阅读说明：本技术 一种肾病早筛小型全自动分析仪配套的尿液检测试剂盒 (Urine detection kit matched with kidney disease early-screening small-sized full-automatic analyzer ) 是由 张乐之 吴敏华 余铭恩 王梦娜 梁春蕾 孙颖 于 2019-10-24 设计创作，主要内容包括：本发明公开了一种能够在同一小型全自动分析仪平台上配套定量测定尿液微量白蛋白、转铁蛋白、β2微球蛋白、α1微球蛋白、免疫球蛋白G和肌酐的肾病早筛小型全自动分析仪配套的尿液微量蛋白检测试剂盒,包括尿液微量蛋白检测试剂盒和肌酐测定试剂盒；所述尿液微量蛋白检测试剂盒包括尿液微量蛋白R1试剂和尿液微量蛋白R2试剂。本发明具有检测质量高、检测快速高效(15分钟出结果)、检测全面系统、使用方便价廉的优点,为肾脏病早期筛查检测,提供了一个有用平台,且可广泛应用于基层医院及大医院门诊检验科,并带来较大的社会效益和经济效益。(The invention discloses a urine trace protein detection kit matched with a nephropathy early screening small-sized full-automatic analyzer, which can quantitatively detect urine trace albumin, transferrin, beta 2 microglobulin, alpha 1 microglobulin, immunoglobulin G and creatinine in a matched manner on the same small-sized full-automatic analyzer platform, and comprises a urine trace protein detection kit and a creatinine determination kit; the urine trace protein detection kit comprises a urine trace protein R1 reagent and a urine trace protein R2 reagent. The invention has the advantages of high detection quality, rapid and efficient detection (15-minute result), comprehensive detection system, convenient use and low cost, provides a useful platform for early screening and detection of the kidney diseases, can be widely applied to primary hospitals and outpatient clinical laboratories of large hospitals, and brings great social and economic benefits.)

1. A nephropathy early-screening small-sized full-automatic analyzer matched urine detection kit is characterized by comprising a urine trace protein detection kit and a creatinine determination kit;

the urine trace protein detection kit comprises a urine trace protein R1 reagent and a urine trace protein R2 reagent;

the urine micro-protein R1 reagent comprises the following components: polyethylene glycol 6000, PBS buffer solution, surfactant and preservative, wherein the mass of the PBS buffer solution is taken as a reference, the content of the polyethylene glycol 6000, the content of the surfactant and the content of the preservative in the urine trace protein R1 reagent are respectively 1-5%, 0.02-0.1% and 0.02-0.2%; the urine trace protein R2 reagent is prepared by the following method:

(1) adding the carboxyl latex microspheres into MES buffer solution or PBS buffer solution, adding EDC solution and NHS solution, and uniformly stirring to obtain carboxyl latex microsphere solution;

(2) adding an EDC solution and an NHS solution into the carboxyl latex microsphere solution, stirring uniformly, centrifuging to obtain a centrifugal solid, and resuspending the centrifugal solid by using a PBS buffer solution to obtain a carboxyl latex microsphere activation solution;

(3) adding a urine trace protein antibody into a carboxyl latex microsphere activation solution, uniformly mixing, carrying out incubation reaction, adding a bovine serum albumin solution into a reaction product after reaction, carrying out incubation reaction again, centrifuging to obtain a centrifugal solid, carrying out heavy suspension by using PBS (phosphate buffer solution), centrifuging again to obtain the centrifugal solid, and adding a latex microsphere preservative solution to obtain a urine trace protein R2 reagent;

the creatinine kit comprises a creatinine R1 reagent and a creatinine R2 reagent;

the creatinine R1 reagent comprises the following components: the reagent comprises PBS buffer solution, creatinase, sarcosine oxidase, catalase, ascorbate oxidase, surfactant and preservative, wherein in the creatinine R1 reagent, the content of creatinase, sarcosine oxidase, catalase, ascorbate oxidase is 0-0.05%, the content of surfactant is 0.02-0.1% and the content of preservative is 0.02-0.2% based on the mass of the PBS buffer solution;

the creatinine R2 reagent comprises the following components: PBS buffer solution, creatinin, peroxidase, potassium ferrocyanide, 4-aminoantipyrine, surfactant and preservative, wherein the weight of the PBS buffer solution is taken as a reference, in the creatinine R2 reagent, the content of creatinin is 0-0.05%, the content of peroxidase is 0-0.05%, the content of potassium ferrocyanide is 0-0.05%, the content of 4-aminoantipyrine is 0-0.05%, the content of surfactant is 0.02-0.1%, and the content of preservative is 0.02-0.2%.

2. The urine detection kit matched with the small-sized automatic early-screening kidney disease analyzer according to claim 1, wherein in a urine trace protein R1 reagent, the pH value of the PBS buffer solution is 6.5-8.5, the concentration is 50mmol/mL, the surfactant is at least one of Tween-20, Triton-100 and polyether, and the preservative is at least one of sodium azide, p-hydroxybenzoic acid and Proclin 300.

3. The urine detection kit matched with the small-sized automatic early-screening kidney disease analyzer according to claim 1, wherein in the step (1), the pH value of the MES buffer solution is 5.0-6.0, the concentration is 50-200 mmol/mL, the pH value of the PBS buffer solution is 6.5-7.5, and the concentration is 100-200 mmol/mL; the concentration of the carboxyl latex micro-particles in the carboxyl latex microsphere solution is 0.02-0.1%.

4. The urine detection kit matched with the small-sized automatic early-screening kidney disease analyzer according to claim 1, wherein in the step (2), the concentration of EDC solution is 0.02-0.1%, and the concentration of NHS solution is 0.05-0.2%; the micro concentration of the carboxyl latex in the carboxyl latex microsphere solution is 0.05-0.5%; the concentration of the PBS buffer solution is 20-200 mmol/mL, and the pH value is 6.5-8.5.

5. The urine detection kit matched with the kidney disease early-screening small-sized full-automatic analyzer according to claim 1, wherein in the step (3), the urine trace protein antibody is urine trace albumin, transferrin, beta 2-microglobulin, alpha 1-microglobulin and immunoglobulin G.

6. The urine detection kit matched with the small-sized automatic early-screening kidney disease analyzer according to claim 1 or 6, wherein in the step (3), according to the standard that 0.5-2.0 mg of urine trace protein antibody is added into 10mg of carboxyl latex microsphere activation solution, the urine trace protein antibody is added into the carboxyl latex microsphere solution, and the incubation reaction is carried out for 2-4 h at room temperature.

7. The urine detection kit matched with the kidney disease early-screening small-sized full-automatic analyzer according to claim 1, wherein in the step (3), the mass concentration of the bovine serum albumin solution is 5%, according to the standard that 200 μ L of the bovine serum albumin solution is added to 1mL of the reaction product, the bovine serum albumin solution is added to the reaction product, and then the incubation reaction is carried out for 1-2 h at room temperature; the concentration of the PBS buffer solution is 20-200 mmol/mL, and the pH value is 6.5-8.5.

8. The urine detection kit matched with the small-sized automatic early-screening kidney disease analyzer according to claim 1, wherein the latex microsphere preservation solution comprises the following components: the pH value is 6.5-8.5, the concentration is 50-200 mmol/mL PBS buffer solution, preservative and stabilizer, and the preservative content in the latex microsphere preservation solution is 0.05-0.1% and the stabilizer content is 0.1-0.5% by mass of the PBS buffer solution.

9. The urine test kit matched with the small-sized automatic kidney disease early-screening analyzer according to claim 8, wherein the preservative is at least one of sodium azide, p-hydroxybenzoic acid and Proclin300, and the stabilizer is at least one of casein, bovine serum albumin, gelatin, mannitol, sucrose and dextran.

10. The urine detection kit matched with the kidney disease early-screening small-sized full-automatic analyzer according to claim 1, wherein in a creatinine R1 reagent and a creatinine R2 reagent, the pH value of a PBS buffer solution is 8.0-8.2, and the concentration is 50 mmol/mL; the surfactant is at least one of tween-20, triton-100 and polyether, and the preservative is at least one of sodium azide, p-hydroxybenzoic acid and Proclin 300.

Technical Field

The invention relates to the field of in-vitro diagnosis technology immunity and biochemical detection, in particular to a urine detection kit matched with a nephropathy early-screening small-sized full-automatic analyzer.

Background

The kidney disease is a common frequently encountered disease. Over 5 million people worldwide suffer from different kidney diseases. The incidence rate of chronic kidney diseases of common people in China is 10% -13%, so that more than 1 hundred million people in China are presumed to suffer from the chronic kidney diseases.

In the early stage of chronic kidney disease, patients generally have no uncomfortable symptoms and are often neglected. However, seemingly healthy patients with this disease are more than ten times as at risk of dying from cardiovascular and cerebrovascular disease as normal. When uremia develops, the patient is not only harmful to health and even life, but also two to three patients have an irreversible renal impairment when the patient first goes to a hospital.

The international association of renal diseases and the international association of renal disease foundation have called for everyone to care for his "magic kidneys" to discover kidney damage as early as possible and to receive the necessary treatment in order to avoid causing serious symptoms.

In view of the current increasing incidence rate of chronic kidney diseases in the world and the general lack of public knowledge for preventing and treating the chronic kidney diseases, the international association of renal diseases and the international association of renal disease fund propose that the second thursday of 3 months per year is determined as the world kidney day from 2006, aiming at improving the understanding of people on the chronic kidney diseases and the related cardiovascular diseases and mortality and paying attention to the urgent global demand on early detection and prevention of the chronic kidney diseases.

The early screening of kidney diseases has few means and methods, the B-ultrasonic and image examination are not helpful, and the current common method is to determine trace protein in urine. The urine trace protein measurement can reflect the early nephropathy and kidney injury conditions. The increase of trace protein in urine is often seen in the early stage of nephropathy complicated with diseases such as diabetes, hypertension and gestational eclampsia. The early detection of elevated urine trace protein is an early sign and precursor of nephropathy, and kidney damage is in a reversible period, such as timely treatment, to stop or reverse the progression of nephropathy. The urine trace protein joint detection can be used as renal function indexes (such as early renal pathological changes caused by urinary tract infection and the like and prediction indexes of acute pancreatitis complications, understanding and judgment of medicines which influence renal functions after being taken and the like) of systemic or local inflammatory reactions, so that the renal function condition can be observed early and treatment measures can be taken early.

Urine trace protein assay items commonly used for kidney disease early screening are: albumin, transferrin, beta 2 microglobulin, alpha 1 microglobulin, and immunoglobulin G, as well as urine creatinine assay, a biochemical item for the calculated ratio.

Albumin has a molecular weight of about 6.7 ten thousand. Normal human urine contains very little Microalbumine (MA), which increases significantly when the volume barrier of the glomerular basement membrane is compromised. The MA assay is an early indicator that clinically reflects changes in the glomerular and cardiovascular systems.

Transferrin has a molecular weight of about 7.7 ten thousand. Urine Transferrin (TRF) detection is a reliable indicator of damage to the glomerular basement membrane charge barrier, TRF being more sensitive than MA.

The molecular weight of the beta 2 microglobulin is about 3.3 ten thousand. Urinary β 2 microglobulin (β 2MG) is primarily associated with renal tubular function. Detecting beta 2MG as a sensitive and specific index for assisting diagnosis of proximal tubular injury; the urinary protein/β 2MG ratio helps identify glomerular or renal tubule pathologies; the kit is used for identifying the upper and lower urinary tract infection, the beta 2MG is obviously increased when the upper urinary tract is infected, and the beta 2MG is basically normal when the lower urinary tract is infected; used for judging rejection reaction of kidney transplantation; the early beta 2MG of the diabetes and hypertension patients is obviously related to the renal function damage degree; when malignant tumor and autoimmune disease kidney damage, beta 2MG is obviously increased; epidemic investigation of heavy metal poisoning kidney damage, and the beta 2MG can be used as a screening and detecting item.

The molecular weight of the alpha 1 microglobulin is about 1.18 ten thousand. The urine alpha 1 microglobulin (alpha 1MG) measurement is a sensitive index reflecting the renal tubule damage and the reabsorption function thereof, and is superior to beta 2 MG.

Immunoglobulin g (igg) has a molecular weight of about 16 ten thousand. IgG usually does not readily pass through glomerular filtration membranes and is minimally secreted in urine. The detection of urine IgG is mainly used to identify selective and non-selective glomerular proteinuria, and to determine the severity and prognosis of glomerular damage.

Creatinine molecular weight was 113.1. The daily output of the creatinine in the normal human urine is quite stable, the daily output of adult men is 1.0-1.8 g, and the daily output of adult women is 0.7-1.0 g, and the urinary creatinine output is not influenced by the content of food protein and the urine volume. Therefore, the ratio calculation can be carried out by using the urine trace protein and creatinine measurement results, and the renal function condition can be reflected more accurately.

The prior urine trace protein quantitative determination method comprises an enzyme immunoassay method, an immunoturbidimetry method, a colloidal gold immunochromatography method and the like. The quantitative determination CV of the enzyme immunoassay is large and is not commonly used in hospital clinical laboratory; the immunoturbidimetry method is generally applied to the clinical laboratory of large hospitals for detecting urine trace protein in batches by using a large-scale automatic instrument; the colloidal gold immunochromatography is usually applied to primary hospitals, but the quantitative detection quality is poor; the Chinese invention patent CN105911291A discloses a urine trace protein detection kit and a detection method thereof, wherein seven trace proteins in urine are simultaneously and jointly detected on a liquid-phase chip detector, so that the detection sensitivity and the detection speed are improved, and the kit is suitable for high-throughput detection of urine trace proteins in large hospitals, but is not suitable for outpatients of primary hospitals and large hospitals.

Disclosure of Invention

The invention aims to provide a urine trace protein detection kit matched with a nephropathy early-screening small-sized full-automatic analyzer, which can be used for quantitatively determining urine trace albumin, transferrin, beta 2 microglobulin, alpha 1 microglobulin, immunoglobulin G and creatinine on the same small-sized full-automatic analyzer platform, can be widely applied to outpatient inspection departments of primary hospitals and large hospitals, and brings great social benefits and economic benefits.

In order to achieve the purpose, the invention adopts the following technical scheme:

the urine detection kit matched with the kidney disease early-screening small-sized full-automatic analyzer comprises a urine trace protein detection kit and a creatinine determination kit;

the urine trace protein detection kit comprises a urine trace protein R1 reagent and a urine trace protein R2 reagent;

the urine micro-protein R1 reagent comprises the following components: polyethylene glycol 6000, PBS buffer solution, surfactant and antiseptic, by taking the quality of PBS buffer solution as a benchmark, in the urine trace protein R1 reagent, the content of polyethylene glycol 6000 is 1-5%, the content of surfactant is 0.02-0.1%, and the content of antiseptic is 0.02-0.2%;

the urine trace protein R2 reagent is prepared by the following method:

(1) adding the carboxyl latex microspheres into MES buffer solution or PBS buffer solution, adding EDC solution and NHS solution, and uniformly stirring to obtain carboxyl latex microsphere solution;

(2) adding an EDC solution and an NHS solution into the carboxyl latex microsphere solution, stirring uniformly, centrifuging to obtain a centrifugal solid, and resuspending the centrifugal solid by using a PBS buffer solution to obtain a carboxyl latex microsphere activation solution;

(3) adding urine trace protein antibody into the carboxyl latex microsphere activation solution, uniformly mixing, carrying out incubation reaction, adding bovine serum transferrin solution into a reaction product after reaction, carrying out incubation reaction again, centrifuging to obtain a centrifugal solid, carrying out heavy suspension by using PBS buffer solution, centrifuging again to obtain the centrifugal solid, and adding latex microsphere preservative solution to obtain a urine trace protein R2 reagent;

the creatinine kit comprises a creatinine R1 reagent and a creatinine R2 reagent;

the creatinine R1 reagent comprises the following components: the reagent comprises PBS buffer solution, creatinase, sarcosine oxidase, catalase, ascorbate oxidase, surfactant and preservative, wherein in the creatinine R1 reagent, the content of creatinase, sarcosine oxidase, catalase, ascorbate oxidase is 0-0.05%, the content of surfactant is 0.02-0.1% and the content of preservative is 0.02-0.2% based on the mass of the PBS buffer solution;

the creatinine R2 reagent comprises the following components: PBS buffer solution, creatinin, peroxidase, potassium ferrocyanide, 4-aminoantipyrine, surfactant and preservative, wherein the weight of the PBS buffer solution is taken as a reference, in the creatinine R2 reagent, the content of creatinin is 0-0.05%, the content of peroxidase is 0-0.05%, the content of potassium ferrocyanide is 0-0.05%, the content of 4-aminoantipyrine is 0-0.05%, the content of surfactant is 0.02-0.1%, and the content of preservative is 0.02-0.2%.

Preferably, in the urine trace protein R1 reagent, the pH value of the PBS buffer solution is 6.5-8.5, the concentration is 50mmol/mL, the surfactant is at least one of Tween-20, Triton-100 and polyether, and the preservative is at least one of sodium azide, p-hydroxybenzoic acid and Proclin 300.

Preferably, in the step (1), the pH value of the MES buffer solution is 5.0-6.0, the concentration is 50-200 mmol/mL, and the pH value of the PBS buffer solution is 6.5-7.5, the concentration is 100-200 mmol/mL; the concentration of the carboxyl latex micro-particles in the carboxyl latex microsphere solution is 0.02-0.1%.

Preferably, in the step (2), the concentration of the EDC solution is 0.02-0.1%, and the concentration of the NHS solution is 0.05-0.2%; the micro concentration of the carboxyl latex in the carboxyl latex microsphere solution is 0.05-0.5%; the concentration of the PBS buffer solution is 20-200 mmol/mL, and the pH value is 6.5-8.5.

Preferably, in the step (3), the urine trace protein antibody is urine trace transferrin, beta 2-microglobulin, alpha 1-microglobulin and immunoglobulin G.

Preferably, in the step (3), according to the standard that 0.5-2.0 mg of urine trace protein antibody is added into 10mg of carboxyl latex microsphere activation solution, the urine trace protein antibody is added into the carboxyl latex microsphere solution, and the incubation reaction is carried out for 2-4 h at room temperature.

Preferably, in the step (3), the mass concentration of the bovine serum transferrin solution is 5%, according to the standard that 200 mul of bovine serum transferrin solution is added into 1mL of reaction product, the bovine serum transferrin solution is added into the reaction product, and then the incubation reaction is carried out for 1-2 h at room temperature; the concentration of the PBS buffer solution is 20-200 mmol/mL, and the pH value is 6.5-8.5.

Preferably, the latex microsphere preservation solution comprises the following components: the pH value is 6.5-8.5, the concentration is 50-200 mmol/mL PBS buffer solution, preservative and stabilizer, and the preservative content in the latex microsphere preservation solution is 0.05-0.1% and the stabilizer content is 0.1-0.5% by mass of the PBS buffer solution.

Preferably, the preservative is at least one of sodium azide, p-hydroxybenzoic acid and Proclin300, and the stabilizer is at least one of casein, bovine serum transferrin, gelatin, mannitol, sucrose and dextran.

Preferably, in the creatinine R1 reagent and the creatinine R2 reagent, the pH value of a PBS buffer solution is 8.0-8.2, and the concentration is 50 mmol/mL; the surfactant is at least one of tween-20, triton-100 and polyether, and the preservative is at least one of sodium azide, p-hydroxybenzoic acid and Proclin 300.

Therefore, the invention has the following beneficial effects:

(1) the kit can be based on the same small-sized full-automatic analyzer platform, and urine micro transferrin, beta 2 microglobulin, alpha 1 microglobulin, immunoglobulin G and creatinine are matched and quantitatively measured, and the kit is used, so that the method of latex enhancement and scattering turbidimetry can be adopted in the detection of the urine micro transferrin and the creatinine, thereby greatly improving the detection sensitivity and being suitable for the requirements of outpatients of primary hospitals and major hospitals;

(2) the urine micro transferrin quantitative determination kit provided by the invention has a determination linear range of 6.26-400.8mg/L, a linear correlation coefficient of r-0.9986 and a sensitivity of 3.1 mg/L; the precision is 3.64 percent averagely, and the accuracy average recovery rate is 100.72 percent; highly correlated with siemens specific protein analyzer assay results (r-0.9820); the urine transferrin quantitative determination kit has a determination linear range of 0.789-50.49mg/L, a linear correlation coefficient of r being 0.9990, a sensitivity of 0.088mg/L, an average precision of 4.57%, and an average accuracy recovery rate of 100.94%; highly correlated with siemens specific protein analyzer assay results (r-0.9720); the urine beta 2 microglobulin quantitative determination kit has a determination linear range of 0.311-19.93mg/L, a linear correlation coefficient r of 0.9979, a sensitivity of 0.0479mg/L, an average precision of 3.31%, an average accuracy recovery rate of 101.96%, and is highly correlated with the determination result of a Siemens specific protein analyzer (r of 0.9641); the urine alpha 1 microglobulin quantitative determination kit has a determination linear range of 3.119-199.6mg/L, a linear correlation coefficient of r-0.9979, a sensitivity of 0.1433mg/L, an average precision of 3.455%, an average accuracy recovery rate of 101.86%, and is highly correlated with a determination result of a Siemens specific protein analyzer (r-0.9689); the urine immunoglobulin G quantitative determination kit has a determination linear range of 1.567-100.28mg/L, a linear correlation coefficient of r-0.9986, a sensitivity of 0.0352mg/L, an average precision of 4.99%, an average accuracy recovery rate of 101.63%, and is highly related to the determination result of a Siemens specific protein analyzer (r-0.9724); the urine creatinine quantitative determination kit has a determination linear range of 31.361-2007.1umol/L, a linear correlation coefficient of r-0.9998, a sensitivity of 3.574umol/L, an average precision of 3.08%, an average accuracy recovery rate of 101.85%, and is highly correlated with the determination result of a Hitachi full-automatic biochemical analyzer (r-0.9818).

(3) According to the design requirements of high quality, rapidness, high efficiency (15 min results), comprehensive system, small-size, full automation, convenience and low price, a useful platform is provided for the early screening and detection of the kidney diseases, and the method can be widely applied to primary hospitals and outpatient clinical laboratories of large hospitals and bring greater social benefits and economic benefits.

Drawings

Figure 1 is a line fit plot of the microalbumin reagent of example 1.

Figure 2 is a linear fit plot of transferrin microalbumin reagent of example 2.

FIG. 3 is a line fit plot of the α 1-microglobulin reagent of example 3.

FIG. 4 is a line fit plot of the β 2-microglobulin reagent of example 4.

FIG. 5 is a line fit plot of the immunoglobulin G reagent of example 5.

Figure 6 is a linear fit plot of creatinine reagents from example 6.

FIG. 7 is a scattergram of correlation analysis of the microalbumin reagent in example 1.

FIG. 8 is a scattergram of correlation analysis of the transferrin microalbumin reagent of example 2.

FIG. 9 is a scatter plot of the correlation analysis of the α 1-microglobulin reagent of example 3.

FIG. 10 is a scatter plot of the correlation analysis of the β 2-microglobulin reagent of example 4.

FIG. 11 is a scattergram of correlation analysis of the immunoglobulin G reagent in example 5.

FIG. 12 is a scattergram of correlation analysis of creatinine reagents in example 6.

Detailed Description

The invention is further described with reference to the following figures and detailed description.