] Table 3. Results of sFlt-1 single and combined tests in samples at different gestational weeks.

[0076]

[0077] Table 4. Results of single and combined PlGF tests in samples at different gestational weeks.

[0078]

[0079] Table 5 shows the results of single and combined sFlt-1 / PlGF tests in samples at different gestational weeks.

[0080]

[0081] According to Tables 1-5, the average CV (CV) for sFlt-1 single-sample and combined-sample concentrations in the healthy group was 2.7%, while the average CV for the preeclampsia group was 1.2%. The average CV for PlGF single-sample and combined-sample concentrations in the healthy group was 4.0%, while the average CV for the preeclampsia group was 2.1%. The average CV for the sFlt-1 / PlGF single-sample ratio and the sFlt-1 / PlGF combined-sample ratio in the healthy group was 5.4%, and the average CV for the preeclampsia group was 2.7%. Specifically, the CVs for sFlt-1, PlGF, and sFlt-1 / PlGF single-sample and combined-sample results were all less than 8%, indicating consistency between the combined and single-sample test results. This demonstrates the reliability of the combined-sample results. Meanwhile, under the premise that the results of sFlt-1 and PlGF single detection and combined detection are highly consistent, there are some cases where the ratio CV>10% between sFlt-1 / PlGF single detection and combined detection. The combined detection data is detected synchronously, which can effectively avoid data differences caused by factors such as machine time, equipment and personal operation, which may be the main reason for the difference in results.

[0082] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0083] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A dual-marker detection kit for PLGF and sFlt-1, characterized in that, The kit includes: sFlt-1 labeled antibody, PlGF labeled antibody, antibody-coated fluorescent magnetic bead working solution, antibody calibrator, sample diluent, washing solution, enzyme solution and enzyme substrate solution.

2. The combined detection kit according to claim 1, characterized in that, The antibody-coated fluorescent magnetic bead working solution includes PlGF antibody-coated fluorescent magnetic beads and sFlt-1 antibody-coated fluorescent magnetic beads, which are respectively coated with fluorescent dyes with different emission wavelengths.

3. The combined detection kit according to claim 1, characterized in that, Both the sFlt-1 labeled antibody and the PlGF labeled antibody are labeled with biotin; the molar ratio of the sFlt-1 labeled antibody and the PlGF labeled antibody to the biotin is 1:10-80.

4. The combined detection kit according to claim 1, characterized in that, The enzyme solution contains 10-200 pM streptavidin-labeled β-galactosidase.

5. The combined detection kit according to claim 1, characterized in that, The enzyme substrate solution contains 10-200 nM halogen-β-galactoside.

6. The combined detection kit according to claim 1, characterized in that, The sample diluent contains 1-10% BSA, 1-10mM EDTA, 0.05-1% preservative, 50-500μg / mL blocking agent, and 0.1-1% surfactant.

7. The combined detection kit according to claim 1, characterized in that, The antibody calibrators include PlGF calibrators and sFlt-1 calibrators; the cleaning solutions include cleaning solution A and cleaning solution B.

8. The combined detection kit according to claim 7, characterized in that, The washing solution A contains 10-100 mM PBS, 1-10% sucrose, 0.1-1% Tween 20, and 0.05-1% Proclin 300; The washing solution B contains 10-100 mM PBS, 1-10% sucrose and 0.05-1% Proclin 300.

9. The use of the combined detection kit according to any one of claims 1-8 in the preparation of products for the identification and treatment of preeclampsia.

10. The application according to claim 9, characterized in that, The subjects were pregnant, with gestational weeks ranging from 8 to 38 weeks, and the study aimed to detect the levels of sFlt-1 and PlGF in their blood.

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