Method for detecting active soluble urokinase receptor and detection kit

Abstract

The present invention relates to a method for detecting active soluble urokinase receptor and a detection kit, the active soluble urokinase receptor is also called active soluble urokinase-type plasminogen activator receptor (suPAR). The method comprises the following steps: contacting a capture reagent with a sample to be tested to form a complex of the capture reagent and active suPAR; combining the formed complex with a reagent that specifically binds to the captured active suPAR, and detecting the captured active suPAR by detecting the reagent that specifically binds to the captured active suPAR, wherein the capture reagent is a fusion protein containing ATF. The present invention also relates to a kit used in the above detection method. The method of the present invention presents excellent technical effects as described in the specification.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical detection, and specifically relates to a method for detecting active urokinase receptors, and in particular to a method for detecting active soluble urokinase receptors, which are also called active soluble urokinase-type plasminogen activator receptors (Active soluble uPAR, active suPAR). The present invention also relates to a kit used in the above detection method. Background Art

[0002] CN105954522B (Chinese patent application No. 201610541379.6) discloses a method for detecting an active urokinase receptor, and the entire contents of which are incorporated herein by reference. Urokinase receptor (urokinase type plasminogenacitivator receptor, urokinase type plasminogen activator receptor, uPAR) is a cell surface receptor. The receptor was discovered by Stoppelli et al. in 1985, Roldan et al. cloned its cDNA in 1989, and Behrendt et al. purified the protein from the cell membrane extract of human lymphoma cell U937 by affinity chromatography in 1990. At the Fifth International Leukocyte Differentiation Antigen Conference, uPAR was named cluster of differentiation antigen 87 (cluster of differentiation 87, CD87). uPAR is a highly glycosylated surface membrane protein that is widely expressed on the surface of immune cells, such as activated neutrophils, monocytes, activated T lymphocytes, macrophages, and a variety of malignant tumor cells, but is expressed at a low level on the surface of most normal cells [A. Estreicher, et al. The Journal of cell biology 111 (2) (1990) 783-92; C. Pyke, et al. Histopathology 24 (2) (1994) 131-8; M. Thuno, et al. Disease markers 27 (3) (2009) 157-72; F. Blasi, et al. Molecular cell biology 3 (12) (2002) 932-43; T. Plesner, et al. Stem cells 15 (6) (1997) 398-408].

[0003] As a flexible molecule, uPAR can interact with a variety of ligands or receptors, such as vitronectin, urokinase-type plasminogen activator (uPA), low density lipoprotein receptor-related protein 1 (LRP1), integrin, G protein coupled receptor (GPCR), etc. These different interactions play a wide range of important roles in various physiological and pathological processes in the human body, including plasminogen activation, cell adhesion and migration, cell differentiation, chemoactivation, chemokine receptor regulation, immune response, inflammatory response, etc. uPAR is composed of three cysteine-rich Ly6 / uPAR domains (D1, D2, D3) with a size of 81-87 amino acids, and a molecular weight of about 55kDa. uPAR is anchored to the cell membrane surface through the glycosylphosphatidylinositol (GPI) at its C-terminus [F. Blasi, et al. Nature reviews. Molecular cell biology 3(12)(2002)932-43; HW Smith, et al. Nature reviews. Molecular cell biology 11(1)(2010)23-36].

[0004] Urokinase receptors exist in various forms, including full-length membrane receptors, soluble urokinase receptors without transmembrane domains (soluble uPAR, suPAR), and various degradation fragments. Full-length uPAR membrane receptors are susceptible to hydrolysis by phosphatidylase C, causing uPAR to fall off the cell membrane surface to form soluble urokinase receptors without glycosylated phosphatidylinositol (soluble uPAR, suPAR). In addition, uPAR is also sensitive to a variety of hydrolases and can be further hydrolyzed into D1 and D2-D3 fragments [M. Thuno, et al. suPAR: the molecular crystal ball, Disease markers 27 (3) (2009) 157-72].

[0005] Existing crystal structure studies have shown that uPAR forms a bowl-shaped structure through its three domains, and it is this bowl-shaped structure that can efficiently bind to its ligand uPA [C. Yuan, M. Huang, Cellular and molecular lifesciences: CMLS 64 (9) (2007) 1033-7], enrich uPA on the cell surface, activate plasminogen to plasmin, and then degrade the extracellular matrix, playing an important role in cell migration. In addition, it has been proven that the binding of uPA to uPAR can greatly enhance the binding of uPAR to vitronectin [Q. Huai, et al. Nature structural & molecular biology 15 (4) (2008) 422-3], and a large number of literatures have also proven the importance of the binding of uPA to uPAR for its interaction with integrins [HW Smith, et al. Molecular cell biology 11 (1) (2010) 23-36; C. Yuan, M. Huang, Cellular and molecular life sciences: CMLS 64 (9) (2007) 1033-7]. These proteins adhere to and accumulate with other proteins such as caveolin in the lesion, and the integrin receptors initiate intracellular signals, thereby transmitting signals from the extracellular space into the cell, activating intracellular protein kinases, and promoting cell division and cell migration. This type of uPAR is defined as active suPAR. Only active uPAR can conduct signals, which is closely related to tumor invasion and metastasis, while other uPAR fragments are inactive. There is currently no method for measuring active suPAR.

[0006] Since the content of uPAR is closely related to the disease, there are many diagnostic methods for uPAR on the market. They are mainly tested by double antibody sandwich method, and generally measure the total amount of active and inactive suPAR in the blood. For example, the enzyme-linked immunosorbent assay (ELISA) kit for detecting suPAR launched by ViroGates of Denmark has passed the European CE-IVD (in vitro diagnostic reagent) certification and is used to guide clinical practice. This method is based on the fact that an increased concentration of total suPAR in the blood indicates that the patient's immune system is continuously activated [M. Thuno, et al. Disease markers 27 (3) (2009) 157-72]. The concentration level of total suPAR in the blood can also be used as an assessment indicator of disease progression and for clinical decision-making on patient risk status [O. Slot, et al. Annals of therheumatic diseases 58 (8) (1999) 488-92; M. Persson, et al. Atherosclerosis 220 (2) (2012) 502-5; N. Sidenius, et al. Blood 96 (13) (2000) 4091-5]. In addition, suPAR can also be used to screen the severity of patients in intensive care units and evaluate the effectiveness of treatment for patients [I. Casagranda, et al. Internal and emergency medicine 10 (6) (2015) 725-30; R. Uusitalo-Seppala, et al. Journal of internal medicine 272 (3) (2012) 247-56]. Specifically, a suPAR concentration range of 0.1-4.0 μg / L indicates that the subject is normal and has no infection or inflammatory response; 4-6 μg / L indicates that the subject may have an infection or abnormal activation of the immune system and requires further examination; >6 μg / L indicates that the disease is rapidly progressing and requires close monitoring and follow-up treatment. However, currently, methods similar to these antibody double sandwich methods detect the total value of multiple forms of suPAR in the blood and cannot directly detect the content of active suPAR in the blood.

[0007] Although the method for detecting active urokinase receptor disclosed in the prior application CN105954522B (Chinese patent application No. 201610541379.6) can directly detect the content of active suPAR in the blood, it still has aspects that need to be improved. Summary of the invention

[0008] The object of the present invention is to provide a method for detecting active urokinase receptor, or to provide a method for detecting soluble urokinase receptor, which is also called active soluble urokinase-type plasminogen activator receptor (Active soluble urokinase-type plasminogen activator receptor, Active soluble uPAR, Active suPAR). Alternatively, one object of the present invention is to provide a kit used in the above detection method, and a detection reagent used in the kit.

[0009] To this end, the first aspect of the present invention provides a method for detecting soluble urokinase-type plasminogen activator receptor in a biological sample, comprising the following steps:

[0010] 1) contacting the capture reagent with the sample to be tested under conditions suitable for the capture reagent to capture active suPAR in the sample to be tested, thereby forming a complex of the capture reagent and the active suPAR;

[0011] 2) allowing the formed complex to bind to a reagent that specifically binds to the captured active suPAR, and detecting the captured active suPAR by detecting the reagent that specifically binds to the captured active suPAR,

[0012] in:

[0013] The capture reagent is a fusion protein containing ATF;

[0014] The reagent that specifically binds to the captured active suPAR is a monoclonal antibody that binds to the outside of the active suPAR.

[0015] According to the method of the first aspect of the present invention, the monoclonal antibody that binds to the outside of active suPAR is a monoclonal anti-suPAR antibody obtained by immunizing mice with uPAR-D2D3 (amino acids 88-283) and screening using hybridoma technology.

[0016] According to the method of the first aspect of the present invention, the monoclonal antibody that binds to the outside of active suPAR is antibody ATN-658.

[0017] According to the method of the first aspect of the present invention, sodium glycerophosphate is also added along with the monoclonal antibody, and the mass ratio of the two additions is 1:50-100, for example, 1:75.

[0018] According to the method of the first aspect of the present invention, the reagent that specifically binds to the captured active suPAR further comprises a secondary antibody that binds to the monoclonal antibody, and the secondary antibody is, for example, anti-mouse IgG labeled with alkaline phosphatase.

[0019] According to the method of the first aspect of the present invention, the fusion protein of ATF is a fusion protein of ATF and another protein or polypeptide or its fragment. The another protein or polypeptide or its fragment can be serum albumin, such as human serum albumin (HSA), bovine serum albumin (BSA), or ovalbumin (OVA). In a preferred embodiment, the fusion protein containing ATF is an ATF-HSA fusion protein.

[0020] According to the method of the first aspect of the present invention, the fusion protein of ATF is fixed on a solid matrix. The solid matrix includes but is not limited to a multi-well plate (such as a 96-well plate), a protein chip bottom membrane (such as a nitrocellulose membrane, a nylon membrane, etc.), a magnetic bead, a fluorescent microsphere, etc.

[0021] The method according to the first aspect of the present invention comprises the following steps:

[0022] (1) Adding ATF-HSA protein solution diluted with coating solution to the wells of a multi-well plate (in any embodiment of the present invention, unless otherwise specified, the multi-well plate (also known as ELISA plate) can be replaced by magnetic beads, fluorescent microspheres, protein chip bottom membrane (e.g., nitrocellulose membrane, nylon membrane, etc.) and other conventional carriers in the art as solid matrix) for coating (overnight at 4°C), washing and drying; (2) Adding blocking solution to the wells for blocking, incubating at room temperature, washing and drying; (3) Adding a series of concentrations of active suPAR standard, add sample diluent to other wells as blank control, add the sample to be tested to other wells (for example, diluent diluted 10 times with sample diluent); incubate at room temperature, wash and dry; (4) add mouse anti-human ssuPAR monoclonal antibody such as ATN-658 antibody diluted with sample diluent to all wells, incubate at room temperature, wash and dry; (5) add alkaline phosphatase-labeled anti-mouse IgG (for example, alkaline phosphatase-labeled secondary antibody (Anti-mouse IgG, AP-linked Antibody)) to all wells, incubate at room temperature, wash and dry; (6) add color development solution to each well, then place the multi-well plate on a microplate reader and read the absorbance at 405 nm; (7) obtain a regression equation based on the kinetic process (enzyme reaction rate) of the absorbance change of different concentrations of active suPAR standards over time and the concentration of the standards, and use the regression equation to calculate the concentration of active suPAR in the sample.

[0023] The method according to the first aspect of the present invention comprises the following steps:

[0024] (1) Add 100 μl of ATF-HSA protein solution (100-150 μg / ml, e.g., 120 μg / ml) diluted with coating solution to the wells of the multi-well plate for coating, incubate at 4°C overnight, wash and dry; (2) Add 100 μl of blocking solution to the wells for blocking, incubate at room temperature at 80 rpm for 1 hour, wash and dry; (3) Add 100 μl of active suPAR standard of different concentrations to some wells, wherein the different concentrations are distributed in the concentration range of 0.01-2 μg / L, e.g., 0. 03~1μg / L concentration range, for example, 1μg / L, 0.5μg / L, 0.25μg / L, 0.125μg / L, 0.0625μg / L, 0.03125μg / L; add 100μl of sample diluent to other wells as blank control, the blank control does not contain uPAR; add 10 times dilution of the sample to be tested with sample diluent to other wells; incubate at room temperature 80 rpm for 1 hour, wash and dry; (4) add 100μl to all wells 5-15 μg / ml, for example, 12-15 μg / ml, of a mouse anti-human suPAR monoclonal antibody diluted with a sample diluent, incubated at room temperature at 80 rpm for 1 hour, washed and dried (for example, the monoclonal antibody is a monoclonal anti-suPAR antibody obtained by immunizing mice with uPAR D2D3 (amino acids 88-283) and screening with hybridoma technology, such as ATN-658 antibody); or, (a) 100 μl of a 5 μg / ml mouse anti-human suPAR monoclonal antibody diluted with a sample diluent and (b) 25 μl of a 1.5 mg / ml sodium glycerophosphate solution are added to all wells, incubated at room temperature at 80 rpm for 1 hour, washed and dried (for example, the monoclonal antibody is a monoclonal anti-suPAR antibody obtained by immunizing mice with uPAR D2D3 (amino acids 88-283) and screening with hybridoma technology, such as ATN-658 antibody). D2D3 (amino acids 88-283) immunized mice, and a monoclonal anti-suPAR antibody obtained by hybridoma technology screening, such as ATN-658 antibody); (5) adding 100 μl of alkaline phosphatase-labeled anti-mouse IgG (such as alkaline phosphatase-labeled secondary antibody (Anti-mouse IgG, AP-linked Antibody)) diluted 500 times with sample diluent to all wells, incubating at room temperature at 80 rpm for 1 hour, washing and drying; (6) adding 100 μl of color development solution to each well, then placing the ELISA plate on an ELISA reader and reading the absorbance at 405 nm for a total of 60 minutes, with the absorbance value read every 1 minute; (7) obtaining a regression equation based on the kinetic process (enzyme reaction rate) of the absorbance change of different concentrations of active suPAR standards over time and the concentration of the standards, and using the regression equation to calculate the concentration of active suPAR in the sample.

[0025] The method according to the first aspect of the present invention comprises the following steps:

[0026] (1) Treatment of plasma samples: Place peripheral blood collected from the test subject in an EDTA-2Na anticoagulant tube, centrifuge to separate plasma, dilute with sample diluent, aliquot, and set aside; (2) Coating: Add 100 μl of ATF-HSA protein solution (120 μg / ml) diluted with coating solution to all wells 1A-12H of the ELISA plate (i.e., multi-well plate, 96 wells), incubate at 4°C overnight; wash with detergent, and dry; (3) Blocking: Add 100 μl of blocking solution to all wells 1A-12H of the ELISA plate, seal, incubate, wash with detergent, and dry; (4) Sample addition: Add 100 μl of active suPAR standard of different concentrations to ELISA plates 1A-1F, add 100 μl of sample diluent to G1 and G2 as blank controls, add diluted plasma samples of the test subject to other wells in the ELISA plate, seal, incubate at room temperature, wash with detergent, and dry; (5) Add primary antibody: Add (a) 100 μl of 5 μg / ml ATN-658 antibody diluted with sample diluent and (b) 25 μl of 1.5 mg / ml sodium glycerophosphate solution to all wells of ELISA plates 1A-12H, seal, incubate at room temperature, wash with detergent, and dry; (6) Add enzyme-labeled secondary antibody: add 100 μl of alkaline phosphatase-labeled secondary antibody diluted 500 times with sample diluent to all wells of ELISA plates 1A-12H, seal, incubate at room temperature, wash with detergent, and dry; (7) Add colorimetric solution: quickly add 100 μl of colorimetric solution to each well, then place the ELISA plate on an ELISA reader and read the absorbance at 405 nm for a total of 60 minutes, with the absorbance value read every 1 minute; obtain a regression equation based on the kinetic process (enzyme reaction rate) of the absorbance change of active suPAR standards of different concentrations over time and the concentration of the standards, and use the regression equation to calculate the concentration of active suPAR in the sample.

[0027] The method according to the first aspect of the present invention comprises the following steps:

[0028] (1) Treatment of plasma samples: Place peripheral blood collected from the test subject in an EDTA-2Na anticoagulant tube, centrifuge at 4°C and 1500g for 30 min, collect plasma, dilute 10 times with sample diluent, dispense into 1.5 ml centrifuge tubes, and store at -80°C for later use; (2) Coating: Add 100 μl of ATF-HSA protein solution (120 μg / ml) diluted with coating solution to all wells 1A to 12H of the ELISA plate (i.e., multi-well plate, 96 wells), seal the 96-well plate with sealing tape to prevent liquid evaporation, and store at 4°C overnight; wash and dry (remove the sealing tape, spin dry the liquid in the wells of the ELISA plate, and use a spray gun to absorb the washing solution. Wash each well 6 times. After the last wash, gently pat the ELISA plate dry on absorbent paper and ensure that there are no bubbles in the wells); (3) Blocking: Add 100 μl of ATF-HSA protein solution (120 μg / ml) diluted with coating solution to all wells 1A to 12H of the ELISA plate (i.e., multi-well plate, 96 wells), seal the 96-well plate with sealing tape to prevent liquid evaporation, and store at 4°C overnight; wash and dry (remove the sealing tape, spin dry the liquid in the wells of the ELISA plate, and use a spray gun to absorb the washing solution. Wash each well 6 times. After the last wash, gently pat the ELISA plate dry on absorbent paper and ensure that there are no bubbles in the wells); (4) Blocking: Add 100 μl of ATF-HSA protein solution (120 μg / ml) diluted with coating solution to all wells 1A to 12H of the ELISA plate (i.e., multi-well plate, 96 wells), seal the 96- Add 100 μl of blocking solution to all wells 1A-12H in the ELISA plate, and seal the 96-well plate with sealing tape to prevent evaporation of the liquid. Incubate on a shaker at room temperature and 80 rpm for 1 hour. Remove the sealing tape, shake dry the liquid in the wells of the ELISA plate, and use a spray gun to absorb the washing solution and wash each well 6 times. After the last wash, gently pat the ELISA plate dry on absorbent paper to ensure that there are no bubbles in the wells. (4) Sample addition: Add 100 μl of active suPAR standard of different concentrations to ELISA plates 1A-1F, respectively, with two replicate wells for each concentration (A1, A2: 1 μg / L; B1, B2: 0.5 μg / L; C1, C2: 0.25 μg / L; D1, D2: 0.125 μg / L; E1, E2: 0.0625 μg / L; F1, F2: 0.03125 μg / L). Add 100 μl of sample diluent to G1 and G2 as blank controls. Then add the diluted plasma samples of the test subjects to the other wells in the ELISA plate, with 2 replicates for each plasma sample, and then seal the 96-well plate with sealing tape and incubate on a shaker at room temperature at 80 rpm for 1 hour. Remove the sealing tape, spin dry the liquid in the wells of the ELISA plate, and use a spray gun to absorb the washing solution. Wash each well 6 times. After the last wash, gently pat the ELISA plate dry on absorbent paper and ensure that there are no bubbles in the wells; (5) Add primary antibody: Add (a) 100 μl of 5 μg / ml ATN-658 antibody diluted with sample diluent and (b) 25 μl of 1.5 mg / ml sodium glycerophosphate solution to all wells of ELISA plates 1A-12H, seal the 96-well plate with sealing tape, and incubate on a shaker at room temperature at 80 rpm for 1 hour.Remove the sealing tape, shake dry the liquid in the wells of the ELISA plate, and use a pistol to absorb the washing solution. Wash each well 6 times. After the last wash, gently pat the ELISA plate dry on absorbent paper and ensure that there are no bubbles in the wells. (6) Add enzyme-labeled secondary antibody: Add 100 μl of alkaline phosphatase-labeled secondary antibody (Anti-mouse IgG, AP-linked Antibody) diluted 500 times with sample diluent to all wells 1A-12H of the ELISA plate, seal the 96-well plate with sealing tape, and incubate on a shaker at room temperature at 80 rpm for 1 hour. Remove the sealing tape, shake dry the liquid in the wells of the ELISA plate, and use a pistol to absorb the washing solution. Wash each well 6 times, and then use a pistol to absorb deionized water to wash each well 3 times. Gently pat the ELISA plate dry on absorbent paper and ensure that there are no bubbles in the wells; (7) Add colorimetric solution: Then use a dispenser to quickly add 100 μl of colorimetric solution to each well, then place the ELISA plate on an ELISA reader and read the absorbance at 405 nm for a total of 60 minutes, with the absorbance value read every 1 minute; obtain a regression equation based on the kinetic process (enzyme reaction rate) of the absorbance change of active suPAR standards of different concentrations over time and the concentration of the standards, and use the regression equation to calculate the concentration of active suPAR in the sample.

[0029] According to the method of the first aspect of the present invention, the sample to be tested is a plasma sample. In one embodiment, the plasma sample is processed as follows: peripheral blood collected from the subject to be tested is placed in an EDTA-2Na anticoagulant tube, centrifuged at 4°C and 1500g for 30 minutes, the plasma is separated, diluted 10 times with a sample diluent, and dispensed into 1.5 ml centrifuge tubes, and stored at -80°C for use.

[0030] According to the method of the first aspect of the present invention, the ATF-HSA fusion protein is prepared according to the method described in Example 1 of the present invention; it can also be purchased from commercial sources or prepared by referring to methods in other literature.

[0031] According to the method of the first aspect of the present invention, the active suPAR standard is obtained by purifying the recombinant suPAR expressed in S2 Drosophila embryonic cells via an affinity column combined with an ion column.

[0032] According to the method of the first aspect of the present invention, the ATN-658 antibody is a product purchased from Antibody System with the product number FHF99110, with a specification of 100 ug / vial and 1 mg / ml.

[0033] According to the method of the first aspect of the present invention, the sodium glycerophosphate is β-sodium glycerophosphate.

[0034] According to the method of the first aspect of the present invention, the coating solution is an aqueous solution at pH 9.6 containing 40 mM NaHCO 3 and 10 mM Na 2 CO 3 .

[0035] According to the method of the first aspect of the present invention, the blocking solution is a solution comprising 10% w / v bovine serum albumin / 10 mM sodium phosphate / 150 mM NaCl; for example, the blocking solution is Blocker TM BSA / PBS (10X) solution, i.e. 10% w / v bovine serum albumin / 10 mM sodium phosphate / 150 mM NaCl solution, pH 7.4, Thermo Fisher Scientific, catalog number: 37525.

[0036] According to the method of the first aspect of the present invention, the washing solution / sample diluent is a PBS solution with a pH of 7.4 containing 0.5‰ Tween-20, wherein the PBS formula is NaCl 8g / L, KCl 0.2g / L, Na2HPO4·12H2O 3.58g / L, and KH2PO4 0.27g / L.

[0037] According to the method of the first aspect of the present invention, the color developing solution comprises: 100 mM Tris·HCl (pH 9.5), 100 mM NaCl, 5 mM MgCl2, and 2 mg / ml PNPP.

[0038] According to the method of the first aspect of the present invention, the alkaline phosphatase-labeled secondary antibody can be any alkaline phosphatase-labeled secondary antibody available on the market, such as Anti-mouse IgG, AP-linked Antibody, Cell Signaling Technology, Inc., catalog number #7056.

[0039] According to the method of the first aspect of the present invention, the biological sample is selected from the group consisting of blood, serum, plasma, cell culture fluid, saliva and urine.

[0040] The various detection materials used in the method of the present invention can be configured in a kit. To this end, the second aspect of the present invention provides a kit, such as a kit for detecting soluble urokinase receptor, such as a kit for any method described in the first aspect of the present invention.

[0041] The kit according to the second aspect of the present invention comprises:

[0042] ELISA plate (i.e., multi-well plate, such as 96-well plate), ATF-HSA protein (dry powder or solution, such as 1 ml, such as a solution with a concentration of 1 to 1.5 mg / ml), active suPAR standard (dry powder or diluent, such as 1 ml, such as a solution with a concentration of 1 mg / ml), ATN-658 antibody (e.g., 1 mg / ml, such as 100 μl), alkaline phosphatase-labeled secondary antibody (e.g., 25 μl, such as 500× alkaline phosphatase-labeled secondary antibody), sodium glycerophosphate solution (e.g., 5 ml), coating solution (e.g., 10 ml), diluent (e.g., 20 ml), washing solution (e.g., 20 ml), blocking solution (e.g., 10 ml), color developing solution (e.g., 10 ml), several optional anticoagulation tubes for blood sample processing, and optional instructions for use of the kit (which describes the detection method described in any embodiment of the present invention).

[0043] In one / some embodiments of the present invention, the various detection materials used in the method of the present invention can be configured in a kit. In one embodiment, the kit further comprises one or more reagents for detecting captured active suPAR, wherein the capture reagent is labeled with one of the detectable paired label components, and the one or more reagents for detecting captured active suPAR are labeled with the other of the paired label components. The detection of captured active suPAR can be achieved by detecting the interaction between the paired label components. In a preferred embodiment, the one or more reagents for detecting captured active suPAR include a monoclonal antibody that binds to the outside of active suPAR and / or a secondary antibody that binds to the monoclonal antibody, such as anti-mouse IgG labeled with alkaline phosphatase.

[0044] In one / some embodiments of the present invention, the captured active suPAR can be detected by a specific detection system and / or a specific detection method well known to those skilled in the art. The specific detection method selected can be a heterogeneous or homogeneous assay. A heterogeneous assay is an assay that includes one or more washing steps, while in a homogeneous assay, such washing steps are not necessary, and only the detection reagent and the sample to be detected are mixed and measured. The assay method includes, but is not limited to, an ELISA (enzyme-linked immunosorbent assay)-based assay, dry fluorescent immunochromatography, chemiluminescence, DELFIA (dissociation enhanced lanthanide fluorescent immunoassay), SPA (scintillation proximity assay), Flashplate assay, FRET (fluorescence resonance energy transfer) assay, TR-FRET (time resolved fluorescence resonance energy transfer) assay, FP (fluorescence polarization) assay, ALPHA (amplified chemiluminescent affinity homogeneous detection), EFC (enzyme fragment complementation) assay, double hybrid assay or co-immunoprecipitation assay.

[0045] In one / some embodiments of the present invention, in the above-mentioned method for detecting active suPAR, step 2) may include binding the formed complex with a reagent that specifically binds to the captured active suPAR, and detecting the captured active suPAR by detecting the reagent that specifically binds to the captured active suPAR. In this type of detection process, the captured active suPAR can be detected by measuring the detection components contained in the reaction system. The detection components include a capture reagent (e.g., ATF or its fusion protein contained in the capture reagent) and / or a reagent that specifically binds to the captured active suPAR. The determination of the detection components can be performed by labeling with a detectable marker component and detecting the marker component. The detectable marker component varies depending on the specific determination method used. In the present invention, the detection component can be labeled in a variety of ways, such as but not limited to biotin, enzymes, fluorescent dyes (such as FITC, fluorescein, rhodamine, Cy dyes or Alexa fluor), and radioactive markers (such as 3H, 32P, 35S, 125I or 14C) and common enzyme markers such as horseradish peroxidase and alkaline phosphatase. For example, the labeling component can be labeled on a reagent that specifically binds to the captured active suPAR, and the captured active suPAR can be detected by detecting the labeling component; one of the paired labeling components can also be labeled on a capture reagent, and the other can be labeled on a reagent that specifically binds to the captured active suPAR, and the captured active suPAR can be detected by detecting the interaction between the paired labeling components; the labeling component can also be labeled on a capture reagent, and the binding of the capture reagent to the active suPAR will cause the labeling component on the capture reagent to change, and the captured active suPAR can be detected by detecting the change of the labeling component; these labeling components and their detection methods are well known to those skilled in the art.

[0046] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may exist, and the monoclonal antibody is highly specific for a single antigenic epitope. Methods for preparing monoclonal antibodies to specific antigens are well known in the art, for example, by hybridoma methods. The monoclonal antibody that binds to the outside of active suPAR described in the present invention refers to a monoclonal antibody that targets an antigenic epitope located on the outside of active suPAR. The outside of active suPAR is a site on the outside of active suPAR relative to the inside of the active hydrophobic pocket into which ATF is inserted. When active suPAR is bound to ATF, the monoclonal antibody that binds to its outside site can bind to the complex formed, thereby enabling the detection of captured active suPAR. Methods for identifying the antigenic epitope targeted by a monoclonal antibody are well known in the art, so a person skilled in the art can determine whether a monoclonal antibody binds to the outside of active suPAR by methods well known to them.

[0047] Those skilled in the art know that the determination of the monoclonal antibody can be carried out by labeling the detection component with a detectable label component and detecting the label component. The detection component includes a capture reagent (containing ATF or a fusion protein containing ATF), a monoclonal antibody bound to the outside of active suPAR, and / or a secondary antibody bound to the monoclonal antibody. For example, the label component can be labeled on the monoclonal antibody, and the captured active suPAR is detected by detecting the label component; the monoclonal antibody can also be bound to the secondary antibody, and the label component can be labeled on the secondary antibody, and the captured active suPAR is detected by detecting the label component; one of the paired label components can also be labeled on the capture reagent, and the other can be labeled on the monoclonal antibody or the secondary antibody bound to the monoclonal antibody, and the captured active suPAR is detected by detecting the interaction between the paired label components; the label component can also be labeled on the capture reagent, and the binding of the capture reagent to the active suPAR will cause the label component on the capture reagent to change, and the captured active suPAR is detected by detecting the change of the label component; these label components and their detection methods are well known to those skilled in the art. Detectable labeling components include, but are not limited to, biotin, enzymes, fluorescent dyes (such as FITC, fluorescein, rhodamine, Cy dyes or Alexa fluor), radioactive labels (such as 3H, 32P, 35S, 125I or 14C), and common enzyme labels such as horseradish peroxidase and alkaline phosphatase.

[0048] The amino acid sequence of the active suPAR trap ATF of the present invention is shown in SEQ ID NO.2.

[0049] In the present invention, the term "uPA" used refers to urokinase-type plasminogen activator.

[0050] In the present invention, the term "uPAR" used refers to urokinase-type plasminogen activator receptor, also known as urokinase receptor.

[0051] In the present invention, the term "suPAR" is used to refer to the soluble form of urokinase-type plasminogen activator receptor (uPAR).

[0052] In the present invention, the term "active suPAR" used refers to uPAR that contains three complete domains D1 and D2D3 in structure and is functionally capable of binding to natural ligands uPA and vitronectin. The "active suPAR" described herein includes the full-length uPAR membrane receptor and the soluble urokinase receptor (soluble uPAR, suPAR) without the transmembrane region, but does not include various degradation fragments of suPAR.

[0053] In one / some embodiments of the present invention, the detection method of the present invention is for non-diagnostic purposes and is performed in vitro.

[0054] In one / some embodiments of the present invention, the capture agent is labeled with a detectable labeling component. The binding of the capture agent to the active suPAR will cause the labeling component on the capture agent to change, and the captured active suPAR can be detected by detecting the change in the labeling component on the capture agent.

[0055] In one / some embodiments of the present invention, the kit further comprises one or more reagents for detecting captured active suPAR, wherein the capture reagent is labeled with one of the detectable paired label components and the one or more reagents for detecting captured active suPAR are labeled with the other of the paired label components. Detection of captured active suPAR can be achieved by detecting the interaction between the paired label components. In a preferred embodiment, the one or more reagents for detecting captured active suPAR include a monoclonal antibody that binds to the outside of active suPAR and / or a secondary antibody that binds to the monoclonal antibody, such as anti-mouse IgG labeled with alkaline phosphatase.

[0056] In one / some embodiments of the present invention, the kit further comprises any detectable labeling component suitable for detecting active suPAR captured by the capture agent.

[0057] In the present invention, detectable labeling components include but are not limited to biotin, enzymes, fluorescent dyes (such as FITC, fluorescein, rhodamine, Cy dyes or Alexa fluor), radioactive labels (such as 3H, 32P, 35S, 125I or 14C) and common enzyme labels such as horseradish peroxidase and alkaline phosphatase.

[0058] In one / some embodiments of the present invention, in the kit of the present invention, the capture reagent can be fixed on a solid phase matrix, for example, a multi-well plate (such as a 96-well plate), a protein chip bottom membrane (such as a nitrocellulose membrane, a nylon membrane, etc.), or a magnetic bead. The kit can also include one or more of a coating solution, a blocking solution, a washing solution, a sample diluent, and a reagent for detecting a labeled component.

[0059] In one / some embodiments of the present invention, the kit of the present invention comprises: an ELISA plate; an ATF-HSA protein solution; an active suPAR standard; a monoclonal anti-suPAR antibody obtained by immunizing mice with human uPAR D2D3 (amino acids 88-283) and screening using hybridoma technology; anti-mouse IgG labeled with alkaline phosphatase; a coating solution; a washing solution / sample diluent; a blocking solution and a color developing solution.

[0060] In one / some embodiments of the present invention, the kit of the present invention comprises: an ELISA plate; an ATF-HSA protein solution (e.g., 1 ml, concentration 1-1.5 mg / ml); an active suPAR standard (e.g., 1 ml, concentration 1 mg / ml); a monoclonal anti-suPAR antibody (e.g., 1 mg / ml, 100 μl) obtained by immunizing mice with uPAR D2D3 (amino acids 88-283) and screening with hybridoma technology; 25 μl 500× alkaline phosphatase-labeled anti-mouse IgG; 10 ml of coating solution; 20 ml of 30× washing solution / sample diluent; 10 ml of BSA blocking solution; and 10 ml of PNPP color developing solution.

[0061] In one / some embodiments of the present invention, the coating solution is preferably an aqueous solution containing 40 mM NaHCO3, 10 mM Na2CO3, pH 9.6.

[0062] In one or some embodiments of the present invention, the blocking solution can be a 5% BSA solution prepared with the coating solution; TM BSA / PBS (10X) solution, i.e. 10% w / v bovine serum albumin / 10 mM sodium phosphate / 150 mM NaCl solution, pH 7.4, Thermo Fisher Scientific, catalog number: 37525.

[0063] In one / some embodiments of the present invention, the washing solution / sample diluent is preferably a PBS solution containing 0.5‰ Tween-20 pH 7.4, wherein the PBS formula is NaCl 8g / L, KCl 0.2g / L, Na2HPO4·12H2O 3.58g / L, and KH2PO4 0.27g / L.

[0064] In one / some embodiments of the present invention, the color developing solution preferably contains 0.1M Tris·HCl (pH 9.5), 0.1M NaCl, 5mM MgCl2, and 2mg / ml PNPP.

[0065] The detection method and detection kit of the present invention can be used to specifically detect active suPAR in various samples.

[0066] The "include", "comprising" or "containing" used in the present invention may mean "including but not limited to", "consisting essentially of" or "mainly consisting of". The technical solution using "include", "comprising" or "containing" in the present invention may also be a technical solution consisting of the listed components.

[0067] Any embodiment of any aspect of the present invention can be combined with other embodiments, as long as they do not conflict. In addition, in any embodiment of any aspect of the present invention, any technical feature can be applied to the technical features in other embodiments, as long as they do not conflict. The present invention is further described below.

[0068] Soluble urokinase plasminogen activator receptor (suPAR) is a protein derived from the cell membrane-bound urokinase plasminogen activator receptor (uPAR). suPAR and its fragments are present in body fluids, including blood and urine [C. Wei, et al. Nature medicine 17 (8) (2011) 952-60; H. De Witte, et al. Journal international du cancer 77 (2) (1998) 236-42; RW Stephens, et al. Clinical chemistry 43 (10) (1997) 1868-76; K. Wahlberg, et al. Cancer research 58 (15) (1998) 3294-8; CF Sier, et al. Laboratory investigation; a journal of technical methods and pathology 79 (6) (1999) 717-22].Elevated levels of suPAR in plasma or serum have been documented in many pathological conditions, including systemic lupus erythematosus (SLE), human immunodeficiency virus type 1 (HIV-1) infection, type 2 diabetic nephropathy, active tuberculosis (TB), sepsis, malaria, and various solid tumors (such as non-small cell lung cancer, breast cancer, and colorectal cancer) [N. Sidenius, et al. Blood 96(13)(2000)4091-5; CZ Wu, et al. Clinical biochemistry (2015); SROstrowski, et al. The Journal of infectious diseases 191(8)(2005)1331-41; H. Pappot, et al. European journal of cancer 33(6)(1997)867-72; J. Eugen-Olsen, et al. The international journal of tuberculosis and lungdisease: the official journal of the International Union against Tuberculosis and Lung Disease 6(8)(2002)686-92;K.Kofoed,et al.European journal of clinicalmicrobiology&infectious diseases:official publication of the European Society of Clinical Microbiology 27(5)(2008)375-83;RWStephens,et al.Journal of the National Cancer Institute 91(10)(1999)869-74;H.Enocsson,et al. Translational research: the journal of laboratory and clinical medicine 162(5)(2013)287-96].

[0069] High levels of suPAR were found to have strong prognostic value in predicting the severity and prognosis of cancer patients, HIV-infected patients, and patients suspected of infection in the emergency department [N. Sidenius,, et al. Blood 96(13)(2000)4091-5; RW Stephens, et al. Journal of the National Cancer Institute 91(10)(1999)869-74; R. Uusitalo-Seppala, et al. Journal of internal medicine 272(3)(2012)247-56]. In addition, suPAR is considered to be a stable biomarker in critically ill patients with focal segmental glomerulosclerosis (FSGS) and patients at different stages of diabetic nephropathy (DN) [C. Wei, et al. Nature medicine 17 (8) (2011) 952-60; CZ Wu, et al. Clinical biochemistry (2015); A. Koch, et al. Critical care 15 (1) (2011) R63]. Recently, elevated suPAR levels in a large number of patients (1335) were independently associated with chronic kidney disease and accelerated decline in estimated glomerular filtration rate [SSHayek, et al. The New England journal of medicine 373 (20) (2015) 1916-25]. Persson first applied uPAR to PET imaging in human cancer patients [M. Persson, et al. Theranostics 5 (12) (2015) 1303-16].

[0070] It has been found that uPAR-targeted imaging agents are significantly taken up by primary tumor lesions and lymph node metastases, which is paralleled by the high expression of uPAR in removed tumor tissue. uPAR contains three Ly6 / uPAR-type (LU) domains, which are linked to the cell surface by glycophosphatidylinositol (GPI)-anchors. Existing crystal structure studies have shown that the LU domains are tightly clustered in a globular protein and have a central concave cleft, which is formed by the contribution of all three domains [Q. Huai, et al. Science 311 (5761) (2006) 656-9; P. Llinas, et al. The EMBO journal 24 (9) (2005) 1655-63]. The central cavity of uPAR binds to the primary ligand urokinase-type plasminogen activator (uPA) at the so-called amino-terminal fragment (ATF; uPA residues 1-134).

[0071] ATF contains all the structural elements for uPAR recognition and binds to the receptor with an affinity indistinguishable from that of full-length uPA (0.1-1 nM) [Q. Huai, et al. Science 311 (5761) (2006) 656-9; M. Ploug, Current pharmaceutical design 9 (19) (2003) 1499-528; L. Lin, et al. J Biol Chem 285 (14) (2010) 10982-92; R. Mazzieri, et al. Molecular Biology of the Cell 17 (1) (2006) 367-378]. In addition to the main ligand of uPA, uPAR also recognizes a series of other different ligands [G. Eden, et al. Current pharmaceutical design 17 (19) (2011) 1874-89], such as vascular nectin [Q. Huai, et al. Nature structural & molecular biology 15 (4) (2008) 422-3], which is consistent with the broad functional role of uPAR [O. Cunningham, et al. The EMBO journal 22 (22) (2003) 5994-6003; HW Smith, et al. Molecular cell biology 11 (1) (2010) 23-36]. However, uPA remains the main and tightest binder of uPAR. It has been found that uPAR can be cleaved into smaller fragments in vitro and in vivo [G. Hoyer-Hansen, et al. Eur J Biochem 243 (1-2) (1997) 21-6; BK Pliyev, Mol Cell Biochem 321 (1-2) (2009) 111-122; N. Behrendt, et al. J Biol Chem 266 (12) (1991) 7842-7], and many uPAR fragments have been identified in vivo

[28] , namely the suPAR D1 domain and the suPAR D2D3 domain. In addition, recent biochemical and structural studies have shown that the conformation of suPAR is highly unstable, especially its D1 domain [H. Gardsvoll, et al. J Biol Chem 286 (38) (2011) 33544-56]. These new results pose a challenge to the selection of appropriate conformation-specific antibodies for uPAR ELISA and the measurement of uPAR forms.

[0072] ATF-HSA as a capture agent: It is well known that ATF contains full binding capacity to suPAR, indistinguishable from full-length uPA. In the present invention, ATF is fused with human serum albumin, and the recombinant protein ATF-HSA is used to capture suPAR, rather than just ATF itself. This scheme can achieve two purposes: 1) increase the exposure of the ATF receptor binding epitope through the spacing effect of HSA and avoid ATF being too close to the plate surface. ATF is a peptide with a molecular weight of about 16kDa, while HSA is 8nm in size and has a much higher molecular weight of 66kDa; 2) it helps to coat the capture agent on the microwell plate. The large size of HSA promotes the binding of the capture agent to the Maxisorp plate wells, thereby ensuring the high binding capacity of the plate. HSA (Human Serum Albumin) is the most abundant protein in human blood, accounting for about 60% of the total blood protein. HSA is very stable both in vitro and in vivo, with a half-life of 19 days in the blood and can be heat pasteurized at 60°C for 10 hours. This is due to the presence of 17 disulfide bonds in a single HSA molecule, which maintains the tight folding of its tertiary structure. In addition, HSA generally has no specific interaction with other proteins and should not affect the determination. Therefore, the present invention prepares recombinant ATF-HAS and expects it to maintain a stability similar to that of HSA itself.

[0073] ATN-658 as a capture agent: ATN-658 is known to not bind to non-human uPAR, does not block the binding of uPA to uPAR, and can bind to uPAR even if uPAR is occupied by uPA, therefore, it is beneficial to select ATN-658 to detect suPAR.

[0074] There are different forms of suPAR in the blood, and their possible physiological significance is also different. It is known that uPAR is a multifunctional receptor expressed on the surface of various types of cells, involved in extracellular matrix proteolysis, cell adhesion, migration and proliferation, and cell signaling events. uPAR is connected to the cell surface via a GPI anchor connected to the D3 domain of uPAR, which is further connected to the D2 domain and then connected to the D1 domain via a long linker. uPAR fragments of different lengths can be formed by cutting at the GPI anchor or long linker region between D1 and D2D3, producing fragments including suPAR (D1-D3), suPAR-D1 or suPAR-D2D3 [M. Thuno, et al, Disease markers 27 (3) (2009) 157-72; F. Fazioli, et al, The EMBO journal 16 (24) (1997) 7279-86.]. These uPAR fragments have been identified as playing different functional roles. uPAR-D1 is derived from the cleavage of the long linker region of uPAR and cannot bind to its ligand uPA (1000 times weaker than full-length uPAR) [N. Sidenius, et al, Febs Lett 470 (1) (2000) 40-46]. suPAR-D1 is only found in urine, probably due to its rapid clearance from the blood. suPAR-D2D3 can be produced by various types of cells, including human neutrophils, and exhibit chemotactic function due to the presence of a chemotactic epitope (amino acids SRSRY in the linker region). All three domains of suPAR are known to be required for uPA binding [Q. Huai, et al, Science 311 (5761) (2006) 656-9]. suPAR can specifically bind to uPA and vitronectin, so it can compete with membrane-bound uPAR and act as a scavenger of uPA or Vitroneptin.

[0075] It is known that for the recombinant protein of the D2D3 fragment of suPAR, no signal is detected even at a concentration of up to 120 ng / ml, indicating that the full-length suPAR needs to bind to its ligand uPA or ATF, while the D2D3 fragment of suPAR or the D1 fragment of suPAR does not show obvious binding [G. Hoyer-Hansen, et al, Febs Lett 420 (1) (1997) 79-85; N. Sidenius, et al, Febs Lett 470 (1) (2000) 40-46; M. Ploug, et al, Biochemistry-Us 33 (30) (1994) 8991-8997]. There are also literatures showing that the affinity of human ATF binding to human suPAR is two orders of magnitude higher than that of mouse suPAR [L. Lin, et al, J Biol Chem 285 (14) (2010) 10982-92]. Therefore, the assay using the ATF-HAS fusion protein will have very good specificity with no cross-reactivity to mouse uPAR or human uPAR fragments. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 : 12% SDS-PAGE was used to identify the expression of ATF-HSA fusion protein, the relative molecular weight was about 84 kDa, M1 was the SDS-PAGE Marker, and 1 was the sample.

[0077] Figure 2 : Principle of the ELISA assay for active suPAR.

[0078] Figure 3 : Schematic diagram of 96-well plate for ELISA detection. DETAILED DESCRIPTION

[0079] The present invention can be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. The present invention provides general and / or specific descriptions of the materials and test methods used in the test. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described in as much detail as possible herein. The following examples further illustrate the present invention, rather than limiting the present invention.

[0080] The present invention is further explained or illustrated by various embodiments below: unless otherwise specified, the solutions described below are all aqueous solutions; when it comes to percentages, the percentages of mixed materials prepared as liquid / liquid are all volume / volume percentages, the percentages of mixed materials prepared as solid / liquid are all mass / volume percentages, and the percentages of mixed materials prepared as solid / solid are all mass / mass percentages.

[0081] With reference to CN105954522B, some reagents and experimental materials used in the specific examples of the present invention are summarized as follows:

[0082] ATF-HSA fusion protein: prepared according to the method described in Example 1 of the present invention.

[0083] Active suPAR standard: Recombinant suPAR expressed in S2 Drosophila embryonic cells, purified by affinity column combined with ion column. The preparation method thereof can also be referred to the method disclosed by the inventor in

[0020] -

[0021] of CN112180103A (application number 202011147152.6).

[0084] ATN-658 antibody: anti-human uPAR antibody. In the specific examples of the present invention, if not otherwise specified, the product used is the product FHF99110 purchased from Antibody System, with a specification of 100ug / tube, 1mg / ml; it can also be obtained from Attenuon, LLC (San Diego, Calif) as described in the literature. The ATN-658 antibody is a monoclonal anti-suPAR antibody obtained by immunizing mice with uPAR D2D3 (amino acids 88-283) and screening with hybridoma technology [TW Bauer, et al, Cancer research 65 (17) (2005) 7775-81]. Sodium glycerophosphate (the example of the present invention uses β-sodium glycerophosphate, purchased from Sigma-Aldrich, analytical grade) is prepared with water for injection.

[0085] Alkaline phosphatase labeled secondary antibody (Anti-mouse IgG, AP-linked Antibody, Cell Signaling Technology, Inc., Catalog #7056): Enzyme-labeled secondary antibody specifically used in Western blotting and ELISA. It is an affinity-purified goat anti-mouse IgG (H&L) antibody that can be conjugated to calf intestinal alkaline phosphatase and can be used as a secondary antibody in protein immunoblotting experiments and ELISA applications; the recommended antibody dilution is 1:500-3000. Alkaline phosphatase (AP)-conjugated secondary antibodies are used in protein blotting detection to bind to specific chemiluminescence or other substrates. One advantage of AP conjugation is that the reaction rate can remain linear over a long period of time.

[0086] 96-well ELISA plate: 96 wells, polystyrene, transparent, Nunc-Immuno TM MicroWell TM , purchased from Merck.

[0087] Coating solution: Aqueous solution containing 40 mM NaHCO3 and 10 mM Na2CO3 at pH 9.6.

[0088] Blocking solution: Blocker TM BSA / PBS (10X) solution, i.e. 10% w / v bovine serum albumin / 10 mM sodium phosphate / 150 mM NaCl solution, pH 7.4, Thermo Fisher Scientific, catalog number: 37525.

[0089] Washing solution / sample diluent: PBS solution with pH 7.4 containing 0.5‰ Tween-20, where the PBS formula is NaCl 8g / L, KCl 0.2g / L, Na2HPO4·12H2O 3.58g / L, KH2PO4 0.27g / L.

[0090] Color development solution: 100 mM Tris·HCl (pH 9.5), 100 mM NaCl, 5 mM MgCl2, 2 mg / ml PNPP. PNPP (4-nitrophenylphosphate disodium hexahydrate, Sigma) can be used as an enzyme immunoassay substrate for AP.

[0091] Recombinant suPAR was expressed in Drosophila Schneider 2 cells and purified using the method described in the literature [C. Yuan, Q. Huai, CB Bian, MD Huang, Progress in Biochemistry and Biophysics 33 (3) (2006) 277-281]. Recombinant soluble mouse uPAR (smuPAR) and suPAR D2D3 were provided by the Finsen laboratory. BSA powder used as blocking solution can also be purchased from Sangong Biotech.

[0092] Example 1: Construction, expression, purification and characterization of ATF-HSA fusion protein

[0093] The ATF-HSA fusion protein was constructed, expressed, purified and characterized with reference to CN104800855B (Chinese patent application number 201410034942.1).

[0094] The amino acid sequence of the ATF-HSA fusion protein described herein is shown in SEQ ID NO.1 at the end of the text. In the SEQ ID NO.1, the amino acid sequence portion from ATF is shown in SEQ ID NO.2 at the end of the text, and the amino acid sequence portion from HAS is shown in SEQ ID NO.3 at the end of the text. The remaining amino acid sequence portions in the SEQ ID NO.1 are enzyme cleavage sites (EcoR I and Sal I), amino acids (EF and VD), and amino acid tags (glycine tag G and histidine tag H) that must be introduced when constructing the fusion protein.

[0095] The nucleotide sequence of the ATF-HSA fusion protein described herein is shown in SEQ ID NO.4 at the end of the text. In SEQ ID NO.4, the nucleotide sequence portion SEQ ID NO.5 at the end of the text encodes ATF, and the nucleotide sequence portion SEQ ID NO.6 at the end of the text encodes HSA.

[0096] The construction, expression and purification process of the fusion protein are as follows:

[0097] The ATF-HSA fusion protein gene (SEQ ID NO. 4) pPICZαA expression vector (purchased from Invitrogen, USA) was constructed by gene cloning;

[0098] After identification by gene sequencing, the expression vector was electroporated into Pichia pastoris X-33 (purchased from Invitrogen, USA) competent cells;

[0099] After small-scale expression and identification, the fusion protein was expressed in large quantities in the eukaryotic expression system, and methanol was added every 24 hours to a final concentration of 0.8%;

[0100] After 4 days of induction, the cells were centrifuged at 4°C and 8000 rpm for 10 min to remove the bacteria, and the pH was adjusted to pH 4.5 using sodium acetate buffer (liquid A, i.e., 20 mM AB-pH 4.5);

[0101] Then, the culture medium was centrifuged at 4°C and 12000 rpm for 30 min, and vacuum filtered through 0.45 μm and 0.22 μm filter membranes, and the treated culture medium was diluted 5 times with deionized water (keeping its pH at 4.5).

[0102] At 10°C, flow through a cation exchange column SPFF (purchased from GE, USA) that has been balanced (balanced with solution A, i.e., 20 mM AB-pH4.5), first use solution A to balance until the reading of the protein detector (Beckman Coulter IMMAGE 800) is stable, and then use the NaCl concentration continuous elution method for elution and purification, that is, solution B (solution A supplemented with 1 M NaCl) and solution A (20 mM AB-pH4.5) are continuously mixed and eluted, and the detector elution peak is collected (12% SDS-PAGE is used to detect the collection of the target protein);

[0103] The collected elution peak fractions were placed in a dialysis bag at 4°C, concentrated with PEG (Mw = 10000-12000), and freeze-dried to obtain ATF-HSA fusion protein.

[0104] The concentration of the target protein in the elution peak fraction or concentrate after purification was detected using an ultra-micro spectrophotometer (UV5Nano, METTLER TOLEDO). The purity of the purified target protein was detected using a molecular sieve superdex200 gel column (purchased from Cytiva). The SDS-PAGE identification results are shown in Figure 2. Figure 1 As shown, the results show that the expression level of ATF-HSA fusion protein is high, and the relative molecular weight is about 84kDa, which is consistent with the theoretical value of 84339.44g / mol. The binding activity of ATF-HSA with uPAR in vitro has been confirmed by the literature; the purity of the obtained ATF-HSA fusion protein is 93.4%.

[0105] The ATF-HSA fusion protein obtained above can also be purified using a nickel-nitrilotriacetic acid (Ni-NTA) column [R. Li, M. Huang, et al. A Novel Tumor Targeting Drug Carrier for Optical Imaging and Therapy, Theranostics 4(6)(2014)642-659].

[0106] As described in this embodiment, the present invention relates to an expression vector comprising the ATF-HSA fusion protein gene of the present invention, such as a pPICZαA expression vector comprising the ATF-HSA fusion protein gene. The expression vector is used to express the ATF-HSA fusion protein.

[0107] As described in this embodiment, the present invention relates to an expression system comprising the ATF-HSA fusion protein gene of the present invention or the expression vector of the present invention, and the expression system is preferably a eukaryotic cell, such as Pichia pastoris X-33. The cell is used to express the ATF-HSA fusion protein. As described in this embodiment, the present invention relates to a fusion protein gene encoding the fusion protein ATF-HSA. For example, the nucleotide sequence of the fusion protein gene is shown in SEQ ID NO.4. As described in this embodiment, the present invention relates to a novel fusion protein comprising an amino-terminal fragment of urokinase (uPA), such as uPA amino-terminal Ser1-Glu143 (ATF). The present invention also relates to a fusion protein comprising an amino-terminal fragment of u2A and human serum albumin (Human Serum Albumin, HSA), such as ATF-HSA. As described in this embodiment, the present invention relates to a novel fusion protein comprising an amino-terminal fragment of uPA, such as uPA amino-terminal Ser1-Glu143 (ATF). As described in this embodiment, the present invention relates to a fusion protein comprising an amino terminal fragment of uPA and human serum albumin (HSA). As described in this embodiment, the present invention relates to a fusion protein, the amino acid sequence of which is shown in SEQ ID NO.1. As described in this embodiment, the present invention relates to a gene encoding the above fusion protein, the gene having a nucleotide sequence as shown in SEQ ID NO.4. As described in this embodiment, the present invention relates to an expression vector of the above gene. As described in this embodiment, the present invention relates to the above expression vector being a pPICZαA expression vector. As described in this embodiment, the present invention relates to an expression system of the above gene or the above expression vector. As described in this embodiment, the present invention relates to the above expression system being a eukaryotic cell. As described in this embodiment, the present invention relates to the above eukaryotic cell being Pichia pastoris X-33.

[0108] Example 2: Detection of active suPAR in human blood samples using ATF-HSA

[0109] The principle of the ELISA detection method for active suPAR in this embodiment is similar to that described in CN105954522B. Figure 2 The specific steps include:

[0110] (1) Processing of plasma samples: Peripheral blood collected from the test subject was placed in an EDTA-2Na anticoagulant tube and centrifuged at 4°C and 1500 g for 30 min. The plasma was collected and diluted 10 times with sample diluent, divided into 1.5 ml centrifuge tubes, and stored at -80°C until use.

[0111] (2) Coating: Coat all wells 1A to 12H of the ELISA plate (see Figure 3 ) was added with 100 μl of ATF-HSA protein solution (120 μg / ml) diluted with coating solution, and the 96-well plate was sealed with sealing tape to prevent the liquid from evaporating, and kept at 4°C overnight. The sealing tape was removed, the liquid in the wells of the ELISA plate was shaken dry, and the washing solution was sucked out with a spray gun. Each well was washed 6 times. After the last wash, the ELISA plate was gently patted dry on absorbent paper to ensure that there were no bubbles in the wells.

[0112] (3) Blocking: Add 100 μl of blocking solution to all wells 1A-12H in the ELISA plate, seal the 96-well plate with sealing tape to prevent evaporation of the liquid, and incubate on a shaker at 80 rpm at room temperature for 1 hour. Remove the sealing tape, shake dry the liquid in the wells of the ELISA plate, and use a spray gun to absorb the washing solution and wash each well 6 times. After the last wash, gently pat the ELISA plate dry on absorbent paper and ensure that there are no bubbles left in the wells.

[0113] (4) Sample addition: Add 100 μl of active suPAR standard of different concentrations to ELISA plates 1A-1F, with two replicate wells for each concentration (A1, A2: 1 μg / L; B1, B2: 0.5 μg / L; C1, C2: 0.25 μg / L; D1, D2: 0.125 μg / L; E1, E2: 0.0625 μg / L; F1, F2: 0.03125 μg / L). Add 100 μl of sample diluent to G1 and G2 as blank controls. Then add the diluted plasma samples of the subjects to be tested to the other wells in the ELISA plate, with two replicates for each plasma sample. Then seal the 96-well plate with sealing tape and incubate on a shaker at 80 rpm for 1 hour at room temperature. Remove the sealing tape, shake dry the liquid in the wells of the ELISA plate, and use a spray gun to absorb the washing solution. Wash each well 6 times. After the last wash, gently pat the ELISA plate dry on absorbent paper and ensure that there are no bubbles left in the wells.

[0114] (5) Adding primary antibody: Add (a) 100 μl of 5 μg / ml ATN-658 antibody diluted in sample diluent and (b) 25 μl of 1.5 mg / ml sodium glycerophosphate solution to all wells of ELISA plate 1A-12H, seal the 96-well plate with sealing tape, and incubate on a shaker at 80 rpm at room temperature for 1 hour. Remove the sealing tape, shake dry the liquid in the wells of the ELISA plate, and use a spray gun to absorb the washing solution. Wash each well 6 times. After the last wash, gently pat the ELISA plate dry on absorbent paper and ensure that there are no bubbles in the wells.

[0115] (6) Add enzyme-labeled secondary antibody: Add 100 μl of alkaline phosphatase-labeled secondary antibody (Anti-mouse IgG, AP-linked Antibody) diluted 500 times with sample diluent to all wells 1A-12H of the ELISA plate, seal the 96-well plate with sealing tape, and incubate on a shaker at 80 rpm at room temperature for 1 hour. Remove the sealing tape, shake dry the liquid in the wells of the ELISA plate, and use a spray gun to absorb the washing liquid. Wash each well 6 times, and then use a spray gun to absorb deionized water to wash each well 3 times. Gently pat the ELISA plate dry on absorbent paper and ensure that there are no bubbles in the wells.

[0116] (7) Add colorimetric solution: Use a dispenser to quickly add 100 μl of colorimetric solution to each well. Then place the plate on an ELISA reader and read the absorbance at 405 nm for 60 min, with the absorbance value read every 1 min. Prepare a standard curve based on the absorbance value and the concentration of the standard, and then calculate the concentration of active suPAR in the blood sample.

[0117] The above standard curve was prepared with reference to the method described in CN105954522B. Specifically, the absorbance of different concentrations of active suPAR standard was read at 405 nm on a microplate reader over time. The horizontal axis was the monitoring time for 60 minutes, and the vertical axis was the absorbance value at 405 nm. The active suPAR concentrations corresponding to the kinetic curve from top to bottom were from high to low. The absorbance value at 405 nm at the 60th minute corresponding to different active suPAR concentrations was subtracted from the absorbance value at the 1st minute. The absorbance value at 405nm is used to obtain the change value X (mOD, milliAbs) of OD405nm within 60 minutes; X is divided by 60 minutes to obtain the enzyme reaction rate (milliAbs / min, mOD / min) corresponding to different active suPAR concentrations; then, a standard curve is made based on a series of active suPAR concentrations (X axis, μg / L) and the corresponding enzyme reaction rate (Y axis, mOD / min), and the regression equation of the standard curve can be obtained; then, the enzyme reaction rate corresponding to the plasma sample to be tested is substituted into the regression equation to obtain the active suPAR content in the corresponding plasma sample. The present invention can use Synergy TM The absorbance was measured using a BioTek 4 plate reader (BioTek Instruments), and the data were processed using Gen 5 software.

[0118] The regression equation obtained by measuring / calculating the active suPAR standard concentration and the enzyme reaction rate corresponding to each concentration is: Y=5.1372X+0.7326, R2=0.9997, where Y is the enzyme reaction rate (mOD / min), X is the uPAR concentration (μg / L), and the enzyme reaction rate in the uPAR concentration range of 0.03125 to 1 μg / L is in the range of 0.893 to 5.871.

[0119] Example 3: Performance of the Active suPAR Detection Method

[0120] Proceed according to the method described in Example 2.

[0121] 1. Precision

[0122] The method of Example 2 was used to repeatedly measure the active suPAR content in the same plasma sample on the same plate on the same day (n=16 times) to obtain the corresponding average value (AVE), standard deviation (SD), and intra-assay coefficient of variation (%). The result of a plasma sample to be tested was:

[0123] AVE = 0.937 μg / L, SD = 0.072, intra-assay coefficient of variation = 7.68%

[0124] In addition, the active suPAR content in the above-mentioned plasma samples to be tested was repeatedly measured every day for 6 consecutive days to obtain the corresponding 6-day average value (AVE), standard deviation (SD), and inter-batch coefficient of variation (%). The results of the above-mentioned plasma samples to be tested are:

[0125] AVE = 0.924 μg / L, SD = 0.091, inter-assay coefficient of variation = 9.85%

[0126] The above results show that the intra-batch coefficient of variation is less than 10% and the inter-batch coefficient of variation is less than 15%, indicating that the established new ELISA method has good reproducibility.

[0127] 2. Recovery rate

[0128] 10 μl of a series of known concentrations of active suPAR standard samples were added to 100 μl of sample diluent or 10-fold diluted plasma, and the final added active suPAR concentration in the 110 μl system was calculated (this concentration was recorded as the active suPAR added concentration). The active suPAR concentration in the sample diluent and diluted plasma to which active suPAR was added was detected using the method of Example 2 (this concentration was recorded as the active suPAR detection concentration).

[0129] With the detection concentration as the Y axis and the added concentration as the X axis, the regression equations obtained by the two dilution methods were calculated respectively; the regression equation for the sample dilution solution was Y=1.0238X+0.0163, R2 =0.9986, the regression equation for diluted plasma is Y=1.0164X+0.0734, R 2 =0.9973; the percentage obtained by dividing the slope of the regression equation for diluted plasma by the slope of the regression equation for sample dilution is the recovery rate R = 99.28%, indicating that the method has a high recovery rate and excellent accuracy. This shows that the method has a high recovery rate of active suPAR added to the sample, and there is very little loss of specific active suPAR after addition to the plasma sample. The definition of 100% recovery is: the slope of the curve between active suPAR added to the sample and the test suPAR, where active suPAR is added to the dilution buffer.

[0130] 3. Detection limit and linear range

[0131] The detection limit of active suPAR content detected by the method of Example 2 was confirmed by referring to the method contained in CN105954522B. The corresponding enzyme reaction rate in 6 groups of blank controls (samples were added as sample diluents, and the active suPAR concentration was 0 μg / L) was measured, and then substituted into the standard curve of active suPAR concentration and enzyme reaction rate to obtain the corresponding active suPAR content. The average value (AVE) and the corresponding standard deviation (SD) of the active suPAR content in the 6 groups of blank controls were calculated, and the detection limit of the method was obtained by the formula AVE±3SD. The detection limit of the active suPAR content of the method of Example 2 was obtained as 18 ng / L. In addition, by processing the linear relationship curve of the active suPAR concentration corresponding to the enzyme reaction rate of 0-96 μg / L, the linear range of the method was obtained to be 0.024-12 μg / L (the linear equation satisfies R 2 >0.99, good linear relationship).

[0132] There are differences in the levels of active suPAR in the plasma of healthy people and pregnant women. As the main receptor for pericellular proteolytic activity, suPAR is known to be related to placental and fetal development [M. Uszynski, et al, European journal of obstetrics, gynecology, and reproductive biology 114 (1) (2004) 54-8; C. Floridon, et al, Placenta 20 (8) (1999) 711-21]. Using the method of Example 2 of the present invention, the average plasma active suPAR level of the citric acid anticoagulated plasma samples obtained from 18 pregnant women (24 to 32 years old) was measured to be 1.32 ± 0.67 μg / L. The average plasma active suPAR level of the plasma samples obtained from 15 normal female healthy blood donors (25 to 36 years old) was measured by the method of Example 2 to be 0.92 ± 0.61 μg / L. The concentration of active suPAR in the plasma of the former is slightly higher than that of the healthy donors.

[0133] Example 4: Detection of active suPAR in human blood samples using ATF-HSA

[0134] In step "(5) adding primary antibody" of Example 2, sodium glycerophosphate was added. The inventors have found that if this chemical reagent is not added, the precision of the methodology will be significantly deteriorated or even unacceptable. The specific experiments are shown in the following Examples 41 to 44. Example 41: The method was carried out in accordance with Example 2, except that "(b) 25 μl of 1.5 mg / ml sodium glycerophosphate solution" was not added in the operation of step "(5) adding primary antibody" of Example 2. When Example 3 was used to investigate the performance of the methodology, the precision results of the plasma samples to be tested in the same batch as those in Example 2 were: intra-batch AVE = 0.893 μg / L, SD = 0.142, coefficient of variation = 15.90%, and inter-batch AVE = 0.906 μg / L, SD = 0.161, coefficient of variation = 17.77%; it can be seen that the precision results are unacceptable if sodium glycerophosphate is not used. Example 42: The method was carried out in accordance with Example 2, except that in the operation of step "(5) adding primary antibody" of Example 2, "(a) 100 μl of 8 μg / ml ATN-658 antibody diluted with sample diluent was added" and "(b) 25 μl of 1.5 mg / ml sodium glycerol phosphate solution" was not added. When Example 3 was used to conduct a methodological performance investigation, the precision results of the plasma samples of the same batch as those of Example 2 were: intra-batch AVE = 0.907 μg / L, SD = 0.124, coefficient of variation = 13.67%; inter-batch AVE = 0.913 μg / L, SD = 0.145, coefficient of variation = 15.88%. It can be seen that if sodium glycerol phosphate is not used but the amount of ATN-658 antibody added is appropriately increased, the precision result is still unacceptable but is better than the 5 μg / ml concentration added in Example 41. Example 43: The method was carried out in accordance with Example 2, except that in the operation of step "(5) adding primary antibody" of Example 2, "(a) 100 μl of 12 μg / ml ATN-658 antibody diluted with sample diluent was added" and "(b) 25 μl of 1.5 mg / ml sodium glycerol phosphate solution" was not added. When Example 3 was used to conduct a methodological performance investigation, the precision results of the plasma samples from the same batch as those measured in Example 2 were: intra-batch AVE = 0.927 μg / L, SD = 0.088, coefficient of variation = 9.49%; inter-batch AVE = 0.919 μg / L, SD = 0.114, coefficient of variation = 12.40%. It can be seen that if sodium glycerol phosphate is not used but the amount of ATN-658 antibody added is increased to 12 μg / ml, the precision can meet the general requirements of intra-batch coefficient of variation less than 10% and inter-batch coefficient of variation less than 15, but it is still worse than the solution of adding sodium glycerol phosphate in Example 2.Example 44: The method was carried out in accordance with Example 2, except that in the operation of step "(5) adding primary antibody" in Example 2, "(a) 100 μl of 15 μg / ml ATN-658 antibody diluted with sample diluent was added" and "(b) 25 μl of 1.5 mg / ml sodium glycerol phosphate solution" was not added. When Example 3 was used to conduct a methodological performance investigation, the precision results of the plasma samples from the same batch as those measured in Example 2 were: intra-batch AVE = 0.935 μg / L, SD = 0.077, coefficient of variation = 8.24%; inter-batch AVE = 0.927 μg / L, SD = 0.096, coefficient of variation = 10.36%. It can be seen that if sodium glycerol phosphate is not used but the amount of ATN-658 antibody added is increased to 15 μg / ml, the precision can meet the general requirements of intra-batch coefficient of variation less than 10% and inter-batch coefficient of variation less than 15, which is equivalent to the solution of adding sodium glycerol phosphate in Example 2. The results of Examples 41 to 44 above also seem to indicate that, without the addition of sodium glycerophosphate, the less the amount of ATN-658 antibody added, the lower the measured active suPAR content. Although the inventors are not yet clear about the mechanism by which sodium glycerophosphate achieves the above technical effects in the present invention, since the ATN-658 antibody is expensive, adding an appropriate amount of cheap sodium glycerophosphate during the detection process can greatly reduce the amount of ATN-658 antibody used, which will be extremely beneficial.

[0135] The amino acid sequence and nucleotide sequence involved in the present invention are summarized as follows.

[0136] The amino acid sequence SEQ ID NO.1 of the ATF-HSA fusion protein described in this article is as follows: EFSNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGKKPSSPPEEVDGGGGDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLVDHHHHHH. In this SEQ ID NO.1, the part from the ATF amino acid sequence (SEQ ID NO.2) is: SNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGKKPSSPPEE.In the SEQ ID NO.1, the amino acid sequence portion from HAS (SEQ ID NO.3) is: The remaining amino acid sequence portion in the SEQ ID NO.1 is the enzyme cleavage site (EcoR I and Sal I), amino acids (EF and VD) and amino acid tags (glycine tag G and histidine tag H) that must be introduced when constructing the fusion protein.

[0137]

Claims

1. A method for detecting active suPAR in a human blood sample using ATF-HSA, comprising the following steps: (1) Treatment of plasma samples: Peripheral blood collected from the test subject was placed in an EDTA-2Na anticoagulant tube, centrifuged at 4°C and 1500 g for 30 min, and plasma was collected. The plasma was diluted 10 times with sample diluent, divided into 1.5 ml centrifuge tubes, and stored at -80°C for later use; (2) Coating: Add 100 μl of capture reagent ATF-HSA protein solution (120 μg / ml) diluted with coating solution to all wells 1A to 12H of a 96-well ELISA plate, seal the 96-well plate with sealing tape to prevent evaporation of the liquid, and incubate at 4°C overnight. Remove the sealing tape, spin dry the liquid in the wells of the ELISA plate, and use a spray gun to absorb the washing solution. Wash each well 6 times. After the last wash, gently pat the ELISA plate dry on absorbent paper and ensure that there are no bubbles in the wells. (3) Blocking: Add 100 μl of blocking solution to all wells 1A to 12H in the ELISA plate, seal the 96-well plate with sealing tape to prevent evaporation of the liquid, and incubate on a shaker at 80 rpm at room temperature for 1 hour; remove the sealing tape, shake dry the liquid in the wells of the ELISA plate, and use a spray gun to absorb the washing solution and wash each well 6 times. After the last wash, gently pat the ELISA plate dry on absorbent paper and ensure that there are no bubbles left in the wells; (4) Sample addition: Add 100 μl of active suPAR standard of different concentrations to ELISA plates 1A-1F, with two replicate wells for each concentration: A1, A2: 1 μg / L; B1, B2: 0.5 μg / L; C1, C2: 0.25 μg / L; D1, D2: 0.125 μg / L; E1, E2: 0.0625 μg / L; F1, F2: 0.03125 μg / L; add 100 μl of active suPAR standard to G1, G2, respectively. μl of sample diluent was used as a blank control; then the diluted plasma samples of the test subjects were added to the other wells in the ELISA plate, with 2 replicates for each plasma sample, and then the 96-well plate was sealed with sealing tape and incubated on a shaker at room temperature at 80 rpm for 1 hour; the sealing tape was removed, the liquid in the wells of the ELISA plate was shaken dry, and the washing liquid was sucked up with a spray gun, and each well was washed 6 times. After the last wash, the ELISA plate was gently patted dry on absorbent paper to ensure that there were no bubbles in the wells; (5) Add primary antibody: Add to all wells 1A-12H of the ELISA plate (a) 100 μl of 5 μg / ml ATN-658 antibody diluted in sample diluent and (b) 25 μl of 1.5 mg / ml sodium glycerophosphate solution, Seal the 96-well plate with sealing tape and incubate it on a shaker at 80 rpm at room temperature for 1 hour; remove the sealing tape, shake dry the liquid in the wells of the ELISA plate, and use a spray gun to absorb the washing solution. Wash each well 6 times. After the last wash, gently pat the ELISA plate dry on absorbent paper and ensure that there are no bubbles in the wells; (6) Adding enzyme-labeled secondary antibody: Add 100 μl of alkaline phosphatase-labeled secondary antibody diluted 500 times with sample diluent to all wells 1A-12H of the ELISA plate, seal the 96-well plate with sealing tape, and incubate on a shaker at room temperature at 80 rpm for 1 hour; remove the sealing tape, shake dry the liquid in the wells of the ELISA plate, and use a spray gun to absorb the washing solution, wash each well 6 times, and then use a spray gun to absorb deionized water to wash each well 3 times; gently pat the ELISA plate dry on absorbent paper and ensure that there are no bubbles in the wells; (7) Add colorimetric solution: Use a dispenser to quickly add 100 μl of colorimetric solution to each well. Then place the plate on an ELISA reader and read the absorbance at 405 nm for 60 min, with the absorbance value read every 1 min. Prepare a standard curve based on the absorbance value and the concentration of the standard, and then calculate the concentration of active suPAR in the blood sample.

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