CN118068002A

CN118068002A - Active urokinase receptor suPAR and its use in kidney disease detection

Info

Publication number: CN118068002A
Application number: CN202311474480.0A
Authority: CN
Inventors: 陈曦; 黄仁杰; 陈佳宇; 黄明东; 江龙光; 袁彩
Original assignee: Fujian Yitong Biotechnology Co ltd
Current assignee: Fujian Yitong Biotechnology Co ltd
Priority date: 2023-11-08
Filing date: 2023-11-08
Publication date: 2024-05-24

Abstract

The present invention relates to the active urokinase receptor suPAR and its use in the detection of kidney disease. In one aspect, there is provided a use of suPAR in a method of detecting renal disease, e.g. chronic kidney disease, the detection comprising the steps of: contacting a capture reagent with a test sample under conditions suitable for the capture reagent to capture active suPAR in the test sample to form a complex of the capture reagent and the suPAR; binding the formed complex to an agent that specifically binds to the captured suPAR and detecting the captured suPAR by detecting the agent that specifically binds to the captured suPAR. Wherein the capture reagent is a fusion protein comprising an ATF; the agent that specifically binds to the captured suPAR is a monoclonal antibody that binds to the outside of the suPAR. Also relates to a method for detecting the amount of a soluble urokinase-type plasminogen activator receptor in a biological sample, e.g. blood, e.g. venous blood, of a subject suffering from or suspected of suffering from kidney disease, and to a corresponding kit.

Description

Active urokinase receptor suPAR and its use in kidney disease detection

Technical Field

The present invention is in the biomedical field, in particular to a method for detecting an active urokinase receptor, in particular an active soluble urokinase receptor, also called soluble urokinase type plasminogen activator receptor (soluble urokinase-type plasminogen activator receptor, suPAR), sometimes also called active soluble urokinase type plasminogen activator receptor (Active soluble urokinase-type plasminogen activator receptor, active soluble uPAR, active suPAR). The invention also relates to a kit used for the detection method. Furthermore, the invention also relates to the application of the active urokinase receptor suPAR in the detection of kidney diseases such as chronic kidney diseases, which can be embodied in the form of the kit.

Background

CN116008557a of the inventor's research team (chinese patent application No. 202211149120.9), the entire contents of which are incorporated herein by reference, discloses a method of detecting active soluble urokinase-type plasminogen activator receptor in a biological sample, and a detection kit for use in the method. In addition, CN105954522B (chinese patent application No. 201610541379.6), the entire contents of which are incorporated herein by reference, discloses a method for detecting an active urokinase receptor.

Urokinase receptor (urokinase type plasminogen acitivator receptor, urokinase-type plasminogen activator receptor, uPAR) is well known as a cell surface receptor. The receptor was found from Stoppelli equal to 1985, cDNA was cloned from Roldan et al, 1989, and the protein was purified from cell membrane extracts of human lymphoma cells U937 by affinity chromatography, behrendt, 1990. In the fifth international leukocyte differentiation antigen conference uPAR was named differentiation antigen cluster 87 (cluster of differentiation, cd 87). 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 on the surface of many malignant cells, but is expressed in 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 markers27(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].

As a flexible molecule, uPAR can interact with a variety of ligands or receptors. Such as vitronectin (vitronectin), urokinase-type plasminogen activator (urokinase-type plasminogen activator, uPA), low density lipoprotein receptor-related protein 1 (low density lipoprotein receptor-relatedprotein 1, LRP 1), integrin (integrin), G-protein coupled receptor (G protein coupled receptor, GPCR), and the like. These various interactions play a wide range of important roles in a variety of physiological and pathological processes in the human body, including activation of plasminogen, cell adhesion and migration, cell differentiation, chemo-activation phenomena, chemokine receptor modulation, immune responses, inflammatory responses, and the like. uPAR consists of three cysteine-rich Ly6/uPAR domains (D1, D2, D3) of 81-87 amino acids in size and has a molecular weight of about 55kDa. uPAR is anchored to cell membrane surface by its C-terminal glycosylated phosphatidylinositol (glycosylphosphatidylinositol, GPI) [F. Blasi, et al. Nature reviews. Molecular cell biology 3(12) (2002)932-43;H. W. Smith, et al. Nature reviews. Molecular cell biology 11(1) (2010)23-36].

Urokinase receptors exist in a variety of forms, including full-length membrane receptors, soluble uPAR (suPAR) receptors without a transmembrane region, and various degradation fragments. The full-length uPAR membrane receptor is susceptible to hydrolysis by phosphatidyl enzyme C, allowing the uPAR to shed from the cell membrane surface, forming a soluble urokinase receptor free of glycosylated phosphatidylinositol (solubleuPAR, suPAR). In addition, uPAR is also sensitive to various hydrolases and can be further hydrolyzed into D1 and D2-D3 fragments [ M. Thuno, et al, suPAR: the molecular crystal ball, DISEASE MARKERS27 (3) (2009), 157-72].

The existing crystal structure research shows that the uPAR forms a bowl-shaped structure through three domains of the uPAR, and the bowl-shaped structure can be combined with a ligand uPA [ C. Yuan, M. Huang, cellular and molecular LIFE SCIENCES: CMLS 64 (9) (2007) 1033-7] of the uPAR efficiently, so that the uPA is enriched on the cell surface, and the plasminogen is activated to plasmin, so that the extracellular matrix is degraded, and the uPAR plays an important role in cell migration. In addition, it has been demonstrated that binding of uPA to uPAR greatly enhances binding of uPAR to vitronectin [ Q. Huai, et al Nature structural & molecular biology 15 (4) (2008) 422-3], and a number of references also demonstrate the importance of uPA binding to uPAR for its interaction with integrins [H. W. 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 locally to and accumulate on lesions with other proteins such as caveolin and the like, and integrin receptors initiate intracellular signals which are transmitted from outside the cell into the cell, activate intracellular protein kinases, promote cell division and cell migration. This uPAR is defined as active uPAR. Only active uPAR can signal, but is closely related to tumor invasion and metastasis, while other uPAR fragments are inactive.

Chronic Kidney Disease (CKD) is a chronic kidney structure and dysfunction (history of kidney damage greater than 3 months) caused by a variety of causes, including normal and abnormal pathological lesions of Glomerular Filtration Rate (GFR), abnormal blood or urine composition, and imaging abnormalities, or a decline in GFR (< 60ml/min 1.73m 2) of unknown cause for more than 3 months, i.e., CKD.

Diseases causing chronic kidney disease include various primary, secondary glomerulonephritis, tubular injury, and renal vascular lesions, among others. According to GFR, chronic kidney diseases can be divided into 5 stages, complications of CKD patients can be obviously reduced by early discovery and early intervention, survival rate is obviously improved, treatment of CKD comprises primary disease treatment, treatment of various dangerous factors and delay of progression of chronic renal insufficiency, and kidney replacement treatment should be timely carried out when the CKD patients progress to 5 stages. Diabetes mellitus, hypertension are important factors in exacerbation of kidney disease. Inappetence, nausea, vomiting, oral cavity urosis and the like are common symptoms of chronic kidney disease.

Glomerular filtration rate GFR is an index indicating kidney function, and the unit of the amount of two kidneys producing primary urine per minute is ml/min.1.73 m 2, and the normal range of GFR is usually considered to be 90-140 ml/min.1.73 m 2 clinically, and more usually 90-120 ml/min.1.73 m 2 clinically.

In recent years, a new proposal has been made for a method for the staging of CKD by the K/DOQI guideline (KIDNEY DISEASE Outcome Quality Initiative, guideline for prognosis of kidney disease) developed by the national kidney foundation (National Kidney Fondation, NKF), as shown in the following table:

The staging method treats patients with GFR more than or equal to 90 ml/min.1.73 m 2 and kidney diseases as stage 1 CKD, and aims to strengthen cognition and early prevention and treatment of early CKD.

The cause of CKD mainly includes primary glomerulonephritis, hypertensive glomerulonephritis, diabetic nephropathy, secondary glomerulonephritis, tubular interstitial lesions (chronic pyelonephritis, chronic uric acid nephropathy, obstructive nephropathy, drug-induced nephropathy, etc.), ischemic nephropathy, hereditary nephropathy (polycystic kidney disease, hereditary nephritis), etc. In developed countries, diabetic nephropathy, hypertensive renal arteriosclerotic disease have become a major cause of chronic kidney disease; in China, these two diseases remain after primary glomerulonephritis in various etiologies, but there is a significant trend in recent years. According to relevant statistics, the prevalence of CKD in american adults (about 2 billion total) has been as high as 11.3%. The prevalence of CKD is reported in the chinese section to be about 10%. The susceptibility to CKD is mainly: age (e.g., elderly), family history of CKD (including hereditary and non-hereditary kidney disease), diabetes, hypertension, obesity-metabolic syndrome, high protein diet, hyperlipidemia, hyperuricemia, autoimmune disease, urinary or systemic infection, hepatitis virus (e.g., hepatitis b or c virus) infection, urinary calculus, urinary obstruction, urinary or systemic tumor, history of application of nephrotoxic drugs, cardiovascular disease, anemia, smoking, low body weight at birth, and the like. Other risk factors are environmental pollution, low economic level, low medical insurance level, low education level, etc.

Clinical manifestations vary among stages of CKD. Before the CKD3 stage, the patient can have no symptoms or only slight discomfort such as hypodynamia, soreness of waist, nocturia and the like; few patients may have anorexia, metabolic acidosis, and mild anemia. After stage 3 CKD, the symptoms become more obvious, and the symptoms become more serious after entering the renal failure stage, and hypertension, heart failure, severe hyperkalemia, acid-base balance disorder, digestive tract symptoms, anemia, mineral bone metabolism abnormality, hyperparathyroidism, central nervous system disorder and the like can sometimes occur, so that the life is even dangerous. The most common clinical manifestations of chronic kidney disease are gastrointestinal symptoms, mainly manifested by loss of appetite, nausea, vomiting, and oral cavity having a urine taste. The occurrence rate of inflammation, ulcer and hemorrhage of stomach and duodenum is higher than that of normal people. Abnormalities in the blood system of CKD patients are mainly manifested by renal anemia and bleeding tendencies. Most patients generally have mild to moderate anemia, which is mainly due to erythropoietin deficiency and is called renal anemia. Respiratory symptoms such as shortness of breath, shortness of breath and shortness of breath can appear in case of excessive body fluid or acidosis, and severe acidosis can lead to deep breathing. Excessive body fluids and cardiac insufficiency can cause pulmonary oedema or pleural effusion. Some severe patients may be accompanied by uremia, pulmonary edema, uremic pleurisy, uremic lung calcification, etc. Cardiovascular lesions are one of the major complications and the most common cause of death in CKD patients. Along with the continuous deterioration of renal function, the prevalence rate of heart failure is obviously increased to 65-70% of uremia stage. Heart failure is the most common cause of death in uremic patients. Hemodialysis patients have a greater degree of atherosclerosis and vascular calcification than pre-dialysis patients, and atherosclerosis tends to progress more rapidly. Uremic cardiomyopathy is mainly related to retention of metabolic waste and anemia, and pericardial effusion is also quite common in CKD patients.

Symptoms of neuromuscular system may be insomnia, inattention, hypomnesis, etc. in the early stages of CKD. With the progress of the disease, there are often reaction apathy, convulsion, hallucination, somnolence, coma, mental abnormality, etc. Peripheral neuropathy is also common. Hypocalcemia, hyperphosphatemia, active vitamin D deficiency, etc. can induce secondary hyperparathyroidism (abbreviated as hyperparathyroidism); these factors in turn lead to renal osteodystrophy (i.e., renal bone disease), including fibrocystic osteosis (high turnover bone disease), osteomalacia (low turnover bone disease), osteogenesis failure, osteoporosis, and mixed bone disease.

In addition, CKD patients often develop endocrine dysfunction, kidney self endocrine dysfunction, including: 1,25 (OH) 2 vitamin D3, erythropoietin deficiency and elevated levels of renin-angiotensin II; hypothalamic-pituitary endocrine dysfunction may also be caused: elevated levels of e.g. prolactin, melanin-stimulating hormone (MSH), luteinizing hormone (FSH), follicle-stimulating hormone (LH), adrenocorticotropic hormone (ACTH); most patients have secondary hyperthyroidism, insulin receptor disorders, elevated glucagon levels, etc. About 1/4 of patients had a slight thyroxine level reduction. Some patients may be accompanied by skin symptoms such as pigmentation, calpain, itching, difficult sweating, ulcers, etc. Some patients may have hypogonadism manifested by gonadal maturation disorder or atrophy, hyposexuality, amenorrhea, infertility, etc., and may be associated with abnormal serum sex hormone levels, uremic toxin effects, certain nutrient (e.g., zinc) deficiencies, etc.

To clarify the prevention and treatment targets of different stages of CKD, three-stage prevention concepts are proposed for this purpose. Primary prevention, also known as primary prevention, refers to the timely and effective treatment of existing kidney disorders or disorders that may cause kidney damage (e.g., diabetes, hypertension, etc.), preventing the occurrence of Chronic Renal Failure (CRF). The second-stage prevention means that the existing patients with mild and moderate CRF are treated in time, the progress of chronic renal failure is delayed, stopped or reversed, and the occurrence of uremia is prevented. And the third stage of prevention refers to early treatment measures for uremic patients, prevents certain serious complications of uremia, and improves the survival rate and life quality of the patients.

The end result of chronic renal insufficiency progression is end-stage renal failure (ESRF), and the patient will have to rely on renal replacement therapy to sustain life. Despite the great progress in dialysis treatment, patients with ESRF still have a high mortality rate and a low quality of life. Thus, treatment of CKD patients includes treatment that delays progression of renal insufficiency and treatment for various complications. In one aspect, the primary pathogenesis of CKD may be treated. On the other hand, the following measures may be taken to delay the progression of chronic renal insufficiency: 1) Controlling blood pressure: actively controlling blood pressure can reduce proteinuria, reduce glomerular hyperfiltration, and slow down the progress of chronic renal failure lesions. The selection principle of the antihypertensive drugs varies according to the stage of CKD, and when CCr is more than 30ml/min, an Angiotensin Converting Enzyme Inhibitor (ACEI) or an angiotensin II receptor 1 Antagonist (ARB) can be selected first, and other antihypertensive drugs can be used in combination if necessary. When the Ccr of the patient falls below 30ml/min, the application of ACEI and ARB may cause low intraglomerular perfusion pressure and thus too low glomerular filtration rate, so that the patient should be cautiously treated with non-dialysis CKD; 2) Diet: the low protein diet can reduce intraglomerular high perfusion, hypertension and hyperfiltration, reduce proteinuria, thereby slowing down the progression of glomerulosclerosis and interstitial fibrosis in CRF patients, when GFR is less than 25 ml/(min.1.73 m 2), the protein intake should be limited to 0.6 g/(kg.d), it should be ensured that enough heat-cationed is greater than 35 kcal/(kg.d) to make maximum use of the protein in the diet, additionally essential amino acids or keto acid amino acid mixtures can be supplemented, furthermore, salt intake should be limited for patients with hypertension and edema, dyslipidemia patients should be subjected to diet adjustment, if necessary, lipid-lowering drugs should be used; 3) The factor CRF, which corrects the acute exacerbation of chronic renal failure, is a slowly evolving disease, but may deteriorate renal function during the course of the disease due to the high susceptibility of patients to a variety of risk factors. Common risk factors are: ① Hypovolemia, including hypotension, dehydration, shock, etc.; ② Severe infection, sepsis; ③ Tissue trauma or major bleeding; ④ Kidney damage by endogenous or exogenous toxins; ⑤ Obstruction of urinary tract; ⑥ Severe hypertension and malignant hypertension that are not controlled. Carefully identify the cause of the accelerated progression of renal function and take targeted treatments to help improve renal function. Finally, when CKD patient disease progresses to ESRD, renal replacement therapy including hemodialysis, peritoneal dialysis and renal transplantation should be actively performed, and the manner of renal replacement therapy is determined according to the specific condition of the patient.

However, it would be of great benefit to intervene in CKD treatment as early as possible, if a prognosis could be made by measuring certain indicators of the body, for example by detecting in vivo suPAR levels to predict the presence or absence of CKD, before CKD presents with clinical symptoms.

Accordingly, it would be highly desirable by those skilled in the art to provide a method for predicting the presence or absence of a kidney disease, such as chronic kidney disease, by detecting the level of suPAR in a subject.

Disclosure of Invention

It is an object of the present invention to provide a method for detecting an active urokinase receptor, or a method for detecting a soluble urokinase receptor, also known as an active soluble urokinase type plasminogen activator receptor (Active soluble urokinase-type plasminogen activator receptor, active soluble uPAR, active suPAR). Or it is an object of the present invention to provide a kit for use in the above-mentioned detection method, and a detection reagent for use in the kit. Further, it is an object of the present invention to provide the use of the active urokinase receptor suPAR in the detection of renal diseases, such as chronic kidney disease, which can be embodied in the form of the above-described kit.

To this end, a first aspect of the invention provides the use of a soluble urokinase-type plasminogen activator receptor in a method of detecting kidney disease, e.g. chronic kidney disease, in a subject suffering from kidney disease (e.g. chronic kidney disease) or in a biological sample, e.g. blood, e.g. venous blood, suspected of suffering from kidney disease (e.g. chronic kidney disease), the detection method comprising the steps of:

1) Contacting a capture reagent with a test sample under conditions suitable for the capture reagent to capture active suPAR in the test sample to form a complex of the capture reagent and active suPAR;

2) Binding the formed complex to an agent that specifically binds to the captured active suPAR and detecting the captured active suPAR by detecting the agent that specifically binds to the captured active suPAR,

Wherein:

The capture reagent is a fusion protein comprising an ATF;

the agent which specifically binds to the captured active suPAR is a monoclonal antibody which binds to the outside of the active suPAR;

The sample to be measured is a plasma sample, which is obtained from a subject to be measured, for example, in the range of 10 to 140 ml/(min.1.73 m 2), for example, in the range of 10 to 130 ml/(min.1.73 m 2), and which has a GFR value of 0 to 140 ml/(min.1.73 m 2), and is processed as follows: placing collected venous blood of a to-be-detected subject into a potassium oxalate anticoagulation tube, centrifuging at 0-10 ℃ (e.g. 4 ℃) (e.g. centrifuging at 1500g for 30 min), separating plasma, diluting the plasma with a sample diluent (e.g. to a proper concentration, e.g. to a concentration in a linear range of a standard curve), sub-packaging (e.g. in a 1.5ml centrifuge tube), and preserving at low temperature (e.g. at-80 ℃) for later use.

According to the use of the first aspect of the present invention, the potassium oxalate anticoagulant tube is prepared by mixing 1ml of potassium oxalate for venous blood with 1ml of potassium oxalate (blood: potassium oxalate): 1-4 mg, for example 1ml: anticoagulation was carried out at a rate of 2 mg. In one embodiment, the anticoagulant tube is prepared by the following method: a (e.g., 10%) potassium oxalate solution (e.g., 0.2 ml) was placed in a test tube, the solution was dispersed around the tube wall, and dried to obtain an anticoagulant tube.

According to the use of the first aspect of the invention, the sample diluent is a PBS solution of pH7.4 containing 0.5%o Tween-20, wherein the PBS formulation is 8g/L sodium chloride, 0.2g/L potassium chloride, 3.58g/L disodium hydrogen phosphate dodecahydrate, 0.27g/L potassium dihydrogen phosphate.

According to the use of the first aspect of the invention, the GFR value is determined by a method selected from the group consisting of: urinary creatinine clearance method, serum creatinine concentration method, radioisotope labeling method, serum creatinine/urea nitrogen ratio method, inulin clearance method, and combinations thereof.

According to the use of the first aspect of the present invention, the monoclonal antibody binding to the outside of active suPAR is a monoclonal anti-suPAR antibody obtained by immunizing a mouse with uPAR-D2D3 (amino acids 88 to 283) and screening by hybridoma technique.

According to the use of the first aspect of the invention, the monoclonal antibody that binds outside the active suPAR is the antibody ATN-658.

According to the application of the first aspect of the invention, sodium glycerophosphate is added with the monoclonal antibody, and the mass ratio of the sodium glycerophosphate to the monoclonal antibody is 1: 50-100, for example 1:75.

According to the use of the first aspect of the invention, the agent that specifically binds to the captured active suPAR further comprises a secondary antibody that binds to the monoclonal antibody, e.g. alkaline phosphatase-labelled anti-mouse IgG.

According to the use of the first aspect of the invention, the fusion protein of ATF is a fusion protein of ATF with another protein or polypeptide or fragment thereof. The further protein or polypeptide or fragment thereof may be serum albumin, such as Human Serum Albumin (HSA), bovine Serum Albumin (BSA), or Ovalbumin (OVA). In a preferred embodiment, the ATF-containing fusion protein is an ATF-HSA fusion protein.

According to the use of the first aspect of the invention, the fusion protein of ATF is immobilized on a solid substrate. Such solid matrices include, but are not limited to, multiwell plates (e.g., elisa plates), protein chip carrier films (e.g., nitrocellulose films, nylon films, etc.), magnetic beads, fluorescent microspheres, and the like.

According to the use of the first aspect of the invention, the detection method comprises the steps of:

(1) To wells of a multi-well plate (in any of the embodiments of the present invention, the multi-well plate (also referred to as an ELISA plate) which can be replaced with a carrier conventional in the art such as magnetic beads, fluorescent microspheres, protein chip carrier films (e.g., nitrocellulose films, nylon films, etc.) as a solid matrix, etc., an ATF-HSA protein solution diluted with a coating liquid is added to coat (overnight at 4 ℃ C.), washed and dried, as described below; (2) Adding a sealing liquid into the hole for sealing, incubating at room temperature, washing and drying; (3) Adding a series of active suPAR standard substances with a concentration to some holes respectively, adding sample diluent to other holes to serve as blank control, and adding samples to be detected to other holes (for example, diluting the sample diluent by 10 times); incubating at room temperature, washing and drying; (4) Adding mouse anti-human ssuPAR monoclonal antibody such as ATN-658 antibody diluted with sample diluent into all wells, incubating at room temperature, washing and drying; (5) Alkaline phosphatase-labeled Anti-mouse IgG (e.g., alkaline phosphatase-labeled secondary Antibody (AP-linked Antibody)) was added to all wells, incubated at room temperature, washed and dried; (6) Adding a color development liquid into each hole, and then placing the porous plate on an enzyme-labeling instrument to read absorbance at 405 nm; (7) And (3) solving a regression equation according to the dynamic process (enzyme reaction speed) of the absorbance of the active suPAR standard substances with different concentrations along with the time and the concentration of the standard substances, and calculating the concentration of the active suPAR in the sample by using the regression equation.

(1) Adding 100 mu l of ATF-HSA protein solution (100-150 mu g/ml, for example, 120 mu g/ml) diluted by a coating solution into the holes of the porous plate for coating, and washing and drying at 4 ℃ overnight; (2) Adding 100 μl of blocking solution into the well for blocking, incubating at room temperature for 1 hr at 80 rpm, washing and drying; (3) Adding 100 μl of active suPAR standard substances with different concentrations to some wells, wherein the concentration distribution is in the concentration range of 0.01-2 μg/L, for example, in the concentration range of 0.03-1 μg/L, for example, 1 μg/L, 0.5 μg/L, 0.25 μg/L, 0.125 μg/L, 0.0625 μg/L and 0.03125 μg/L respectively; 100 μl of sample dilution was added to the other wells as a blank, which did not contain uPAR; adding a diluent which is 10 times diluted by the sample diluent to other holes; incubation for 1 hour at room temperature 80 rpm, washing and drying; (4) Adding 100 μl of 5-15 μg/ml, e.g., 12-15 μg/ml, of a mouse anti-human suPAR monoclonal antibody diluted by a sample diluent, incubating for 1 hour at room temperature for 80 rpm, washing and drying (e.g., the monoclonal antibody is obtained by immunizing a mouse with uPAR D2D3 (amino acids 88-283), screening by a hybridoma technology, e.g., the ATN-658 antibody); or (a) 100. Mu.l of a monoclonal antibody of 5. Mu.g/ml of a murine anti-human supAR diluted with a sample dilution and (b) 25. Mu.l of a 1.5mg/ml sodium glycerophosphate solution were added to all wells, incubated at room temperature for 80 rpm, washed and dried (for example, the monoclonal antibody was obtained by immunizing a mouse with uPAR D2D3 (amino acids 88-283), and screening by a hybridoma technique, for example, ATN-658 antibody); (5) 100 μl of alkaline phosphatase-labeled Anti-mouse IgG (e.g., alkaline phosphatase-labeled secondary Antibody (AP-linked Antibody)) diluted 500-fold in sample dilution was added to all wells, incubated for 1 hour at room temperature at 80 rpm, washed and dried; (6) Adding 100 mu l of color development liquid into each hole, and then placing the ELISA plate on an ELISA reader to read absorbance at 405nm for 60min, wherein the absorbance value is read every 1 min; (7) And (3) solving a regression equation according to the dynamic process (enzyme reaction speed) of the absorbance of the active suPAR standard substances with different concentrations along with the time and the concentration of the standard substances, and calculating the concentration of the active suPAR in the sample by using the regression equation.

(1) Treatment of plasma samples: placing peripheral blood collected from a subject to be detected into a potassium oxalate anticoagulation tube, centrifuging to separate plasma, diluting with a sample diluent, and sub-packaging for later use; (2) coating: 100. Mu.l of ATF-HSA protein solution (120. Mu.g/ml) diluted with the coating solution was added to all 1A-12H wells of the ELISA plate (i.e., multi-well plate, 96-well) overnight at 4 ℃; washing with a washing liquid and drying; (3) closing: adding 100 μl of sealing solution into all the holes 1A-12H in the ELISA plate, sealing, incubating, washing with washing solution, and drying; (4) sample addition: adding 100 mu l of active suPAR standard products with different concentrations into the ELISA plates 1A-1F respectively, adding 100 mu l of sample diluent into the G1 and G2 respectively to serve as blank control, adding diluted plasma samples of the tested subjects into other holes in the ELISA plates, sealing, incubating at room temperature, washing with a washing solution, and drying; (5) adding primary antibody: adding (a) 100. Mu.l of ATN-658 antibody diluted with sample diluent and (b) 25. Mu.l of 1.5mg/ml sodium glycerophosphate solution into all wells of ELISA plates 1A-12H, sealing, incubating at room temperature, washing with a washing solution, and drying; (6) adding an enzyme-labeled secondary antibody: adding 100 mu l alkaline phosphatase labeled secondary antibodies diluted 500 times by sample diluent into all holes of the ELISA plates 1A-12H, sealing, incubating at room temperature, washing by using a washing solution, and drying; (7) adding a color development liquid: adding 100 mu l of color development liquid into each hole rapidly, and then placing the ELISA plate on an ELISA reader to read absorbance at 405nm for 60min, wherein absorbance values are read once every 1 min; and (3) solving a regression equation according to the dynamic process (enzyme reaction speed) of the absorbance of the active suPAR standard substances with different concentrations along with the time and the concentration of the standard substances, and calculating the concentration of the active suPAR in the sample by using the regression equation.

(1) Treatment of plasma samples: placing peripheral blood collected from a subject to be detected in a potassium oxalate anticoagulation tube, centrifuging at 4 ℃ for 30min at 1500g, separating plasma, diluting 10 times with sample diluent, subpackaging in a 1.5ml centrifuge tube, and preserving at-80 ℃ for later use; (2) coating: 100. Mu.l of ATF-HSA protein solution (120. Mu.g/ml, sealed with a sealing tape to prevent evaporation of the liquid and overnight at 4 ℃) was added to all 1A-12H wells of the ELISA plate (i.e., multiwell plate, 96 well); washing and drying (removing the sealing adhesive tape, spin-drying the liquid in the holes of the enzyme label plate, sucking the washing liquid by a gun, washing each hole for 6 times, and after the last washing, lightly shooting the enzyme label plate on the water absorbing paper to dry and ensure that no bubbles remain in the holes); (3) Closing: adding 100 μl of sealing solution into all the holes 1A-12H in the ELISA plate, sealing the ELISA plate with sealing tape to prevent evaporation of the solution, and incubating on a shaking table at room temperature of 80 rpm for 1 hr; removing the sealing adhesive tape, spin-drying the liquid in the holes of the enzyme label plate, sucking the washing liquid by a gun to wash each hole for 6 times, and gently beating the enzyme label plate on the water absorbing paper after the last washing to ensure that no bubbles remain in the holes; (4) sample addition: 100 μl of active suPAR standard substances with different concentrations are respectively added into the wells A-G of the ELISA plate, and 100 μl of sample diluent is respectively added into the three wells H1, H2 and H3 of each concentration of the three repeated wells (A1、A2、A3：1μg/L,B1、B2、B3：0.5μg/L,C1、C2、C3：0.25μg/L,D1、D2、D3：0.125μg/L,E1、E2、E3：0.0625μg/L,F1、F2、F3：0.03125μg/L,G1、G2、GF3：0.015625μg/L); to serve as blank control; diluted plasma samples of the test subjects were added to the other wells in the elisa plate, 3 replicate wells per plasma sample, and the elisa plate was sealed with sealing tape and incubated for 1 hour on a shaker at room temperature of 80 rpm. Removing the sealing adhesive tape, spin-drying the liquid in the holes of the enzyme label plate, sucking the washing liquid by a discharge gun, washing each hole for 6 times, and after the last washing, gently beating the enzyme label plate on the water absorbing paper to dry and ensure that no bubbles remain in the holes; (5) adding primary antibody: to all wells of the ELISA plate were added (a) 100. Mu.l of ATN-658 antibody diluted with sample dilution and (b) 25. Mu.l of 1.5mg/ml sodium glycerophosphate solution, the ELISA plate was sealed with sealing tape, and incubated on a shaker at 80 rpm at room temperature for 1 hour. Removing the sealing adhesive tape, spin-drying the liquid in the holes of the enzyme label plate, sucking the washing liquid by a discharge gun, washing each hole for 6 times, and after the last washing, gently beating the enzyme label plate on the water absorbing paper to dry and ensure that no bubbles remain in the holes; (6) adding an enzyme-labeled secondary antibody: to all wells of the ELISA plate, 100. Mu.l of alkaline phosphatase-labeled secondary antibody 500-fold diluted in the sample diluent was added, and the ELISA plate was sealed with a sealing tape and incubated on a shaker at room temperature of 80 rpm for 1 hour. Removing the sealing adhesive tape, spin-drying the liquid in the enzyme-labeled plate holes, sucking the washing liquid by a gun, washing each hole for 6 times, and sucking deionized water by the gun for 3 times. Gently beating the ELISA plate on the absorbent paper to dry and ensure no bubbles remain in the holes; (7) adding a color development liquid: adding 100 μl of color development liquid into each well rapidly with a row gun, placing the ELISA plate on an ELISA reader, reading absorbance at 405nm for 60min, and reading absorbance value every 1 min; and (3) preparing a standard curve and a regression equation thereof according to the absorbance value and the standard concentration, and calculating the concentration of active suPAR in the blood sample by using the regression equation.

According to the use of the first aspect of the invention, the sample to be tested is a plasma sample. In one embodiment, the plasma sample is obtained from a subject to be tested having a glomerular filtration rate, i.e., GFR value, in the range of 0 to 140 ml/(min.1.73 m 2), e.g., in the range of 10 to 130 ml/(min.1.73 m 2), and is processed as follows: placing collected venous blood of a to-be-detected subject into a potassium oxalate anticoagulation tube, centrifuging at 0-10 ℃ (e.g. 4 ℃) (e.g. centrifuging at 1500g for 30 min), separating plasma, diluting the plasma with a sample diluent (e.g. to a proper concentration, e.g. to a concentration in a linear range of a standard curve), sub-packaging (e.g. in a 1.5ml centrifuge tube), and preserving at low temperature (e.g. at-80 ℃) for later use.

According to the use of the first aspect of the invention, the ATF-HSA fusion protein is prepared according to the method carried out in example 1 of the invention; or can be obtained by commercial way or prepared by reference to other literature methods.

According to the use of the first aspect of the present invention, the active suPAR standard is obtained by purifying recombinant suPAR expressed in S2 drosophila embryo cells by means of an affinity column combined with an ion column.

According to the use of the first aspect of the invention, the ATN-658 Antibody is a product of the product No. FHF99110, available from the anti-body System company, 100 ug/count, 1 mg/ml.

According to the use of the first aspect of the invention, the sodium glycerophosphate is sodium beta-glycerophosphate.

According to the use of the first aspect of the invention, the coating solution is an aqueous solution comprising 40mM NaHCO3 and 10mM Na2CO3, pH 9.6.

According to the use of the first aspect of the 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 a Blocker ™ BSA/PBS (10X) solution, i.e., 10% w/v bovine serum albumin/10 mM sodium phosphate/150 mM NaCl solution, pH7.4, siemens, cat: 37525.

According to the use of the first aspect of the invention, the wash solution/sample diluent is a PBS solution at pH7.4 comprising 0.5% Tween-20, wherein the PBS formulation is NaCl 8g/L, KCl 0.2g/L, na2 HPO4.12H2O 3.58g/L, KH2PO4 0.27g/L.

According to the use of the first aspect of the invention, the color-developing solution comprises: 100mM Tris-HCl (pH 9.5), 100mM NaCl, 5mM MgCl2, 2mg/ml PNPP.

The alkaline phosphatase-labeled secondary Antibody according to the use of the first aspect of the present invention may be any commercially available alkaline phosphatase-labeled secondary Antibody such as Anti-mouse IgG, AP-linked Anti-body, CELL SIGNALING Technology, inc.

The use according to the first aspect of the invention, wherein the biological sample is selected from the group consisting of blood, serum, plasma, cell culture broth, saliva and urine.

Further, a second aspect of the invention provides a method of detecting the amount of a soluble urokinase-type plasminogen activator receptor in a subject suffering from or suspected of suffering from a kidney disease (e.g. chronic kidney disease), e.g. blood, e.g. venous blood, comprising the steps of:

Wherein:

The capture reagent is a fusion protein comprising an ATF;

According to the method of the second aspect of the present invention, the potassium oxalate anticoagulant tube is prepared by allowing venous blood potassium oxalate to be used in an amount of 1ml of both (blood: potassium oxalate): 1-4 mg, for example 1ml: anticoagulation was carried out at a rate of 2 mg. In one embodiment, the anticoagulant tube is prepared by the following method: a (e.g., 10%) potassium oxalate solution (e.g., 0.2 ml) was placed in a test tube, the solution was dispersed around the tube wall, and dried to obtain an anticoagulant tube.

According to the method of the second aspect of the invention, the sample diluent is PBS solution of pH7.4 containing 0.5%o Tween-20, wherein the PBS formulation is 8g/L sodium chloride, 0.2g/L potassium chloride, 3.58g/L disodium hydrogen phosphate dodecahydrate, 0.27g/L potassium dihydrogen phosphate.

According to the method of the second aspect of the invention, the GFR value is determined by a method selected from the group consisting of: urinary creatinine clearance method, serum creatinine concentration method, radioisotope labeling method, serum creatinine/urea nitrogen ratio method, inulin clearance method, and combinations thereof.

According to the method of the second aspect of the present invention, the monoclonal antibody binding to the outside of active suPAR is a monoclonal anti-suPAR antibody obtained by immunizing a mouse with uPAR-D2D3 (amino acids 88 to 283) and screening by hybridoma technique.

According to the method of the second aspect of the invention, the monoclonal antibody that binds outside the active suPAR is the antibody ATN-658.

According to the method of the second aspect of the invention, sodium glycerophosphate is added along with the monoclonal antibody, and the mass ratio of the sodium glycerophosphate to the monoclonal antibody is 1: 50-100, for example 1:75.

According to the method of the second aspect of the invention, the agent that specifically binds to the captured active suPAR further comprises a secondary antibody that binds to the monoclonal antibody, e.g. alkaline phosphatase-labeled anti-mouse IgG.

According to the method of the second aspect of the present invention, the fusion protein of ATF is a fusion protein of ATF with another protein or polypeptide or a fragment thereof. The further protein or polypeptide or fragment thereof may be serum albumin, such as Human Serum Albumin (HSA), bovine Serum Albumin (BSA), or Ovalbumin (OVA). In a preferred embodiment, the ATF-containing fusion protein is an ATF-HSA fusion protein.

According to the method of the second aspect of the invention, the fusion protein of ATF is immobilized on a solid substrate. Such solid matrices include, but are not limited to, multiwell plates (e.g., elisa plates), protein chip carrier films (e.g., nitrocellulose films, nylon films, etc.), magnetic beads, fluorescent microspheres, and the like.

The method according to the second aspect of the invention comprises the steps of:

According to the method of the second aspect of the invention, the sample to be tested is a plasma sample. In one embodiment, the plasma sample is obtained from a subject to be tested having a glomerular filtration rate, i.e., GFR value, in the range of 0 to 140 ml/(min.1.73 m 2), e.g., in the range of 10 to 130 ml/(min.1.73 m 2), and is processed as follows: placing collected venous blood of a to-be-detected subject into a potassium oxalate anticoagulation tube, centrifuging at 0-10 ℃ (e.g. 4 ℃) (e.g. centrifuging at 1500g for 30 min), separating plasma, diluting the plasma with a sample diluent (e.g. to a proper concentration, e.g. to a concentration in a linear range of a standard curve), sub-packaging (e.g. in a 1.5ml centrifuge tube), and preserving at low temperature (e.g. at-80 ℃) for later use.

In the present invention, the peripheral blood is venous blood, unless otherwise specified.

According to the method of the second aspect of the invention, the ATF-HSA fusion protein is prepared according to the method carried out in example 1 of the invention; or can be obtained by commercial way or prepared by reference to other literature methods.

According to the method of the second aspect of the present invention, the active suPAR standard is obtained by purifying recombinant suPAR expressed in S2 drosophila embryo cells by affinity column binding to ion column.

According to the method of the second aspect of the invention, the ATN-658 Antibody is a product of the product No. FHF99110 from the anti-body System company, 100 ug/count, 1 mg/ml.

According to the method of the second aspect of the invention, the sodium glycerophosphate is sodium beta-glycerophosphate.

According to the method of the second aspect of the invention, the coating liquid is an aqueous solution of pH9.6 comprising 40mM NaHCO3 and 10mM Na2CO 3.

According to the method of the second aspect of the 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 a Blocker ™ BSA/PBS (10X) solution, i.e., 10% w/v bovine serum albumin/10 mM sodium phosphate/150 mM NaCl solution, pH7.4, siemens, cat: 37525.

According to the method of the second aspect of the invention, the wash solution/sample diluent is a PBS solution at pH7.4 comprising 0.5% Tween-20, wherein the PBS formulation is NaCl 8g/L, KCl 0.2g/L, na2 HPO4.12H2O 3.58g/L, KH2PO4 0.27g/L.

According to the method of the second aspect of the present invention, the color-developing solution comprises: 100mM Tris-HCl (pH 9.5), 100mM NaCl, 5mM MgCl2, 2mg/ml PNPP.

The alkaline phosphatase-labeled secondary Antibody according to the method of the second aspect of the present invention may be any commercially available alkaline phosphatase-labeled secondary Antibody such as Anti-mouse IgG, AP-linked Anti-body, CELL SIGNALING Technology, inc.

The method according to the second aspect of the invention, wherein the biological sample is selected from the group consisting of blood, serum, plasma, cell culture broth, saliva and urine.

Various detection materials for use in the methods of the invention may be provided in a kit, for which purpose the third aspect of the invention provides a kit, for example a kit for use in a method of detecting a soluble urokinase-type plasminogen activator receptor in a renal disease (e.g. chronic kidney disease) subject or a biological sample such as blood, e.g. venous blood, of a subject suspected of having a renal disease (e.g. chronic kidney disease), for example a kit for use in a method of any of the second aspects of the invention, comprising:

ELISA plates (i.e., multi-well plates, e.g., 96 wells), ATF-HSA protein (dry powder or solution, e.g., 1ml, e.g., 1 to 1.5mg/ml solution), active supAR standard (dry powder or diluent, e.g., 1ml, e.g., 1mg/ml solution), ATN-658 antibody (e.g., 1mg/ml, e.g., 100. Mu.l), alkaline phosphatase-labeled secondary antibody (e.g., 25. Mu.l, e.g., 500 Xalkaline 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), optionally blood sample-treating anticoagulant tubes (e.g., potassium oxalate anticoagulant tubes), and optionally kit instructions (in which the detection methods of any of the embodiments of the invention are described herein).

In describing the method steps of the present invention, the specific steps described therein may be distinguished in some details or in language description from the steps described in the examples of the detailed description section below, however, the above-described method steps may be summarized by one skilled in the art in light of the detailed disclosure of the present invention as a whole.

Any of the embodiments of any of the aspects of the invention may be combined with other embodiments, provided that they do not contradict. Furthermore, in any of the embodiments of any of the aspects of the present invention, any technical feature may be applied to the technical feature in other embodiments as long as they do not contradict. The present invention is further described below.

All documents cited herein are incorporated by reference in their entirety and are incorporated by reference herein to the extent they are not inconsistent with this invention. Furthermore, various terms and phrases used herein have a common meaning known to those skilled in the art, and even though they are still intended to be described and explained in greater detail herein, the terms and phrases used herein should not be construed to be inconsistent with the ordinary meaning in the sense of the present invention.

In one/some embodiments of the invention, the various detection materials used in the methods of the invention may be configured in a kit. In one embodiment, the kit further comprises one or more reagents for detecting the captured active suPAR, wherein the capture reagent is labeled with one of the detectable pair of labeled components and the one or more reagents for detecting the captured active suPAR is labeled with the other of the pair of labeled components. Detection of the captured active suPAR can be achieved by detecting the interaction between the paired marker components. In a preferred embodiment, the one or more reagents for detecting captured active suPAR comprise a monoclonal antibody that binds to the outside of the active suPAR and/or a secondary antibody that binds to the monoclonal antibody, e.g., alkaline phosphatase-labeled anti-mouse IgG.

In one/some embodiments of the invention, the detection methods of the invention are of non-diagnostic interest and are performed in vitro.

In one/some embodiments of the invention, the capture reagent is labeled with a detectable label component. Binding of the capture reagent to the active suPAR causes a change in the label moiety on the capture reagent, and detection of the captured active suPAR can be achieved by detecting the change in the label moiety on the capture reagent.

Drawings

Fig. 1: principle of ELISA detection method of active suPAR.

Fig. 2: ELISA detection 96-well ELISA plate schematic.

Detailed Description

The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof. The present invention generally and/or specifically describes the materials used in the test as well as the test methods. Although many materials and methods of operation are known in the art for accomplishing the objectives of the present invention, the present invention will be described in as much detail herein. The following examples further illustrate the invention, but do not limit it.

The following further explains or illustrates the contents of the present invention by means of examples: unless otherwise indicated, the solutions described below are aqueous solutions; when referring to percentages, the percentages of the mixture formulated with liquid/liquid are all volume/volume percentages, the percentages of the mixture formulated with solid/liquid are all mass/volume percentages, and the percentages of the mixture formulated with solid/solid are all mass/mass percentages.

Some reagents and experimental materials used in the specific examples of the present application are described in detail in CN116008557a of the research team of the present inventors, and in particular some reagents and example materials are summarized as follows:

ATF-HSA fusion protein: prepared according to the method carried out in example 1 of CN 116008557A.

Active suPAR standard: recombinant suPAR expressed in S2 drosophila embryo cells was purified by affinity column binding to ion column. The preparation process can also be found in the methods disclosed in [0020] to [0021] of CN112180103A (application No. 202011147152.6) of the present inventors.

ATN-658 antibody: anti-human uPAR antibodies, the specific examples of the present invention used, unless otherwise indicated, are those purchased from Antibody System under the product number FHF99110, 100 ug/per standard, 1mg/ml; as described in the literature, the ATN-658 antibody was obtained from Attenuon, LLC (San Diego, calif.), which was a monoclonal anti-supAR antibody obtained by screening a mouse immunized with uPAR D2D3 (amino acids 88-283) by hybridoma technique [ T.W. Bauer, et al, CANCER RESEARCH 65 (17) (2005) 7775-81]. Sodium glycerophosphate (used in the present example as sodium beta-glycerophosphate, available from Sigma-Aldrich, analytical grade) was formulated with water for injection.

Alkaline phosphatase-labeled secondary antibodies (AP-linked antibodies, CELL SIGNALING Technology, inc., # 7056): the enzyme-labeled secondary antibody special for Western blotting and ELISA is an affinity purified goat anti-mouse IgG (H & L) antibody which can be jointed with bovine small intestine alkaline phosphatase and can be used as the secondary antibody in Western immunoblotting experiments and ELISA application; recommended antibody dilution is 1: 500-3000. Alkaline Phosphatase (AP) conjugated secondary antibodies are used in Western blot assays to bind specific chemiluminescent or other substrates. One advantage of AP conjugation is that the reaction rate can remain linear over a long period of time.

96-Well elisa plate: 96-well, polystyrene, transparent, nunc-Immuno ^™ MicroWell^™, available from Merck.

Coating liquid: an aqueous solution of pH9.6 containing 40mM sodium bicarbonate and 10mM sodium carbonate.

Sealing liquid: blocker ™ BSA/PBS (10X) solution, 10% w/v bovine serum albumin/10 mM sodium phosphate/150 mM NaCl solution, pH7.4, siemens, cat: 37525.

Wash/sample dilution: PBS solution containing 0.5%o Tween-20 and having pH7.4, wherein the PBS formulation comprises 8g/L sodium chloride, 0.2g/L potassium chloride, 3.58g/L disodium hydrogen phosphate dodecahydrate and 0.27g/L potassium dihydrogen phosphate.

Color development liquid: 100mM Tris-HCl (pH 9.5), 100mM sodium chloride, 5mM magnesium chloride, 2mg/ml PNPP. PNPP (disodium 4-nitrophenylphosphate hexahydrate, sigma) was used as a substrate for the enzyme immunoassay of AP.

Recombinant suPAR was expressed in Drosophila Schneider cells and purified by the method of literature [C. Yuan, Q. Huai, C.B. Bian, M.D. Huang, Progress in Biochemistry and Biophysics 33(3) (2006)277-281]; recombinant soluble murine uPAR (smopar) and suPAR D2D3 were supplied by Finsen laboratories; BSA powder used as blocking solution is also commercially available from Sangong Biotech.

Example 1: construction, expression, purification and characterization of ATF-HSA fusion proteins

The ATF-HSA fusion protein obtained was of a relative molecular weight of about 84kDa, and a purity of 94.1% for ATF-HSA fusion protein sufficient for ELISA detection of supAR, according to the method carried out in example 1 of CN116008557A of the research team of the present inventors. The amino acid sequences and nucleotide sequences involved in the preparation of the ATF-HSA fusion protein are described in detail in CN 116008557A.

Example 2: detection of active suPAR in human blood samples Using ATF-HSA

The principle of ELISA detection of active suPAR in this example is the same as that described in example 1 of CN116008557A, as shown in FIG. 1. The method specifically comprises the following steps:

(1) Treatment of plasma samples: venous blood collected from a subject to be tested (healthy volunteers or chronic kidney disease patients) is placed in a potassium oxalate anticoagulation tube, centrifuged at 1500g for 30min at 4 ℃, plasma is separated, the plasma is diluted with a sample diluent (for example, diluted to a proper concentration, for example, diluted to a concentration in the linear range of a standard curve), and the diluted plasma is subpackaged in a 1.5ml centrifuge tube for storage at-80 ℃ for later use; [ preparation of potassium oxalate anticoagulant tube ] it is well known to those skilled in the art, for example, that the anticoagulant tube used in the specific experiments of the present invention is prepared as follows, unless otherwise indicated: taking 0.2ml of 10% potassium oxalate solution, placing into a test tube, lightly knocking the test tube to disperse the solution around the tube wall, and drying at 75 ℃ to obtain an anticoagulation tube capable of being used for anticoagulation of 10ml of blood, namely, the dosage of potassium oxalate per 1ml of blood is 2mg; thus, in one embodiment of any aspect of the invention, venous blood collected from a test subject is treated with potassium oxalate at 1ml of both: 2mg proportion anticoagulation ];

(2) Coating: to all wells 1A-12H of the 96-well ELISA plate (see FIG. 2), 100. Mu.l of ATF-HSA protein solution (120. Mu.g/ml) diluted with the coating solution was added, and the ELISA plate was sealed with a sealing tape to prevent evaporation of the liquid, and at 4℃overnight. Removing the sealing adhesive tape, spin-drying the liquid in the holes of the enzyme label plate, sucking the washing liquid by a discharge gun, washing each hole for 6 times, and after the last washing, gently beating the enzyme label plate on the water absorbing paper to dry and ensure that no bubbles remain in the holes.

(3) Closing: 100. Mu.l of a blocking solution was added to all wells 1A-12H in the ELISA plate, and the ELISA plate was sealed with a sealing tape to prevent evaporation of the liquid, and incubated for 1 hour on a shaker at room temperature of 80 rpm. Removing the sealing adhesive tape, spin-drying the liquid in the holes of the enzyme label plate, sucking the washing liquid by a gun to wash each hole for 6 times, and after the last washing, gently beating the enzyme label plate on the water absorbing paper to dry and ensure that no bubbles remain in the holes.

(4) Sample adding: 100 μl of active suPAR standard substances with different concentrations are respectively added into the wells A-G of the ELISA plate, and 100 μl of sample diluent is respectively added into the three wells H1, H2 and H3 of each concentration of the three repeated wells (A1、A2、A3：1μg/L,B1、B2、B3：0.5μg/L,C1、C2、C3：0.25μg/L,D1、D2、D3：0.125μg/L,E1、E2、E3：0.0625μg/L,F1、F2、F3：0.03125μg/L,G1、G2、GF3：0.015625μg/L); to serve as blank control; diluted plasma samples of the test subjects were added to the other wells in the elisa plate, 3 replicate wells per plasma sample, and the elisa plate was sealed with sealing tape and incubated for 1 hour on a shaker at room temperature of 80 rpm. Removing the sealing adhesive tape, spin-drying the liquid in the holes of the enzyme label plate, sucking the washing liquid by a discharge gun, washing each hole for 6 times, and after the last washing, gently beating the enzyme label plate on the water absorbing paper to dry and ensure that no bubbles remain in the holes.

(5) Adding an antibody: to all wells of a 96-well ELISA plate, 100. Mu.l of ATN-658 antibody diluted with sample dilution and 25. Mu.l of 1.5mg/ml sodium glycerophosphate solution were added, and the ELISA plate was sealed with a sealing tape and incubated on a shaker at 80 rpm at room temperature for 1 hour. Removing the sealing adhesive tape, spin-drying the liquid in the holes of the enzyme label plate, sucking the washing liquid by a discharge gun, washing each hole for 6 times, and after the last washing, gently beating the enzyme label plate on the water absorbing paper to dry and ensure that no bubbles remain in the holes.

(6) Adding enzyme-labeled secondary antibodies: to all wells of a 96-well ELISA plate, 100. Mu.l of alkaline phosphatase-labeled secondary antibody 500-fold diluted in the sample diluent was added, and the ELISA plate was sealed with a sealing tape and incubated on a shaker at room temperature of 80 rpm for 1 hour. Removing the sealing adhesive tape, spin-drying the liquid in the enzyme-labeled plate holes, sucking the washing liquid by a gun, washing each hole for 6 times, and sucking deionized water by the gun for 3 times. The ELISA plate is gently patted dry on the absorbent paper and no bubbles remain in the holes.

(7) Adding a color development liquid: adding 100 μl of color development liquid into each well rapidly with a row gun, placing the ELISA plate on an ELISA reader, reading absorbance at 405nm for 60min, and reading absorbance value every 1 min; and (3) preparing a standard curve and a regression equation thereof according to the absorbance value and the standard concentration, and calculating the concentration of active suPAR in the blood sample by using the regression equation.

The standard curve can be prepared by referring to a method carried by CN105954522B, specifically, the dynamic process of the change of the absorbance of active suPAR standard products with different concentrations along with time is read at 405nm on an enzyme-labeled instrument, the time of monitoring 60 minutes is taken as an abscissa, the absorbance value at 405nm is taken as an ordinate, and the concentration of the active suPAR corresponding to the dynamic curve from top to bottom is sequentially from high to low; subtracting the absorbance at the OD405nm of the 1 st minute from the absorbance at the OD405nm of the 60 th minute corresponding to different active supAR concentrations to obtain a variation value X (mOD, milliAbs) of the OD405nm in 60 minutes; dividing X by 60 minutes to obtain enzyme reaction speeds (milliAbs/min, mOD/min) corresponding to different active supAR concentrations; then, a standard curve is made according to the concentration (X axis, mu g/L) of serial active suPAR and the corresponding enzyme reaction speed (Y axis, mOD/min), and a regression equation of the standard curve can be obtained; and substituting the enzyme reaction speed corresponding to the plasma sample to be detected into a regression equation to obtain the active suPAR content in the corresponding plasma sample. The invention can use a Synergy ™ 4 plate reader (BioTek Instruments) to measure absorbance and use Gen5 software to process data.

The regression equation obtained by measuring/calculating the concentration of the active suPAR standard substance and the enzyme reaction speed corresponding to each concentration is as follows: y=5.1418x+0.7835, r2=0.9998, where Y is the enzyme reaction rate (mOD/min), X is uPAR concentration (μg/L), and the enzyme reaction rate in the range of 0.015625 to 1 μg/L is in the range of 0.8638 to 5.9253.

Based on the method detailed in this example 2, a kit comprising the following components may be provided for use in a method of detecting a soluble urokinase-type plasminogen activator receptor in a renal disease (e.g., chronic kidney disease) subject or in a biological sample such as blood, e.g., venous blood, suspected of having a renal disease (e.g., chronic kidney disease) subject, the kit comprising: an ELISA plate (i.e., a multi-well plate, e.g., 96 wells), ATF-HSA protein (dry powder or solution, e.g., 1ml, e.g., 1 to 1.5mg/ml solution), an active supAR standard (dry powder or diluent, e.g., 1ml, e.g., 1mg/ml solution), ATN-658 antibody (e.g., 1mg/ml, e.g., 100. Mu.l), alkaline phosphatase-labeled secondary antibody (e.g., 25. Mu.l, e.g., 500 Xalkaline phosphatase-labeled secondary antibody), sodium glycerophosphate solution (e.g., 5 ml), coating solution (e.g., 10 ml), sample diluent (e.g., 20 ml), washing solution (e.g., 20 ml), blocking solution (e.g., 10 ml), color-developing solution (e.g., 10 ml), an optional blood sample treatment anticoagulant tube (e.g., potassium oxalate anticoagulant tube), and optional kit instructions (in which the assay methods of any one embodiment of the invention or suitable variations of these assay methods are described herein are described). The components of the various kits described above are as detailed herein in the context of the present disclosure, for example, the coating solution may be an aqueous solution having a pH of 9.6 containing 40mM sodium bicarbonate and 10mM sodium carbonate; the wash solution/sample diluent may be a PBS solution of pH7.4 containing 0.5% Tween-20, wherein the PBS formulation is 8g/L sodium chloride, 0.2g/L potassium chloride, 3.58g/L disodium hydrogen phosphate dodecahydrate, 0.27g/L potassium dihydrogen phosphate; the color-developing solution may be an aqueous solution comprising the following composition: 100mM Tris-HCl (pH 9.5), 100mM sodium chloride, 5mM magnesium chloride, 2mg/ml PNPP.

Example 3: performance of the active suPAR assay

This example is a methodology performed according to the method described in example 2, wherein the plasma sample to be tested is obtained from a subject (as not otherwise described, the subject involved in the specific test of the invention is a volunteer) having a GFR value of 45.7 ml/(min.1.73 m 2).

1. Precision of

The method of example 2 was used to repeatedly measure the active suPAR content in the same test plasma sample (n=16 times) on the same day on the same plate to obtain the corresponding Average Value (AVE), standard Deviation (SD), intra-batch coefficient of variation (%), and the result of a certain test plasma sample is: ave=0.902 μg/L, sd=0.068, intra-batch coefficient of variation=6.83%; in addition, the active suPAR content in the above-mentioned plasma samples to be measured was repeatedly measured daily for 6 consecutive days to obtain the Average Value (AVE), standard Deviation (SD), and inter-lot variation coefficient (%) for 6 days, and the above-mentioned plasma samples to be measured gave the following results: ave=0.987 μg/L, sd=0.089, inter-lot variation coefficient=8.74%. The above results show that the variation coefficient in the batch is less than 10% and the variation coefficient between batches is less than 15%, and the reproducibility of the established ELISA new method is good.

2. Recovery rate

A series of 10. Mu.l of active supAR standard samples of known concentration were added to 100. Mu.l of the sample dilutions or 10-fold dilutions of plasma, respectively, and the final concentration of active supAR added to the 110. Mu.l system was calculated (this concentration was referred to as active supAR added concentration). The concentration of active suPAR in the sample dilution to which active suPAR was added and the diluted plasma were separately measured by the method of example 2 (this concentration was referred to as the active suPAR detection concentration).

Respectively calculating regression equations obtained by two dilution modes by taking the detection concentration as a Y axis and the addition concentration as an X axis; the regression equation for the sample dilutions was y=1.0423x+0.0272, r ² = 0.9992, and the regression equation for the diluted plasma was y=1.0327 x+0.0483, r ² =0.9987; the percent obtained by dividing the slope of the dilution plasma regression equation by the slope of the sample dilution regression equation is the recovery rate r=99.08%, which shows that the method has high recovery rate and excellent accuracy. The method is shown to have high recovery of the load active suPAR and very little loss of specific active suPAR after addition to plasma samples. The definition of 100% recovery is: the slope of the curve between loading active suPAR and detecting the suPAR, wherein the active suPAR is added to the dilution buffer.

3. Detection limit and linear range

The detection limit and linear range of the method of example 2 of the present invention can be determined with reference to the method carried out in example 3 of CN 116008557A. By investigation, the detection limit of the method of example 2 of the present invention for determining the active suPAR content is less than 20ng/L, and the linear relationship is good R2 >0.99 in a wide linear range of less than 15. Mu.g/L.

In addition, the inventors have found that the method of example 2 of the present invention exhibits excellent recovery for plasma samples at different GFR value levels, and that precision and detection limits and linear ranges are excellent. However, it was unexpectedly found that when anticoagulation of blood was performed without potassium oxalate, and instead with other commonly used anticoagulants, subjects with different GFR values exhibited significantly different recovery rates, as detailed in examples 4-6.

Example 4: performance of the active suPAR assay

This example uses venous blood from 36-58 year old male subjects whose GFR values were tested on day 3 using urinary creatinine clearance and serum creatinine concentration methods, and reports the GFR values from these subjects as a mean value of the two-way GFR, and it is well known that GFR values can be determined by a number of methods such as, but not limited to, urinary creatinine clearance, serum creatinine concentration, radioisotope labeling, serum creatinine/urea nitrogen ratio, inulin clearance, and combinations thereof, other available GFR value determination methods can also be used in the present invention, blood samples are grouped according to source subject GFR values, and the performance of the present invention for determining the par method is examined for each group of blood samples by reference to the methods of example 2 and example 3 of the present invention, specifically, the following is described. Group a: 3 venous blood obtained from 3 subjects with GFR values ranging from 112 to 127 ml/(min.1.73 m 2) (normally considered as normal kidneys of subjects with GFR ranges) were treated according to the treatment of plasma sample in step (1) of example 2 of the present invention, diluted 10-fold with sample diluent to obtain 10-fold diluted plasma, and then tested and the recovery was calculated according to the method of step "2, recovery" of example 3 of the present invention, resulting in recovery means of 98.81% (98.57%, 99.12%, 98.74%, respectively, the average values given below) for 3 samples; group b: 3 venous blood obtained from 4 subjects with GFR values ranging from 93 to 98 ml/(min.1.73 m 2) (the normal kidneys of the subjects with GFR ranges are generally considered to be normal) are treated according to the treatment of plasma sample in step (1) of example 2 of the present invention, diluted 10 times with sample diluent to obtain 10 times diluted plasma, and then tested and the recovery is calculated according to the method of step "2, recovery" of example 3 of the present invention, resulting in a mean recovery of 99.13% for each sample; group c: 3 venous blood obtained from 4 subjects with GFR values ranging from 72 to 77 ml/(min.1.73 m 2) (subjects with GFR in this range are generally considered to be slightly decreased in GFR), were treated according to the treatment "treatment of plasma sample in step (1) of example 2 of the present invention, diluted 10-fold with sample diluent to obtain 10-fold diluted plasma, and then tested and recovery was calculated according to the method of step" 2, recovery "of example 3 of the present invention, resulting in a recovery mean value of 98.57% for each sample; group d: 3 parts of venous blood obtained from 5 subjects having GFR values ranging from 54 to 58 ml/(min.1.73 m 2) (usually regarded as a moderate decrease in GFR in this GFR-range subject) were treated according to the treatment of plasma sample in step (1) of example 2 of the present invention, diluted 10-fold with a sample diluent to obtain 10-fold diluted plasma, and then tested and the recovery was calculated according to the method of step "2, recovery" of example 3 of the present invention, resulting in a mean recovery of 98.74% for each sample; group e: 3 venous blood obtained from 3 subjects having GFR values ranging from 32 to 36 ml/(min.1.73 m 2) (usually considered as a moderate decrease in GFR in this GFR-range subject) were treated according to the treatment of plasma sample in step (1) of example 2 of the present invention, diluted 10-fold with sample diluent to obtain 10-fold diluted plasma, and then tested and the recovery was calculated according to the method of step "2, recovery" of example 3 of the present invention, resulting in a mean recovery of 99.02% for each sample; group f: venous blood obtained from 4 subjects having GFR values ranging from 18 to 23 ml/(min.1.73 m 2) (usually considered as a severe decrease in GFR in this GFR-range subject) was treated according to the treatment "treatment of plasma sample in step (1) of example 2 of the present invention, diluted 10-fold with sample diluent to obtain 10-fold diluted plasma, and the recovery was measured and calculated according to the method of step" 2, recovery "of example 3 of the present invention, and the average recovery of each sample was 98.36%. The above results demonstrate that example 2 and example 3 of the present invention have excellent performance in the measurement of the suPAR, and that the recovery rates of different blood samples (with a large difference in GFR values) are substantially the same when the suPAR is measured. However, it has been unfortunately found that when blood is treated in the same way using EDTA-2Na anticoagulant tube (1.5 mg:1ml, commercially available) or sodium citrate anticoagulant tube (1:9, commercially available) as commonly used in the art in example 2, step "(1) of treatment of plasma samples", there is a significant difference in recovery of the method when the suPAR is measured for blood samples of different GFR values, particularly when the GFR value is lower. As described in particular in example 5 and example 6.

Example 5: performance of the active suPAR assay

This example was conducted using venous blood from the same subjects as in example 4 (whose GFR values have been previously determined as in example 4), and blood samples were grouped according to the GFR values of the source subjects (group and group names are the same as in example 4), and blood samples of each group were anticoagulated by referring to the methods of the present invention in examples 2 and 3 but using EDTA-2Na anticoagulation tubes (1.5 mg:1ml, commercially available) to examine the performance of the assay of the present invention, as follows. Group a: 3 venous blood obtained from 3 subjects with GFR values ranging from 112 to 127 ml/(min.1.73 m 2) (normally considered as normal kidneys of subjects with GFR ranges) were subjected to the method of "treatment of plasma sample" according to example 2 of the present invention, but anticoagulated blood sample using EDTA-2Na anticoagulation tube, diluted 10-fold with sample diluent to obtain 10-fold diluted plasma, followed by testing and calculation of recovery according to the method of "step 2, recovery" according to example 3 of the present invention, and as a result, the recovery average of 3 samples was 98.43%; group b: 3 venous blood obtained from 4 subjects with GFR values ranging from 93 to 98 ml/(min.1.73 m 2) (the normal kidneys of the subjects with GFR ranges are generally considered), anticoagulated blood samples were treated by the method of step "(1) of example 2 according to the present invention but anticoagulated blood samples were treated with EDTA-2Na anticoagulants, then tested and recovery was calculated by the method of step" 2, recovery "according to example 3 according to the present invention, and as a result, the average recovery of each sample was 98.71%; group c: 3 venous blood obtained from 4 subjects with GFR values ranging from 72 to 77 ml/(min.1.73 m 2) (subjects with GFR in this range are generally considered to be slightly reduced in GFR), anticoagulated blood samples were tested and the recovery was calculated according to the procedure of example 2 of the present invention, procedure "(1) treatment of plasma samples" but using EDTA-2Na anticoagulants, followed by the procedure of example 3, procedure "2, recovery", and the average recovery of each sample was 96.36%; group d: 3 venous blood obtained from 5 subjects with GFR values ranging from 54 to 58 ml/(min.1.73 m 2) (usually considered as moderate decrease of GFR in this GFR-range subject) were subjected to test and calculation of recovery rate according to the method of example 2 of the present invention of "treatment of plasma sample" but anticoagulated blood sample using EDTA-2Na anticoagulation tube, followed by the method of example 3 of "2, recovery rate" of the present invention, resulting in a mean recovery rate of 92.27% for each sample; group e: 3 venous blood obtained from 3 subjects with GFR values ranging from 32 to 36 ml/(min.1.73 m 2) (usually considered as moderate decrease of GFR in this GFR-range subject) were subjected to test and calculation of recovery rate according to the method of example 2 of the present invention of "treatment of plasma sample" but anticoagulated blood sample with EDTA-2Na anticoagulation tube, followed by the method of example 3 of "2, recovery rate" of the present invention, resulting in a mean value of recovery rate of 86.36% for each sample; group f: venous blood obtained from 4 subjects having GFR values ranging from 18 to 23 ml/(min.1.73 m 2) (usually considered as a severe decrease in GFR in this GFR-range subject) was subjected to 3 copies of venous blood obtained by the "treatment of plasma sample" method of example 2 of the present invention but anticoagulated blood sample using EDTA-2Na anticoagulated tube, then tested and recovery was calculated according to the "step 2, recovery" method of example 3 of the present invention, and as a result, the average recovery of each sample was 80.25%.

Example 6: performance of the active suPAR assay

This example was conducted using venous blood from the same subjects as in example 4 (whose GFR values have been previously determined as in example 4), each blood sample was grouped according to the GFR value of the source subject (group and group designation are the same as in example 4), and each group of blood samples was anticoagulated blood samples by referring to the methods of examples 2 and 3 of the present invention but using sodium citrate anticoagulation tube (1:9, commercially available) to examine the performance of the assay suPAR method of the present invention, as follows. Group a: 3 venous blood obtained from 3 subjects with GFR values ranging from 112 to 127 ml/(min.1.73 m 2) (the normal kidneys of the subjects with GFR ranges are generally considered to be normal) are subjected to the method of the invention of example 2, step "(1) treatment of plasma samples, but anticoagulated blood samples are diluted 10 times by using sodium citrate anticoagulation tube, 10 times diluted plasma is obtained, and then the recovery rate is measured and calculated according to the method of the invention of example 3, step 2, recovery rate, and the average recovery rate of 3 samples is 98.74%; group b: 3 venous blood obtained from 4 subjects with GFR values ranging from 93 to 98 ml/(min.1.73 m 2) (the normal kidneys of the subjects with GFR ranges are generally considered), blood samples were anticoagulated by the method of example 2 "(1) plasma sample treatment" according to the present invention, and then tested and recovery was calculated according to the method of example 3, step 2, recovery "according to the present invention, and the average recovery of each sample was 99.27%; group c: 3 venous blood obtained from 4 subjects with GFR values ranging from 72 to 77 ml/(min.1.73 m 2) (subjects with GFR in this range are generally considered to be slightly reduced in GFR), anticoagulated blood samples were tested and the recovery was calculated according to the method of example 2 of the present invention, step "(1) treatment of plasma samples but using sodium citrate anticoagulants, followed by the method of example 3, step 2 of the present invention, and the recovery was found to be 97.43% in average; group d: 3 venous blood obtained from 5 subjects with GFR values ranging from 54 to 58 ml/(min.1.73 m 2) (usually considered as moderate decrease of GFR in this GFR-range subject) were subjected to the method of example 2 of the present invention of "treatment of plasma sample" but anticoagulated with sodium citrate anticoagulated tube, followed by the test and calculation of recovery rate according to the method of example 3 of the present invention of "step 2, recovery rate", and as a result, the average recovery rate of each sample was 94.34%; group e: 3 venous blood obtained from 3 subjects with GFR values ranging from 32 to 36 ml/(min.1.73 m 2) (usually considered as moderate decrease of GFR in this GFR-range subject) were subjected to the method of example 2 of the present invention of "treatment of plasma sample" but anticoagulated with sodium citrate anticoagulated tube, followed by the test and calculation of recovery rate according to the method of example 3 of the present invention of "step 2, recovery rate", and as a result, the average recovery rate of each sample was 87.93%; group f: venous blood obtained from 4 subjects having GFR values ranging from 18 to 23 ml/(min.1.73 m 2) (usually considered as severe GFR drop in this GFR-range subject) was subjected to 3 copies of venous blood obtained by the method of "treatment of plasma sample" in example 2 of the present invention but anticoagulated blood sample using sodium citrate, then tested and recovery was calculated according to the method of "step 2, recovery" in example 3 of the present invention, and as a result, the average recovery of each sample was 84.47%. The results of examples 4-6 above show that, for subjects with low glomerular filtration rate, the method recovery rate was related not only to the source of the blood sample, but also to the anticoagulation method, and that a wide range of GFR blood samples had excellent recovery rates when oxalate was selected as the anticoagulant, whereas the recovery rates of blood samples with low GFR values were significantly reduced when other commonly used anticoagulants such as sodium citrate and disodium edentate were selected, which was not anticipated at all by the prior art.

Example 7: active suPAR assay

The active suPAR concentrations in these blood samples (plasma) were determined using the methods of example 2 and example 3 for all subjects blood samples of groups a to f mentioned in example 4, 3 times per blood sample, and the results of each test of each group were averaged and reported as follows: 3 subjects of group a had a plasma activity suPAR concentration (μg/L) =1.87±0.32, 4 subjects of group b had a plasma activity suPAR concentration (μg/L) =1.93±0.27, 4 subjects of group c had a plasma activity suPAR concentration (μg/L) =3.14±0.41, 5 subjects of group d had a plasma activity suPAR concentration (μg/L) =4.76±0.73, 3 subjects of group e had a plasma activity suPAR concentration (μg/L) =6.83±0.38, and 3 subjects of group f had a plasma activity suPAR concentration (μg/L) =9.11±0.64. These results indicate that the more severe renal disease characterized by GFR values is a higher concentration of suPAR in the patient, and thus the present invention provides a method that can readily detect and reflect the presence or absence of a subject and the severity of renal disease, particularly chronic renal disease.

The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.

Claims

1. Use of a soluble urokinase-type plasminogen activator receptor in a method of detecting kidney disease, e.g., chronic kidney disease, in a biological sample, e.g., blood, e.g., venous blood, of a subject suffering from or suspected of suffering from kidney disease, e.g., chronic kidney disease, comprising the steps of:

Wherein:

The capture reagent is a fusion protein comprising an ATF;

2. Use according to claim 1, wherein:

The potassium oxalate anticoagulant tube is prepared by mixing 1ml of potassium oxalate for venous blood with 1ml of potassium oxalate for venous blood: 1-4 mg, for example 1ml: anticoagulation in a proportion of 2 mg; for example, the anticoagulant tube is prepared as follows: putting potassium oxalate solution into a test tube, dispersing the solution around the tube wall, and drying to obtain an anticoagulant tube;

The sample diluent is PBS solution containing 0.5 per mill Tween-20 and having pH of 7.4, wherein the PBS formula comprises 8g/L of sodium chloride, 0.2g/L of potassium chloride, 3.58g/L of disodium hydrogen phosphate dodecahydrate and 0.27g/L of potassium dihydrogen phosphate;

The GFR value is determined by a method selected from the group consisting of: urinary creatinine clearance method, serum creatinine concentration method, radioisotope labeling method, serum creatinine/urea nitrogen ratio method, inulin clearance method, and combinations thereof;

the monoclonal antibody combined with the outside of the active suPAR is a monoclonal anti-suPAR antibody obtained by immunizing a mouse with uPAR-D2D3 (amino acids 88-283) and screening by a hybridoma technology;

The monoclonal antibody bound to the outside of the active suPAR is the antibody ATN-658;

Sodium glycerophosphate is also added along with the monoclonal antibody, and the mass ratio of the sodium glycerophosphate to the monoclonal antibody is 1: 50-100, for example 1:75;

Reagents that specifically bind the captured active suPAR also include a secondary antibody that binds to the monoclonal antibody, such as alkaline phosphatase-labeled anti-mouse IgG;

The fusion protein of the ATF is a fusion protein of the ATF and another protein or polypeptide or a fragment thereof; for example, the further protein or polypeptide or fragment thereof may be serum albumin, such as human serum albumin, bovine serum albumin, or ovalbumin; in a preferred embodiment, the ATF-containing fusion protein is an ATF-HSA fusion protein;

the fusion protein of the ATF is immobilized on a solid substrate; for example, the solid substrate includes, but is not limited to, a multiwell plate (e.g., an ELISA plate), a protein chip carrier film (e.g., nitrocellulose film, nylon film, etc.), magnetic beads, fluorescent microspheres, etc.

3. Use according to claim 1, said detection method comprising the steps of:

(1) Adding ATF-HSA protein solution diluted by coating liquid into the holes of a porous plate for coating (overnight at 4 ℃), washing and drying; (2) Adding a sealing liquid into the hole for sealing, incubating at room temperature, washing and drying; (3) Adding a series of active suPAR standard substances with a concentration to some holes respectively, adding sample diluent to other holes to serve as blank control, and adding samples to be detected to other holes (for example, diluting the sample diluent by 10 times); incubating at room temperature, washing and drying; (4) Adding mouse anti-human ssuPAR monoclonal antibody such as ATN-658 antibody diluted with sample diluent into all wells, incubating at room temperature, washing and drying; (5) Alkaline phosphatase-labeled Anti-mouse IgG (e.g., alkaline phosphatase-labeled secondary Antibody (AP-linked Antibody)) was added to all wells, incubated at room temperature, washed and dried; (6) Adding a color development liquid into each hole, and then placing the porous plate on an enzyme-labeling instrument to read absorbance at 405 nm; (7) And (3) solving a regression equation according to the dynamic process (enzyme reaction speed) of the absorbance of the active suPAR standard substances with different concentrations along with the time and the concentration of the standard substances, and calculating the concentration of the active suPAR in the sample by using the regression equation.

4. Use according to claim 1, said detection method comprising the steps of:

5. Use according to claim 1, said detection method comprising the steps of:

(1) Treatment of plasma samples: placing peripheral blood collected from a subject to be detected into a potassium oxalate anticoagulation tube, centrifuging to separate plasma, diluting with a sample diluent, and sub-packaging for later use; (2) coating: 100. Mu.l of ATF-HSA protein solution (120. Mu.g/ml) diluted with the coating solution was added to all 1A-12H wells of the ELISA plate (i.e., multi-well plate, 96-well) overnight at 4 ℃; washing with a washing liquid and drying; (3) closing: adding 100 μl of sealing solution into all the holes 1A-12H in the ELISA plate, sealing, incubating, washing with washing solution, and drying; (4) sample addition: adding 100 mu l of active suPAR standard products with different concentrations into the ELISA plates 1A-1G respectively, adding 100 mu l of sample diluent into H1, H2 and H3 respectively to serve as blank control, adding diluted plasma samples of the tested subjects into other holes in the ELISA plates, sealing, incubating at room temperature, washing with a washing solution, and drying; (5) adding primary antibody: adding (a) 100. Mu.l of ATN-658 antibody diluted with sample diluent and (b) 25. Mu.l of 1.5mg/ml sodium glycerophosphate solution into all wells of ELISA plates 1A-12H, sealing, incubating at room temperature, washing with a washing solution, and drying; (6) adding an enzyme-labeled secondary antibody: adding 100 mu l alkaline phosphatase labeled secondary antibodies diluted 500 times by sample diluent into all holes of the ELISA plates 1A-12H, sealing, incubating at room temperature, washing by using a washing solution, and drying; (7) adding a color development liquid: adding 100 mu l of color development liquid into each hole rapidly, and then placing the ELISA plate on an ELISA reader to read absorbance at 405nm for 60min, wherein absorbance values are read once every 1 min; and (3) solving a regression equation according to the dynamic process (enzyme reaction speed) of the absorbance of the active suPAR standard substances with different concentrations along with the time and the concentration of the standard substances, and calculating the concentration of the active suPAR in the sample by using the regression equation.

6. Use according to claim 1, said detection method comprising the steps of:

(1) Treatment of plasma samples: placing peripheral blood collected from a subject to be detected in a potassium oxalate anticoagulation tube, centrifuging at 4 ℃ for 30min at 1500g, separating plasma, diluting 10 times with sample diluent, subpackaging in a 1.5ml centrifuge tube, and preserving at-80 ℃ for later use; (2) coating: 100. Mu.l of ATF-HSA protein solution (120. Mu.g/ml, sealed with a sealing tape to prevent evaporation of the liquid and overnight at 4 ℃) was added to all 1A-12H wells of the ELISA plate (i.e., multiwell plate, 96 well); washing and drying (removing the sealing adhesive tape, spin-drying the liquid in the holes of the enzyme label plate, sucking the washing liquid by a gun, washing each hole for 6 times, and after the last washing, lightly shooting the enzyme label plate on the water absorbing paper to dry and ensure that no bubbles remain in the holes); (3) Closing: adding 100 μl of sealing solution into all the holes 1A-12H in the ELISA plate, sealing the ELISA plate with sealing tape to prevent evaporation of the solution, and incubating on a shaking table at room temperature of 80 rpm for 1 hr; removing the sealing adhesive tape, spin-drying the liquid in the holes of the enzyme label plate, sucking the washing liquid by a gun to wash each hole for 6 times, and gently beating the enzyme label plate on the water absorbing paper after the last washing to ensure that no bubbles remain in the holes; (4) sample addition: 100 μl of active suPAR standard substances with different concentrations are respectively added into the wells A-G of the ELISA plate, and 100 μl of sample diluent is respectively added into the three wells H1, H2 and H3 of each concentration of the three repeated wells (A1、A2、A3：1μg/L,B1、B2、B3：0.5μg/L,C1、C2、C3：0.25μg/L,D1、D2、D3：0.125μg/L,E1、E2、E3：0.0625μg/L,F1、F2、F3：0.03125μg/L,G1、G2、GF3：0.015625μg/L); to serve as blank control; adding diluted plasma samples of the to-be-detected subjects into other holes in the ELISA plate, repeating the holes for 3 times for each plasma sample, sealing the ELISA plate by using a sealing tape, and incubating for 1 hour on a shaking table at room temperature of 80 revolutions per minute; removing the sealing adhesive tape, spin-drying the liquid in the holes of the enzyme label plate, sucking the washing liquid by a discharge gun, washing each hole for 6 times, and after the last washing, gently beating the enzyme label plate on the water absorbing paper to dry and ensure that no bubbles remain in the holes; (5) adding primary antibody: to all wells of the ELISA plate were added (a) 100. Mu.l of 5. Mu.g/ml ATN-658 antibody diluted with the sample diluent and (b) 25. Mu.l of 1.5mg/ml sodium glycerophosphate solution, the ELISA plate was sealed with sealing tape and incubated on a shaker at 80 rpm at room temperature for 1 hour; removing the sealing adhesive tape, spin-drying the liquid in the holes of the enzyme label plate, sucking the washing liquid by a discharge gun, washing each hole for 6 times, and after the last washing, gently beating the enzyme label plate on the water absorbing paper to dry and ensure that no bubbles remain in the holes; (6) adding an enzyme-labeled secondary antibody: adding 100 mu l alkaline phosphatase marked secondary antibodies diluted by 500 times of the sample diluent into all holes of the ELISA plate, sealing the ELISA plate by using a sealing tape, and incubating on a shaking table at room temperature of 80 rpm for 1 hour; removing the sealing adhesive tape, spin-drying the liquid in the holes of the enzyme-labeled plate, sucking the washing liquid by a gun, washing each hole for 6 times, and sucking deionized water by the gun for 3 times; gently beating the ELISA plate on the absorbent paper to dry and ensure no bubbles remain in the holes; (7) adding a color development liquid: adding 100 μl of color development liquid into each well rapidly with a row gun, placing the ELISA plate on an ELISA reader, reading absorbance at 405nm for 60min, and reading absorbance value every 1 min; and (3) preparing a standard curve and a regression equation thereof according to the absorbance value and the standard concentration, and calculating the concentration of active suPAR in the blood sample by using the regression equation.

7. Use according to claim 1, wherein:

The sample to be detected is a plasma sample;

the plasma sample is obtained from a subject to be tested, for example, a subject having a glomerular filtration rate, that is, a GFR value, in the range of 0 to 140 ml/(min.1.73 m 2), for example, in the range of 10 to 130 ml/(min.1.73 m 2), and is processed as follows: placing collected venous blood of a to-be-detected subject into a potassium oxalate anticoagulation tube, centrifuging at 0-10 ℃ (e.g. 4 ℃) (e.g. centrifuging at 1500g for 30 min), separating plasma, diluting the plasma with a sample diluent (e.g. to a proper concentration, e.g. to a concentration in a linear range of a standard curve), sub-packaging (e.g. in a 1.5ml centrifuge tube), and preserving at low temperature (e.g. at-80 ℃) for later use;

the active suPAR standard is obtained by purifying recombinant suPAR expressed in S2 drosophila embryo cells through an affinity column and an ion column;

The coating solution is an aqueous solution of pH9.6 comprising 40mM NaHCO3 and 10mM Na2CO 3;

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 a Blocker ™ BSA/PBS (10X) solution, i.e., 10% w/v bovine serum albumin/10 mM sodium phosphate/150 mM NaCl solution, pH7.4;

The washing solution/sample diluent is a PBS solution of pH7.4 containing 0.5 per mill Tween-20, wherein the PBS formulation is 8g/L, KCl g/L, na2 HPO4.12H2O 3.58g/L, KH2PO4 0.27g/L;

The color-developing solution comprises: 100mM Tris-HCl (pH 9.5), 100mM NaCl, 5mM MgCl2, 2mg/ml PNPP.

8. A method of detecting the amount of a soluble urokinase-type plasminogen activator receptor in a renal disease (e.g., chronic kidney disease) subject or a biological sample, such as blood, e.g., venous blood, suspected of having a renal disease (e.g., chronic kidney disease) subject, comprising the steps of:

Wherein:

The capture reagent is a fusion protein comprising an ATF;

9. The method according to claim 8, which is as claimed in any one of claims 1 to 7.

10. A kit for use in a method of detecting a soluble urokinase-type plasminogen activator receptor in a renal disease (e.g., chronic kidney disease) subject or a biological sample, such as blood, e.g., venous blood, of a subject suspected of having a renal disease (e.g., chronic kidney disease), comprising: ELISA plates (i.e., multi-well plates, e.g., 96 wells), ATF-HSA protein (dry powder or solution, e.g., 1ml, e.g., 1 to 1.5mg/ml solution), active supAR standard (dry powder or diluent, e.g., 1ml, e.g., 1mg/ml solution), ATN-658 antibody (e.g., 1mg/ml, e.g., 100. Mu.l), alkaline phosphatase-labeled secondary antibody (e.g., 25. Mu.l, e.g., 500 Xalkaline 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), optional blood sample processing anticoagulant tube (e.g., potassium oxalate anticoagulant tube), and optional kit instructions.