Joint detection kit for placental growth factor and soluble fms-like tyrosine kinase-1 and application of joint detection kit

Abstract

The invention relates to the technical field of biological detection, and particularly discloses a placental growth factor and soluble fms-like tyrosine kinase-1 joint detection kit and application thereof. The placenta growth factor and soluble fms-like tyrosine kinase-1 joint detection kit comprises a sample pad, a combination pad, a coating film, a water absorption pad and a sample diluent, the coating film is an NC film coating antibody; the preparation method of the conjugate pad comprises the following steps: treating the fiber membrane with a pretreatment solution, drying, and then spraying the time-resolved fluorescent microsphere labeled antibody. The placenta growth factor and soluble fms-like tyrosine kinase-1 are jointly detected by using the detection kit and the detection method provided by the invention, and the sensitivity, the accuracy and the repeatability are relatively high.

Description

Technical Field

[0001] This application relates to the technical field of biodetection, specifically to a combined detection kit for placental growth factor and soluble FMS-like tyrosine kinase-1 and its application. Background Technology

[0002] This method is used for the in vitro quantitative detection of placental growth factor (PLGF) and soluble FMS-like tyrosine kinase-1 (sF1t-1) levels in human serum, plasma, and whole blood. Clinically, it is used as an adjunct to the diagnosis of preeclampsia. Preeclampsia (PE), also known as preeclampsia, is the most common peculiar disease among pregnant women with hypertension. Placental growth factor and soluble FMS-like tyrosine kinase-1 are biomarkers for the auxiliary diagnosis of preeclampsia.

[0003] Placental growth factor (PLGF) is a member of the vascular endothelial growth factor (VEGF) family. It is primarily synthesized by syncytiotrophoblast cells and binds to tyrosinase receptors located on trophoblast cells and vascular endothelial cells. PLGF is a secreted homodimeric glycoprotein that plays a crucial role in inducing vascular endothelial cell activation, proliferation, and migration, as well as promoting angiogenesis. During pregnancy, women synthesize large amounts of PLGF, which promotes trophoblast cell proliferation and differentiation, and regulates placental angiogenesis and physiological remodeling. PLGF can also synergistically enhance the biological activity of other VEGFs, promoting placental angiogenesis and maintaining normal placental blood supply and function during pregnancy. Studies have shown that PLGF levels in women with preeclampsia are lower than in normal pregnancies, and detecting PLGF concentration can be used to aid in the diagnosis of early preeclampsia.

[0004] Soluble FMS-like tyrosine kinase-1 (sF1t-1) is a glycoprotein with tyrosine kinase activity, belonging to the type III receptor tyrosine kinase family. It can inhibit the expression of placental growth factor, causing impaired angiogenesis, accelerating endothelial cell damage, thereby disrupting vascular wall integrity and reducing vascular permeability. In pregnant women with preeclampsia, sF1t-1 expression is abnormally elevated, with concentrations higher than in normal pregnancies. sF1t-1 can serve as a biological indicator for predicting the occurrence and development of preeclampsia.

[0005] The combined measurement of the ratio of sF1t-1 to PLGF in the blood of pregnant women is more valuable in diagnosing the onset of preeclampsia than the measurement of sF1t-1 or PLGF alone. Summary of the Invention

[0006] To address the aforementioned technical issues, this application provides a combined detection kit for placental growth factor and soluble FMS-like tyrosine kinase-1, and its application.

[0007] In a first aspect, this application provides a combined detection kit for placental growth factor and soluble FMS-like tyrosine kinase-1, comprising the following components: sample pad, conjugate pad, coating membrane, absorbent pad, and sample diluent; The coating membrane is an NC membrane coated with antibodies; The method for preparing the conjugate pad is as follows: the fiber membrane is treated with a pretreatment solution and dried, and then time-resolved fluorescent microspheres labeled with antibodies are sprayed on it. The pretreatment solution is a solution of pH 8.0-8.4 + 8-12 mM BS + 1.8-2.2% BSA + 0.4-0.6% polymer + 0.02-0.03% surfactant + 5-7% sucrose + 0.13-0.18 mg / mL HIER-R-009 inhibitor + 0.08-0.12 mg / mL MAb to RBC; the polymer is composed of PEG4000 and dextran T7 in a weight ratio of 10:0.5-5; the surfactant is composed of Tween-20 and Pluronic F-127 in a weight ratio of 10:0.1-1. The sample diluent was a solution with pH 7.2-7.6, 45-55 mM Tris, 1.0-2.0% sodium caseinate, 1.8-2.2% BSA, 0.8-1.2% NaCl, and 0.04-0.06% Proclin 300.

[0008] The kit provided in this application specifically consists of a test card, a sample diluent, and an IC card; the test card consists of a test strip and a plastic card shell; the test strip includes the following components: a sample pad, a conjugate pad, a coating membrane, and an absorbent pad.

[0009] The principle of the detection kit provided in this application is as follows: It is based on the quantitative detection of placental growth factor (PLGF) and soluble fms-like tyrosine kinase-1 (sFlt-1) in human serum, plasma, and whole blood using a fluorescent immunoassay double-antibody sandwich method. The sample to be tested is added to the sample well of the test card. When the sample contains a certain amount of PLGF, PLGF reacts with PLGF antibody I labeled with fluorescent microspheres in the conjugate pad to form an antigen-antibody complex. This complex moves forward along the nitrocellulose membrane under chromatographic action and is captured by PLGF antibody II pre-coated in the detection area (T1) of the nitrocellulose membrane. When the sample contains a certain amount of sFlt-1, sFlt-1 reacts with sFlt-1 antibody I labeled with fluorescent microspheres in the conjugate pad to form an antigen-antibody complex. This complex moves forward along the nitrocellulose membrane under chromatographic action and is captured by sFlt-1 antibody II pre-coated in the detection area (T2) of the nitrocellulose membrane. The more analyte in the sample, the more complex accumulates on the detection region (T), and the signal intensity of the fluorescent antibody reflects the amount of analyte captured. The concentration of the analyte in the sample can be detected using appropriate instruments.

[0010] The purpose of the pretreatment solution designed in this application is to pretreat the conjugate pad. The conjugate pad is the portion of the test strip on which the "detection antibody-label complex" (i.e., the conjugate of monoclonal antibodies of PlGF and sFlt-1 with fluorescent microspheres) is immobilized. The role of the pretreatment solution is to ensure that these "detection antibody-label complexes" are stably and dryly preserved on the conjugate pad, and that they are rapidly, uniformly, and completely reconstituted when the sample (serum / plasma / whole blood) flows through them, and flow forward with the liquid, while minimizing non-specific binding and ensuring the sensitivity and accuracy of the detection.

[0011] In the pretreatment solution, BS is a borax-boric acid buffer solution, which maintains a relatively stable pH value; it creates an optimal microenvironment for the labeled antibody, ensuring that it remains stable during dry storage, can be released rapidly and uniformly during detection, and binds to PIGF and sFlt-1 in the sample with the highest activity and specificity, while minimizing various non-specific interferences, thereby ensuring high sensitivity and high accuracy of the detection results.

[0012] During antibody drying, BSA (bovine serum albumin) can fill around antibody molecules to prevent denaturation and inactivation, acting as a protective agent. It can effectively block non-specific binding sites on the binding pad, reducing background noise. It also helps the dried marker complex to redissolve more quickly and uniformly as the sample flows through.

[0013] The polymer composed of PEG4000 and dextran T7 increases the viscosity of the liquid, which helps to regulate the flow rate of the sample on the nitrocellulose membrane, making it more stable and uniform. This avoids incomplete reaction due to excessively fast flow, resulting in weak and unstable signals; while excessively slow flow is time-consuming and may cause severe diffusion. During the drying process, a protective film can be formed on the surface of the protein molecules, improving thermal stability and extending the shelf life of the reagent.

[0014] The surfactant, composed of Tween-20 and Pluronic F-127, reduces the surface tension of the liquid, helping the sample solution to quickly penetrate and wet the dry binding pad, thereby ensuring that the labeled complex is released instantly and completely. Incomplete reconstitution can lead to weak signals, poor accuracy and repeatability. At the same time, it further helps to reduce non-specific binding caused by hydrophobic interactions.

[0015] When the labeled solution dries, sucrose forms an amorphous, glassy glycosyl matrix that encapsulates the antibody-microsphere complex. This structure fixes the three-dimensional structure of the protein molecule, preventing it from breaking down and denaturing during dehydration, thereby maximizing its biological activity.

[0016] The blocking agent HIER-R-009 is a specialized and highly effective blocking agent that is more targeted than BSA. It is specifically designed to block certain interference sites in the detection system (e.g., to prevent interference from rheumatoid factor, heterophilic antibodies, etc.). It has a high affinity for certain binding sites on nitrocellulose membranes, which can greatly reduce background and improve the signal-to-noise ratio.

[0017] MAb to RBC antibodies can bind to red blood cells in the blood sample, thereby separating blood cells and allowing the clear plasma portion to continue flowing forward along the test strip, preventing interference from red blood cells in the blood sample.

[0018] It is precisely this precise formulation design of the binding pad pretreatment solution provided in this application that ensures that the PLGF / sFlt-1 combined detection kit achieves high sensitivity, high accuracy, and high repeatability.

[0019] Furthermore, the sample diluent provided in this application can maintain the stability and activity of the target analyte in the sample during the detection process, optimize the detection reaction environment, and inhibit microbial growth. During the experiment, the applicant found that the sample diluent does not contain surfactants, which can effectively avoid the slight disturbance to the tertiary structure of proteins caused by surfactants. Theoretically, this is more conducive to maintaining the natural activity of antigens and antibodies and ensuring the specificity and affinity of the binding reaction.

[0020] Preferably, the NC membrane-coated antibody consists of a detection line T1 coated with PLGF monoclonal antibody II, a detection line T2 coated with sFlt-1 monoclonal antibody II, and a control line C coated with MAb to DNP antibody, sequentially arranged on the NC membrane; in the preparation method of the NC membrane-coated antibody, the coating solution is a solution of pH 7.2-7.6 + 8-12mM PBS + 0.8-1.2% trehalose; the coating concentration is 0.8-1.2 mg / mL.

[0021] Preferably, the pretreatment solution is a solution with pH 8.1-8.3, 9-11 mM BS, 1.9-2.1% BSA, 0.45-0.55% polymer, 0.022-0.028% surfactant, 5.5-6.5% sucrose, 0.14-0.17 mg / mL inhibitor HIER-R-009, and 0.09-0.11 mg / mL MAb to RBC.

[0022] Preferably, in the treatment solution, the polymer is composed of a mixture of PEG4000 and dextran T7 in a weight ratio of 10:0.5-1.5.

[0023] In one specific implementation, the weight ratio of PEG4000 to dextran T7 in the surfactant can be 10:0.5, 10:1, 10:1.5, 10:2, 10:3, 10:4, or 10:5.

[0024] Experimental analysis shows that the surfactant composed of PEG4000 and dextran T7 in the above weight ratio can further improve the application performance of the detection kit and detection method.

[0025] Preferably, in the treatment solution, the surfactant is composed of a mixture of Tween-20 and Pluronic F-127 in a weight ratio of 10:0.3-0.8.

[0026] Preferably, in the treatment solution, the surfactant is composed of a mixture of Tween-20 and Pluronic F-127 in a weight ratio of 10:0.4-0.7.

[0027] In one specific implementation, the weight ratio of Tween-20 to Pluronic F-127 in the surfactant can be 10:0.1, 10:0.3, 10:0.4, 10:0.5, 10:0.7, 10:0.8, or 10:1.

[0028] Experimental analysis shows that the surfactant composed of Tween-20 and Pluronic F-127 in the above weight ratio can further improve the application performance of the detection kit and detection method.

[0029] Preferably, the sample diluent is a solution with pH 7.3-7.5, 47-52 mM Tris, 1.2-1.8% sodium caseinate, 1.9-2.1% BSA, 0.9-1.1% NaCl, and 0.045-0.055% Proclin 300.

[0030] Preferably, the method for preparing the time-resolved fluorescent microsphere-labeled antibody is as follows: Activation: Mix 50 μL of fluorescent microspheres, 400-500 μL of 45-55 mM MES solution at pH 5.8-6.2, 3-5 μL of 8-12 mg / mL sulfo-NHS, and 4-6 μL of 8-12 mg / mL EDC, shake for 15-25 min, centrifuge for 5-15 min, and discard the supernatant; Labeling: Add 80-120 μL of 45-55 mM MES coupling solution with pH 5.8-6.2, add 50-70 μg of antibody, and shake for 100-140 min; Blocking: Add 20-30 μL of blocking solution, shake for 100-140 min, centrifuge for 8-12 min, and discard the supernatant; the blocking solution is a solution of pH 7.8-8.2 + 45-55 mM HEPES + 0.8-1.2% BSA + 0.08-0.12% Proclin-300. Washing: Add 180-220 μL of blocking solution, centrifuge for 8-12 min, and discard the supernatant; Preservation: Add 45-55 μL of preservation solution; the preservation solution is a solution with pH 7.2-7.6 + 8-12 mM BS + 0.8-1.2% BSA + 4.5-5.5% trehalose + 0.04-0.06% Proclin 300.

[0031] Secondly, this application also provides a method for the combined detection of placental growth factor and soluble FMS-like tyrosine kinase-1, using the aforementioned combined detection kit for placental growth factor and soluble FMS-like tyrosine kinase-1; specifically, it includes the following steps in sequence: Add the sample to be tested to the sample diluent at a dilution ratio of 1:1-2, then add it to the sample well of the test card, wait for the sample to spread for 8-12 minutes, and then detect the fluorescence value.

[0032] Preferably, the fluorescence value is detected using a fluorescence immunochromatographic analyzer, wherein the fluorescence excitation wavelength is 365 nm and the emission wavelength is 610 nm.

[0033] Thirdly, this application also provides a diagnostic kit for early-onset eclampsia in pregnant women, including a combined detection kit for placental growth factor and soluble FMS-like tyrosine kinase-1.

[0034] In summary, the technical solution of this application has the following effects: The kit provided in this application innovatively offers a novel quantitative detection method based on a combination of immunofluorescence and immunochromatography techniques to achieve the joint detection of placental growth factor and soluble FMS-like tyrosine kinase-1. This technology utilizes fluorescent microspheres to bind with antibodies to form fluorescently labeled antibodies, which are then detected through an antigen-antibody reaction. It retains the advantages of colloidal gold immunochromatography—simple operation, rapid detection, and high portability—while also achieving accurate quantification of the detection results through fluorescence tracing enhancement technology.

[0035] The kit provided in this application prepares a binding pad by pretreating and drying a fiber membrane with a pretreatment solution, followed by spraying time-resolved fluorescent microspheres labeled with antibodies. The precisely designed pretreatment solution formulation helps the subsequently dried labeled complex to be rapidly, uniformly, and completely reconstituted as the sample flows through; at the same time, it minimizes non-specific binding and ensures the sensitivity and accuracy of the detection. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the test strip structure in the combined detection kit for placental growth factor and soluble FMS-like tyrosine kinase-1 in the embodiments of this application. Detailed Implementation

[0037] The present application will be further described in detail below with reference to embodiments, comparative examples and performance test results. These embodiments should not be construed as limiting the scope of protection claimed in this application.

[0038] Information on the raw materials used in this application is shown in Table 1.

[0039] Table 1 Raw Material Information

[0040] Example

[0041] Example 1

[0042] Example 1 provides a combined detection kit and detection method for placental growth factor and soluble FMS-like tyrosine kinase-1.

[0043] In this embodiment, the kit consists of a test card, a sample diluent, and an IC card; the test card consists of a test strip and a plastic card shell; the test strip includes the following components: a sample pad, a conjugate pad, a coating membrane, and an absorbent pad.

[0044] The structural diagram of the test strip in this embodiment is shown below. Figure 1 As shown; it includes a PVC base plate, which supports the "chromatographic structure: sample pad, coating membrane, conjugate pad, and absorbent pad". The chromatographic structure has a sample pad, conjugate pad, coating membrane, and absorbent pad from the sample application end towards the sample chromatography direction; the sample pad, conjugate pad, coating membrane, and absorbent pad overlap sequentially; the conjugate pad presses against the lower end of the coating membrane, the sample pad presses against the conjugate pad, and the absorbent pad presses against the upper end of the coating membrane, with an overlap of 1-2 mm.

[0045] The preparation method of the test strip in this embodiment is as follows: (1) Preparation of coating membrane Coating solution: pH 7.4 + 10mM PBS + 1% trehalose solution, coating concentration 1mg / ml.

[0046] Using coating buffer, PLGF-T1 antibody, sFlt-1-T2 antibody, and C antibody (MAb to DNP) were diluted to 1 mg / ml. Then, using a gold sputtering membrane scribing instrument, PLGF-T1 antibody was streaked onto the detection line T1 of the NC membrane at a flow rate of 1 μL / cm; sFlt-1-T2 antibody was streaked onto the detection line T2 of the NC membrane; and C antibody (MAb to DNP) was streaked onto the control line C of the NC membrane. The membrane was then placed in an oven at 37°C and dried for 24 h to obtain the coated membrane.

[0047] (2) Preparation of binding pad (2.1) Pretreatment of the bonding pad The pretreatment solution was a pH 8.1 solution containing 10 mM BS, 2% BSA, 0.5% polymer, 0.025% surfactant, 6% sucrose, 0.15 mg / mL HIER-R-009 inhibitor, and 0.1 mg / mL MAb to RBC. The polymer was a mixture of PEG4000 and dextran T7 in a weight ratio of 10:1. The surfactant was a mixture of Tween-20 and Pluronic F-127 in a weight ratio of 10:0.5.

[0048] The fiberglass membrane was immersed in the pretreatment solution and soaked at room temperature for 30 minutes. Then the fiberglass membrane was removed and placed in an oven at 37°C for drying for 16 hours.

[0049] (2.2) Preparation of time-resolved fluorescent microsphere labeled antibody Activation: Mix 50 μL of fluorescent microspheres, 450 μL of 50 mM MES solution at pH 6.0, 4 μL of 10 mg / mL sulfo-NHS, and 5 μL of 10 mg / mL EDC, shake for 20 min, centrifuge at 6000 rpm for 10 min, and discard the supernatant. Labeling: Add 100 μL of 50 mM MES coupling buffer at pH 6.0, add 60 μg of antibody, and shake for 120 min. Blocking: Add 25 μL of blocking solution, shake for 120 min, centrifuge at 6000 rpm for 10 min, and discard the supernatant; the blocking solution is a solution with pH 8.0 + 50 mM HEPES + 1% BSA + 0.1% Proclin-300. Washing: Add 200 μL of blocking solution, centrifuge at 6000 rpm for 10 min, and discard the supernatant; Preservation: Add 50 μL of preservation solution; the preservation solution is a solution with pH 7.4 + 10 mM BS + 1% BSA + 5% trehalose + 0.05% Proclin 300; that is, time-resolved fluorescent microsphere-labeled PLGF monoclonal antibody I, time-resolved fluorescent microsphere-labeled sFlt-1-1 monoclonal antibody I, and time-resolved fluorescent microsphere-labeled C (DNP-BSA Conjugated) are prepared respectively.

[0050] (2.3) Spray pad The time-resolved fluorescent microsphere-labeled PLGF monoclonal antibody I, time-resolved fluorescent microsphere-labeled sFlt-1-1 monoclonal antibody I, and time-resolved fluorescent microsphere-labeled C prepared in step (2.2) were mixed in a weight ratio of 1:1:1. The mixture was then sprayed onto the pretreated conjugate pad in step (2.1) using a three-dimensional streak sprayer at a spray rate of 3 μL / cm. The mixture was then placed in an oven at 37°C and dried for 4 hours to obtain the conjugate pad.

[0051] (3) Detection method Turn on the instrument, verify the IC card information, and ensure that the IC card batch number matches the test card batch number. The machine will then read the IC card information.

[0052] Dilute the sample to be tested with sample diluent at a dilution ratio of 1:1, then use a pipette to add 100 μL of the sample solution (serum / plasma / whole blood) into the sample well of the test card, and wait for the sample to spread for 10 minutes; The sample diluent was a solution with pH 7.4, 50 mM Tris, 1.5% sodium caseinate, 1% BSA, 1% NaCl, and 0.05% Proclin 300.

[0053] Insert the test card into the fluorescence immunochromatographic analyzer (FA8000 fluorescence immunochromatographic analyzer manufactured by Qingdao Huajing Biotechnology Co., Ltd.), click "Test" to detect the fluorescence signal intensity of the test card. The IC card contains calibration curve information. The machine can directly output concentration information, that is, the output results are the concentration of PLGF, the concentration of sFIt-1 and the ratio of sFIt-1 / PLGF in the sample to be tested.

[0054] Examples 2-4 Examples 2-4 provide a combined detection kit and detection method for placental growth factor and soluble FMS-like tyrosine kinase-1, respectively.

[0055] The difference between the above embodiments and Embodiment 1 is that the composition of the pretreatment solution in the conjugate pad pretreatment is different.

[0056] In Example 2: The pretreatment solution contains a polymer composed of PEG4000 and dextran T7 in a weight ratio of 10:5 (i.e., 2:1).

[0057] In Example 3: The surfactant in the pretreatment solution is a mixture of Tween-20 and Pluronic F-127 in a weight ratio of 10:0.1.

[0058] In Example 4: The pretreatment solution contains a surfactant composed of Tween-20 and Pluronic F-127 in a weight ratio of 10:1.

[0059] All other process parameters in the above embodiments are the same as those in Embodiment 1.

[0060] Examples 5-6 Examples 5-6 respectively provide a combined detection kit and detection method for placental growth factor and soluble FMS-like tyrosine kinase-1.

[0061] The difference between the above embodiments and Embodiment 1 is that the composition of the sample diluent is different.

[0062] In Example 5: The sample dilution solution was a solution with pH 7.4 + 50mM Tris + 1.0% sodium caseinate + 1% BSA + 1% NaCl + 0.05% Proclin 300.

[0063] In Example 6: The sample dilution solution was a solution with pH 7.4 + 50mM Tris + 2.0% sodium caseinate + 1% BSA + 1% NaCl + 0.05% Proclin 300.

[0064] All other process parameters in the above embodiments are the same as those in Embodiment 1.

[0065] Comparative Example Comparative Examples 1-6 Comparative Examples 1-6 provide a combined detection kit and detection method for placental growth factor and soluble FMS-like tyrosine kinase-1, respectively.

[0066] The differences between the above comparative examples and Example 1 are as follows.

[0067] In Comparative Example 1: In the preparation method of the binding pad, the fiber membrane was not pretreated and time-resolved fluorescent microspheres labeled with antibodies were directly sprayed on.

[0068] In Comparative Example 2: the pretreatment solution contained a polymer composed of PEG5000 and dextran T7 in a weight ratio of 10:1.

[0069] In Comparative Example 3: the pretreatment solution contained a polymer composed of PEG4000 and dextran T7 in a weight ratio of 1:10.

[0070] In Comparative Example 4: the pretreatment solution contained a surfactant composed of Tween-20 and Pluronic F-68 in a weight ratio of 10:0.5.

[0071] In Comparative Example 5: the pretreatment solution contained a surfactant composed of Tween-20 and Pluronic F-127 in a weight ratio of 0.5:10.

[0072] In Comparative Example 6: The sample dilution solution was a solution with pH 7.4 + 50mM Tris + 1.5% sodium caseinate + 0.025% surfactant PEG4000 + 1% BSA + 1% NaCl + 0.05% Proclin 300.

[0073] All other process parameters in the above comparative examples are the same as those in Example 1.

[0074] Performance testing (1) Limit of detection and linear range Using the detection kits and methods of the examples and comparative examples, the lowest detection limit and correlation coefficient of placental growth factor and soluble fms-like tyrosine kinase-1 were determined.

[0075] Limit of Detection (LOD): The test sample standard was diluted to eight concentrations (PLGF: 50 pg / mL, 40 pg / mL, 30 pg / mL, 20 pg / mL, 15 pg / mL, 10 pg / mL, 5 pg / mL, 2 pg / mL; sFIt-1: 60 pg / mL, 50 pg / mL, 40 pg / mL, 30 pg / mL, 20 pg / mL, 15 pg / mL, 10 pg / mL, 5 pg / mL). Each concentration was measured five times. The LOD was determined based on whether the corresponding concentration could be detected.

[0076] Correlation coefficient of linear range: The test sample standard was diluted to 5 concentrations at a certain ratio (PLGF: 2000 pg / mL, 1000 pg / mL, 500 pg / mL, 100 pg / mL, 50 pg / mL; sFIt-1: 20000 pg / mL, 10000 pg / mL, 5000 pg / mL, 1000 pg / mL, 100 pg / mL). Each concentration was measured three times, and the average value was calculated. The average value of the measured concentration was then fitted to the theoretical concentration using the least squares method to form a linear relationship, and the linear correlation coefficient r was calculated.

[0077] (2) Accuracy Using the detection kits and methods of the examples and comparative examples, placental growth factor at a concentration of 100 pg / mL and soluble FMS-like tyrosine kinase-1 standard were used as test samples for detection. The results were substituted into the standard curve of the corresponding examples to calculate the detection values ​​and the accuracy. Each sample was measured 6 times, and the relative deviation of the accuracy was calculated.

[0078] (3) Repeatability Using the detection kits and methods described in the examples and comparative examples, placental growth factor at a concentration of 100 pg / mL and soluble FMS-like tyrosine kinase-1 standard were used as test samples to detect intra-batch and inter-batch coefficients of variation, in order to examine the repeatability of the detection kits and methods in this application.

[0079] Intra-batch coefficient of variation: Each sample was tested 10 times using the same batch of reagent kits, and the intra-batch coefficient of variation of the detection method in this application was calculated.

[0080] Inter-batch coefficient of variation: Each sample was tested using three different batches of reagent kits, and the inter-batch coefficient of variation of the detection method in this application was calculated.

[0081] Test results are shown in Table 2.

[0082] Table 2. Detection results in the examples and comparative examples.

[0083] Analysis of the test results in Table 2 shows that the detection kit and method provided in this application exhibit good performance indicators for the combined detection of placental growth factor and soluble FMS-like tyrosine kinase-1, specifically: 1) Limit of detection: the limit of detection for placental growth factor (PLGF) is no higher than 20 pg / mL; the limit of detection for soluble FMS-like tyrosine kinase-1 (sFlt-1) is no higher than 30 pg / mL; 2) The correlation coefficient r for the linear range of PLGF is ≥0.9910; the correlation coefficient r for the linear range of sFlt-1 is ≥0.9900; 3) Accuracy: the relative deviations of PLGF and sFlt-1 are both within 11.0%; 4) Repeatability: both the intra-assay coefficient of variation and the inter-assay coefficient of variation are less than 12%. These test results demonstrate that the detection kit and method provided in this application have high sensitivity, accuracy, and repeatability for the combined detection of placental growth factor and soluble FMS-like tyrosine kinase-1.

[0084] (4) Specificity The detection kit and detection method provided in this application have good specificity when jointly detecting placental growth factor and soluble fms-like tyrosine kinase-1. Specifically, when the concentration of placental growth factor (PLGF) is not less than 25,000 pg / mL and the concentration of soluble fms-like tyrosine kinase-1 (sFlt-1) is not less than 3,500 pg / mL, the detection result is not higher than 40 pg / mL.

[0085] (5) Anti-interference The detection kit and detection method provided in this application also have good anti-interference properties when jointly detecting placental growth factor and soluble FMS-like tyrosine kinase-1. Triglycerides (<10mg / mL), cholesterol (<5mg / mL), hemoglobin (<5mg / mL), and bilirubin (<0.4mg / mL) in the test sample will not interfere with the test.

[0086] (6) HOOK effect The detection kit and detection method provided in this application do not produce a hook effect when the PLGF concentration in the sample is 10 ng / mL and the sFlt-1 concentration is 100 ng / mL.

[0087] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A combined detection kit for placental growth factor and soluble FMS-like tyrosine kinase-1, characterized in that, It includes the following components: sample pad, conjugate pad, coating membrane, absorbent pad, and sample diluent; The coating membrane is an NC membrane coated with antibodies; The method for preparing the conjugate pad is as follows: the fiber membrane is treated with a pretreatment solution and dried, and then time-resolved fluorescent microspheres labeled with antibodies are sprayed on it. The pretreatment solution is a solution with pH 8.0-8.4, consisting of 8-12 mM BS, 1.8-2.2% BSA, 0.4-0.6% polymer, 0.02-0.03% surfactant, 5-7% sucrose, 0.13-0.18 mg / mL HIER-R-009 inhibitor, and 0.08-0.12 mg / mL MAb to RBC. The polymer is a mixture of PEG4000 and dextran T7 in a weight ratio of 10:0.5-5. The surfactant is a mixture of... Composed of 0.1-1 Tween-20 and Pluronic F-127; The sample diluent was a solution with pH 7.2-7.6, 45-55 mM Tris, 1.0-2.0% sodium caseinate, 1.8-2.2% BSA, 0.8-1.2% NaCl, and 0.04-0.06% Proclin 300.

2. The combined detection kit for placental growth factor and soluble FMS-like tyrosine kinase-1 according to claim 1, characterized in that, The NC membrane-coated antibody consists of a detection line T1 coated with PLGF monoclonal antibody II, a detection line T2 coated with sFlt-1 monoclonal antibody II, and a control line C coated with MAb to DNP antibody, sequentially arranged on the NC membrane. In the preparation method of the NC membrane-coated antibody, the coating solution is a solution of pH 7.2-7.6 + 8-12mM PBS + 0.8-1.2% trehalose; the coating concentration is 0.8-1.2 mg / ml.

3. The combined detection kit for placental growth factor and soluble FMS-like tyrosine kinase-1 according to claim 1, characterized in that, The pretreatment solution is a solution with pH 8.1-8.3, 9-11 mM BS, 1.9-2.1% BSA, 0.45-0.55% polymer, 0.022-0.028% surfactant, 5.5-6.5% sucrose, 0.14-0.17 mg / mL inhibitor HIER-R-009, and 0.09-0.11 mg / mL MAb to RBC.

4. The combined detection kit for placental growth factor and soluble FMS-like tyrosine kinase-1 according to claim 1, characterized in that, In the treatment solution, the polymer is composed of PEG4000 and dextran T7 in a weight ratio of 10:0.5-1.

5.

5. The combined detection kit for placental growth factor and soluble FMS-like tyrosine kinase-1 according to claim 1, characterized in that, In the treatment solution, the surfactant is composed of a mixture of Tween-20 and Pluronic F-127 in a weight ratio of 10:0.3-0.

8.

6. The combined detection kit for placental growth factor and soluble FMS-like tyrosine kinase-1 according to claim 1, characterized in that, In the treatment solution, the surfactant is composed of a mixture of Tween-20 and Pluronic F-127 in a weight ratio of 10:0.4-0.

7.

7. The combined detection kit for placental growth factor and soluble FMS-like tyrosine kinase-1 according to claim 1, characterized in that, The sample diluent was a solution with pH 7.3-7.5, 47-52 mM Tris, 1.2-1.8% sodium caseinate, 1.9-2.1% BSA, 0.9-1.1% NaCl, and 0.045-0.055% Proclin 300.

8. The combined detection kit for placental growth factor and soluble FMS-like tyrosine kinase-1 according to claim 1, characterized in that, The preparation method of the time-resolved fluorescent microsphere labeled antibody is as follows: Activation: Mix 50 μL of fluorescent microspheres, 400-500 μL of 45-55 mM MES solution at pH 5.8-6.2, 3-5 μL of 8-12 mg / mL sulfo-NHS, and 4-6 μL of 8-12 mg / mL EDC, shake for 15-25 min, centrifuge for 5-15 min, and discard the supernatant; Labeling: Add 80-120 μL of 45-55 mM MES coupling solution with pH 5.8-6.2, add 50-70 μg of antibody, and shake for 100-140 min; Blocking: Add 20-30 μL of blocking solution, shake for 100-140 min, centrifuge for 8-12 min, and discard the supernatant; the blocking solution is a solution of pH 7.8-8.2 + 45-55 mM HEPES + 0.8-1.2% BSA + 0.08-0.12% Proclin-300. Washing: Add 180-220 μL of blocking solution, centrifuge for 8-12 min, and discard the supernatant; Preservation: Add 45-55 μL of preservation solution; the preservation solution is a solution with pH 7.2-7.6 + 8-12 mM BS + 0.8-1.2% BSA + 4.5-5.5% trehalose + 0.04-0.06% Proclin 300.

9. A method for the combined detection of placental growth factor and soluble FMS-like tyrosine kinase-1, characterized in that, The detection was performed using the combined detection kit for placental growth factor and soluble FMS-like tyrosine kinase-1 as described in any one of claims 1-8; specifically, the following steps were performed sequentially: Add the sample to be tested to the sample diluent at a dilution ratio of 1:1-2, then add it to the sample well of the test card, wait for the sample to spread for 8-12 minutes, and then detect the fluorescence value.

10. A diagnostic kit for early-onset preeclampsia in pregnant women, characterized in that, The kit includes the combined detection kit for placental growth factor and soluble FMS-like tyrosine kinase-1 as described in any one of claims 1-8.

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