CN110716047B

CN110716047B - Urine detection kit matched with kidney disease early-screening small-sized full-automatic analyzer

Info

Publication number: CN110716047B
Application number: CN201911017662.9A
Authority: CN
Inventors: 张乐之; 吴敏华; 余铭恩; 王梦娜; 梁春蕾; 孙颖
Original assignee: Hangzhou Kangzhi Biomedical Co ltd
Current assignee: Hangzhou Kangzhi Biomedical Co ltd
Priority date: 2019-10-24
Filing date: 2019-10-24
Publication date: 2023-03-17
Anticipated expiration: 2039-10-24
Also published as: CN110716047A

Abstract

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.

Description

Urine detection kit matched with kidney disease early-screening small-sized full-automatic analyzer

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, the seemingly healthy patient is more than ten times as at risk of death from cardiovascular and cerebrovascular disease as normal. If uremia is developed, not only is the health of the patient harmful and even the life of the patient is threatened, but also the renal function damage is developed to an irreversible stage when two to three patients go to a hospital for the first time.

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 measure trace protein in urine. The measurement of urine trace protein can reflect the early nephropathy and kidney injury. The increase of trace protein in urine is often seen in diabetes, hypertension, pregnancy, eclampsia and other diseases and the early stage of nephropathy. 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, prediction indexes of acute pancreatitis complications, understanding and judgment of medicines influencing renal function by taking, and the like) of systemic or local inflammatory reactions, and is convenient for early observing the renal function condition and taking treatment measures as soon as possible.

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 of clinical response to 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 the 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 2 MG) 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/beta 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; beta 2MG in early stage of diabetes and hypertension is obviously related to 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 detection item.

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

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 creatinine is stable in normal people, 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 daily output is not influenced by the content of food protein and urine. 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 uses a large-scale automatic instrument and is generally applied to the mass detection of urine trace protein in the clinical laboratory of a large hospital; 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 detected on a liquid-phase chip detector in a combined manner, 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 primary hospitals and large hospital outpatient clinical laboratories, and brings great social benefit and economic benefit.

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

the urine detection kit matched with the nephropathy 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 trace protein R1 reagent comprises the following components: polyethylene glycol 6000, PBS buffer solution, surfactant active and antiseptic, regard PBS buffer solution quality as the benchmark, in urine trace protein R1 reagent, polyethylene glycol 6000 content is 1-5%, surfactant active content is 0.02-0.1%, antiseptic content 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 a urine trace protein antibody into a carboxyl latex microsphere activation solution, uniformly mixing, carrying out incubation reaction, adding a 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 a 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: PBS buffer solution, creatinase, sarcosine oxidase, catalase, ascorbate oxidase, surfactant and preservative, wherein the creatinine R1 reagent contains 0 to 0.05 percent of creatinase, 0 to 0.05 percent of sarcosine oxidase, 0 to 0.05 percent of catalase, 0 to 0.05 percent of ascorbate oxidase, 0.02 to 0.1 percent of surfactant and 0.02 to 0.2 percent of preservative 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 MES buffer solution has a pH of 5.0-6.0 and a concentration of 50-200 mmol/mL, and the PBS buffer solution has a pH of 6.5-7.5 and a concentration of 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 to 0.1%, and the concentration of the NHS solution is 0.05 to 0.2%; the micro-concentration of the carboxyl latex in the carboxyl latex microsphere solution is 0.05 to 0.5 percent; 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 μ L 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: PBS buffer solution with pH of 6.5-8.5 and concentration of 50-200 mmol/mL, preservative and stabilizer, wherein 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 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.

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 quantitatively measured in a matched manner;

(2) The quantitative determination kit for urine micro transferrin provided by the invention has the linear range of 6.26-400.8mg/L, the linear correlation coefficient is r =0.9986, and the sensitivity is 3.1mg/L; the precision is averagely 3.64 percent, 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 the determination linear range of 0.789-50.49mg/L, the linear correlation coefficient of r =0.9990, the sensitivity of 0.088mg/L, the average precision of 4.57 percent and the average accuracy recovery rate of 100.94 percent; highly correlated with siemens specific protein analyzer assay results (r = 0.9720); the urine beta 2 microglobulin quantitative determination kit has the determination linear range of 0.311-19.93mg/L, the linear correlation coefficient of r =0.9979, the sensitivity of 0.0479mg/L, the average precision of 3.31%, the average accuracy recovery rate of 101.96%, and is highly correlated with the determination result of a Siemens specific protein analyzer (r = 0.9641); the urine alpha 1 microglobulin quantitative determination kit determines a linear range 3.119-199.6mg/L, a linear correlation coefficient is r =0.9979, the sensitivity is 0.1433mg/L, the precision averages 3.455%, the accuracy average recovery rate is 101.86%, and the determination result is highly correlated with a Siemens specific protein analyzer (r = 0.9689); the urine immunoglobulin G quantitative determination kit has the determination linear range of 1.567-100.28mg/L, the linear correlation coefficient of r =0.9986, the sensitivity of 0.0352mg/L, the precision of 4.99% on average, the accuracy average recovery rate of 101.63% and high correlation with the determination result of a Siemens specific protein analyzer (r = 0.9724); the urine creatinine quantitative determination kit determines a linear range 31.361-2007.1umol/L, a linear correlation coefficient is r =0.9998, the sensitivity is 3.574umol/L, the precision is averagely 3.08%, the accuracy average recovery rate is 101.85%, and the kit is highly related to 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 the creatinine reagents of example 6.

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

FIG. 8 is a scatter plot of the correlation analysis of transferrin microalbumin reagent in 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.

Example 1

Preparation of micro transferrin determination kit

Preparing a trace transferrin R1 reagent: adding 1.5% of polyethylene glycol 6000,0.05% of surfactant (Tween 20) and 0.05% of preservative (Proclin 300) into 50mmol/mL PBS buffer solution with the pH value of 7.4 and the concentration of 50mmol/mL, taking the mass of the PBS buffer solution as a reference, uniformly mixing, and filtering by using a 0.4um membrane to obtain an R1 reagent;

preparing a trace transferrin R2 reagent:

(1) Adding the carboxyl latex microspheres into MES buffer solution with pH of 5.0 and concentration of 50mmol/mL to make the concentration of the carboxyl latex microspheres be 1.0% to obtain carboxyl latex microsphere solution;

(2) Adding EDC solution and NHS solution into the carboxyl latex microsphere solution, wherein the concentration of EDC is 0.05% (mass) and the concentration of NHS is 0.1% (mass), stirring for 15 minutes at room temperature, centrifuging to obtain a centrifugal solid, and resuspending the centrifugal solid by PBS buffer solution with the pH of 7.5 and the concentration of 100mmol/mL to obtain the carboxyl latex microsphere activation solution with the concentration of latex microspheres of 0.5%;

(3) Adding 2.0mg of transferrin antibody into 10mg of carboxyl latex microsphere activation solution, fully and uniformly mixing the latex microspheres and the transferrin antibody, incubating for reaction for 3 hours at room temperature, adding 5% (by mass) bovine serum albumin solution into a reaction product according to the standard that 200 mul of bovine serum albumin solution is added into 1mL of the reaction product, incubating for reaction for 1 hour at room temperature, centrifuging to obtain a centrifugal solid, resuspending the centrifugal solid by using PBS (phosphate buffer solution) with the pH of 7.5 and the concentration of 100mmol/mL, centrifuging again to obtain the centrifugal solid, and adding latex microsphere preservation solution to obtain an R2 reagent, wherein the concentration of carboxyl latex microspheres in the R2 reagent is 0.05%; the latex microsphere preservative fluid comprises the following components: the pH value is 7.5, the concentration is 100mmol/mL PBS buffer solution, preservative (Proclin 300) and stabilizer (sucrose), and the content of the preservative in the latex microsphere storage solution is 0.05 percent and the content of the stabilizer is 0.1 percent based on the mass of the PBS buffer solution.

Example 2

Preparation of transferrin determination kit

Preparing a transferrin R1 reagent: adding 1.5% of polyethylene glycol 6000,0.05% of surfactant (Tween 20) and 0.05% of preservative (Proclin 300) into PBS buffer solution with the pH of 7.4 and the concentration of 50mmol/mL by taking the mass of the PBS buffer solution as a reference, uniformly mixing, and filtering by using a 0.4um membrane to obtain a transferrin R1 reagent;

preparation of transferrin R2 reagent:

(3) Adding 2.0mg of transferrin antibody into 10mg of carboxyl latex microsphere activation solution, fully and uniformly mixing the latex microspheres and the transferrin antibody, incubating for reaction for 3 hours at room temperature, adding 5% (by mass) bovine serum albumin solution into a reaction product according to the standard that 200 mul of bovine serum albumin solution is added into 1mL of the reaction product, incubating for reaction for 1 hour at room temperature, centrifuging to obtain a centrifugal solid, resuspending the centrifugal solid by using PBS (phosphate buffer solution) with the pH of 7.5 and the concentration of 100mmol/mL, centrifuging again to obtain the centrifugal solid, and adding latex microsphere preservation solution to obtain transferrin R2, wherein the concentration of carboxyl latex microspheres in the transferrin R2 reagent is 0.05%; the latex microsphere preservative fluid comprises the following components: the pH value is 7.5, the concentration is 100mmol/mL PBS buffer solution, preservative (Proclin 300) and stabilizer (sucrose), and the content of the preservative in the latex microsphere storage solution is 0.05 percent and the content of the stabilizer is 0.1 percent based on the mass of the PBS buffer solution.

Example 3

Preparation of alpha 1-microglobulin determination kit

Preparing an alpha 1-microglobulin R1 reagent: adding 1.5% of polyethylene glycol 6000,0.05% of surfactant (Tween 20) and 0.05% of preservative (Proclin 300) into PBS buffer solution with the pH of 7.4 and the concentration of 50mmol/mL by taking the mass of the PBS buffer solution as a reference, uniformly mixing, and filtering by using a 0.4um membrane to obtain an alpha 1-microglobulin R1 reagent;

preparation of alpha 1-microglobulin R2 reagent:

(3) Adding 2.0mg of alpha 1-microglobulin antibody into 10mg of carboxyl latex microsphere activation solution, fully and uniformly mixing latex microspheres and the alpha 1-microglobulin antibody, incubating for reaction for 3 hours at room temperature, adding 5% (by mass) bovine serum albumin solution into a reaction product according to the standard that 1mL of the reaction product is added with 200 mu L of the bovine serum albumin solution, incubating for reaction for 1 hour at room temperature, centrifuging to obtain a centrifugal solid, re-suspending the centrifugal solid by using PBS buffer solution with the pH of 7.5 and the concentration of 100mmol/mL, centrifuging again to obtain the centrifugal solid, and adding latex microsphere preservative solution to obtain an alpha 1-microglobulin R2 reagent, wherein the concentration of carboxyl latex microspheres in the alpha 1-microglobulin R2 reagent is 0.05%; the latex microsphere preservative fluid comprises the following components: the pH value is 7.5, the concentration is 100mmol/mL PBS buffer solution, preservative (Proclin 300) and stabilizer (sucrose), and the content of the preservative in the latex microsphere storage solution is 0.05 percent and the content of the stabilizer is 0.1 percent based on the mass of the PBS buffer solution.

Example 4

Preparation of beta 2-microglobulin determination reagent kit

Preparing a beta 2-microglobulin R1 reagent: adding 1.5% of polyethylene glycol 6000,0.05% of surfactant (Tween 20) and 0.05% of preservative (Proclin 300) into PBS buffer solution with the pH of 7.4 and the concentration of 50mmol/mL by taking the mass of the PBS buffer solution as a reference, uniformly mixing, and filtering by using a 0.4um membrane to obtain a beta 2-microglobulin R1 reagent;

preparation of alpha 1-microglobulin R2 reagent:

(3) Adding 2.0mg of beta 2-microglobulin antibody into 10mg of carboxyl latex microsphere activation solution, fully and uniformly mixing latex microspheres and the beta 2-microglobulin antibody, incubating for reaction for 3 hours at room temperature, adding bovine serum albumin solution with the concentration of 5% (mass) into a reaction product according to the standard of adding 200 mu L of bovine serum albumin solution into 1mL of the reaction product, incubating for reaction for 1 hour at room temperature, centrifuging to obtain a centrifugal solid, resuspending the centrifugal solid by using PBS buffer solution with the pH of 7.5 and the concentration of 100mmol/mL, centrifuging again to obtain the centrifugal solid, adding latex microsphere preservative solution to obtain a beta 2-microglobulin R2 reagent, wherein the concentration of carboxyl latex microspheres in the beta 2-microglobulin R2 reagent is 0.05%; the latex microsphere preservative fluid comprises the following components: the pH value is 7.5, the concentration is 100mmol/mL PBS buffer solution, preservative (Proclin 300) and stabilizer (sucrose), based on the mass of the PBS buffer solution, the content of the preservative in the latex microsphere storage solution is 0.05 percent, and the content of the stabilizer is 0.1 percent.

Example 5

Preparation of immunoglobulin G determination kit

Preparation of immunoglobulin GR1 reagent: adding 1.5% of polyethylene glycol 6000,0.05% of surfactant (Tween 20) and 0.05% of preservative (Proclin 300) into PBS buffer solution with the pH of 7.4 and the concentration of 50mmol/mL by taking the mass of the PBS buffer solution as the standard, uniformly mixing, and filtering by using a 0.4um membrane to obtain an immunoglobulin GR1 reagent;

preparation of immunoglobulin GR2 reagent:

(2) Adding an EDC solution and an NHS solution into the carboxyl latex microsphere solution, wherein the concentration of EDC is 0.05% (mass) and the concentration of NHS is 0.1% (mass), stirring for 15 minutes at room temperature, centrifuging to obtain a centrifugal solid, and re-suspending the centrifugal solid by using a PBS buffer solution with the pH of 7.5 and the concentration of 100mmol/mL to obtain a carboxyl latex microsphere activation solution with the concentration of latex microspheres of 0.5%;

(3) Adding 2.0mg of immunoglobulin G antibody into 10mg of carboxyl latex microsphere activation solution, fully and uniformly mixing the latex microspheres and the immunoglobulin G antibody, carrying out room-temperature incubation reaction for 3h, adding 5% (by mass) bovine serum albumin solution into a reaction product according to the standard that 200 mul of bovine serum albumin solution is added into 1mL of the reaction product, carrying out room-temperature incubation reaction for 1h, centrifuging to obtain a centrifugal solid, carrying out resuspension by using PBS (phosphate buffer solution) with the pH of 7.5 and the concentration of 100mmol/mL, centrifuging again to obtain the centrifugal solid, and adding latex microsphere preservation solution to obtain an immunoglobulin GR2 reagent, wherein the concentration of carboxyl latex microspheres in the immunoglobulin GR2 reagent is 0.05%; the latex microsphere preservative fluid comprises the following components: the pH value is 7.5, the concentration is 100mmol/mL PBS buffer solution, preservative (Proclin 300) and stabilizer (sucrose), and the content of the preservative in the latex microsphere storage solution is 0.05 percent and the content of the stabilizer is 0.1 percent based on the mass of the PBS buffer solution.

Example 6

Preparation of creatinine kit

Preparing a creatinine R1 reagent: adding 0.02% of creatinase, 0.01% of sarcosine oxidase, 0.05% of catalase, 0.01% of ascorbate oxidase, 0.02% of surfactant (Triton-100) and 0.02% of preservative (KV 600) into PBS (phosphate buffered saline) with the pH of 8.0 and the concentration of 50mmol/mL, taking the mass of the PBS as a reference, uniformly mixing, and filtering by using a 0.4um membrane to obtain an R1 reagent;

preparing a creatinine R2 reagent: adding 0.05% of creatinin, 0.02% of peroxidase, 0.05% of potassium ferrocyanide, 0.04% of 4-aminoantipyrine, 0.02% of surfactant (Triton-100) and 0.02% of preservative (KV 600) into PBS (phosphate buffer solution) with the pH value of 8.2 and the concentration of 50mmol/mL, taking the mass of the PBS as the reference, uniformly mixing, and filtering by using a 0.4um membrane to obtain the R2 reagent.

Preparing a plurality of calibrator diluent, adding the calibrator antigen protein of commercial microalbumin, transferrin, alpha 1-microglobulin, beta 2-microglobulin and immunoglobulin G and creatinine to prepare a mixed solution with the microalbumin concentration of 400mg/L, the transferrin concentration of 50mg/L, the alpha 1-microglobulin concentration of 200mg/L, the beta 2-microglobulin concentration of 20mg/L, the immunoglobulin G concentration of 100mg/L and the creatinine concentration of 2000umol/L, measuring the mixed solution as a sample by using a corresponding contrast reagent, repeatedly measuring for 10 times, and taking the average value as the value assignment of the calibrator.

(II) preparation of Standard Curve

The calibrator in the step (one) is diluted into 7 concentrations in a gradient way, the diluent is used as a zero-concentration calibrator, the reagents in the embodiments 1 to 6 are sequentially used, corresponding parameters are set in a small-sized full-automatic analyzer for calibration and measurement, and a theoretical concentration and a tested signal value are fitted by four parameters or linearity, so that a corresponding calibration curve is automatically obtained in the analyzer.

Example 1 theoretical concentration versus signal values for the microalbumin reagent are shown in table 1 and a linear fit is shown in figure 1.

Example 2 theoretical concentrations of transferrin reagent are shown in table 2 together with signal values, and a linear fit is shown in figure 2.

Example 2	Theoretical concentration (mg/L)	Signal value
			1	50.49	5673
2	25.25	4028.5
			3	12.62	2512
4	6.311	1545
			5	3.156	879.5
6	1.578	423.5
			7	0.789	204
8	0	19.5

Example 3 theoretical concentrations of alpha 1-microglobulin agent versus signal values are shown in table 3, and a linear fit is shown in figure 3.

Example 3	Theoretical concentration (mg/L)	Signal value
			1	199.6	7673
2	99.80	4428.5
			3	49.90	2412
4	24.950	1305
			5	12.475	679.5
6	6.238	363.5
			7	3.119	184
8	0	12

Example 4 theoretical concentrations of β 2-microglobulin agent are shown in table 4 together with signal values, and a linear fit is shown in fig. 4.

Example 4	Theoretical concentration (mg/L)	Signal value
			1	19.93	8227
2	9.97	5528.5
			3	4.98	3112
4	2.491	1545
			5	1.246	679.5
6	0.623	313.5
			7	0.311	154
8	0	22.5

Example 5 theoretical concentration versus signal values for the immunoglobulin G reagent are shown in table 5, and a linear fit is shown in fig. 5.

Example 6 theoretical concentration versus signal values for creatinine reagents are shown in table 6, and a linear fit is shown in figure 6.

Example 6	Theoretical concentration (umol/L)	Signal value
			1	2007.1	0.7984
2	1003.55	0.3998
			3	501.78	0.1986
4	250.888	0.1109
			5	125.444	0.0511
6	62.722	0.0272
			7	31.361	0.0147
8	0	0.0016

(III) evaluation of Performance

The reagents and corresponding standard curves of examples 1 to 6 were taken, and performance indexes such as sensitivity (margin), accuracy (recovery), precision, linearity, and the like were evaluated.

(1) Sensitivity (blank limit)

The evaluation method comprises the following steps: the test was repeated 20 times with blank samples, the signal values of the 20 tests were recorded, the mean (M) and Standard Deviation (SD) were calculated, the value of M +2SD was calculated and substituted into the fitted standard curve, i.e. its sensitivity (blank limit).

(2) Accuracy (recovery)

The evaluation method comprises the following steps: one clinical sample was divided into three 100ul volumes, with the assigned calibrator as the added sample, 10ul of PBS was added to the first sample, 5ul of PBS and 5ul of calibrator were added to the second sample, and 10ul of calibrator was added to the third sample. After mixing, the three recovered samples were tested, each test was repeated three times, the mean value of the test results was calculated, and the recovery rate was calculated by comparing with the added theoretical concentration.

(3) Precision of

The evaluation method comprises the following steps: the test is repeated 10 times by using samples with different concentrations respectively, and the variation times (CV) of the test results of 10 times are calculated.

(4) Linearity

The evaluation method comprises the following steps: and (5) testing the dilution of the sample by using the assigned calibrator, performing linear fitting on the test result and the dilution multiple, and calculating the correlation coefficient r of the sample.

(IV) evaluation of the correlation with control reagents

50 clinical samples were collected, and the control reagent and the reagents prepared in examples 1 to 6 of the present invention were measured on respective instruments, and correlation analysis was performed on the results of the two measurements. The results of the control reagent test were plotted on the abscissa and the measured values of the reagent of the present invention were plotted on the ordinate to prepare scattergrams, and the correlation analysis scattergrams of examples 1 to 6 are shown in FIGS. 7 to 12, respectively.

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

The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.

Claims

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 R1 reagent comprises the following components: polyethylene glycol 6000, a PBS buffer solution, a surfactant and a preservative, wherein the pH value of the PBS buffer solution is 6.5-8.5, the concentration of the PBS buffer solution is 50mmol/mL, and the content of the polyethylene glycol 6000, the content of the surfactant and the content of the preservative in the urine micro-protein R1 reagent are respectively 1~5%, 0.02-0.1% and 0.02-0.2%, respectively;

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

(1) Adding 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, wherein the pH of the MES buffer solution is 5.0-6.0, the concentration of the MES buffer solution is 50-200mmol/mL, the pH of the PBS buffer solution is 6.5-7.5, and the concentration of the PBS buffer solution is 100-200mmol/mL; the concentration of the carboxyl latex micro in the carboxyl latex microsphere solution is 0.02 to 0.1 percent;

(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; the concentration of the EDC solution is 0.02 to 0.1 percent, and the concentration of the NHS solution is 0.05 to 0.2 percent; the micro concentration of the carboxyl latex in the carboxyl latex microsphere solution is 0.05 to 0.5 percent; the concentration of the PBS buffer solution is 20 to 200mmol/mL, and the pH value is 6.5 to 8.5;

(3) Adding 0.5-2.0 mg of urine trace protein antibody into 10mg of carboxyl latex microsphere activation solution, adding the urine trace protein antibody into the carboxyl latex microsphere activation solution, uniformly mixing, carrying out incubation reaction, adding a 5% bovine serum albumin solution into a reaction product according to the standard that 1mL of the reaction product is added with 200 μ L of the bovine serum albumin solution after the reaction, carrying out incubation reaction, centrifuging to obtain a centrifugal solid, carrying out resuspension by using PBS buffer solution, wherein the concentration of the PBS buffer solution is 20-200 mmol/mL, the pH is 6.5-8.5, centrifuging again to obtain the centrifugal solid, and adding latex microsphere preservation solution to obtain a urine trace protein R2 reagent; the latex microsphere preservative fluid comprises the following components: PBS buffer solution with pH of 6.5 to 8.5 and concentration of 50 to 200mmol/mL, preservative and stabilizer, wherein the content of the preservative in the latex microsphere storage solution is 0.05 to 0.1 percent and the content of the stabilizer is 0.1 to 0.5 percent based on the mass of the PBS buffer solution;

the creatinine R1 reagent comprises the following components: the reagent comprises a PBS buffer solution, 0 to 0.05 percent of creatinase, 0 to 0.05 percent of sarcosine oxidase, 0 to 0.05 percent of catalase, 0.02 to 0.1 percent of ascorbate oxidase, 0.02 to 0.2 percent of surfactant and 0.02 to 0.2 percent of preservative in a creatinine R1 reagent by taking the mass of the PBS buffer solution as a reference;

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 to 0.05%, the content of peroxidase is 0 to 0.05%, the content of potassium ferrocyanide is 0 to 0.05%, the content of 4-aminoantipyrine is 0 to 0.05%, the content of surfactant is 0.02 to 0.1%, and the content of preservative is 0.02 to 0.2%.

2. The urine test kit matched with the small-sized automatic early-screening kidney disease analyzer according to claim 1, wherein in the urine trace protein R1 reagent, 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 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.

4. 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), after adding urine trace protein antibody into a carboxyl latex microsphere solution, incubation reaction is carried out at room temperature for 2 to 4h.

5. The urine detection kit matched with the small-sized automatic early-screening kidney disease analyzer according to claim 1, wherein in the step (3), a bovine serum albumin solution is added into a reaction product, and then the incubation reaction is carried out at room temperature for 1 to 2h.

6. The urine test kit matched with the small-sized automatic kidney disease early-screening analyzer according to claim 1, 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.

7. 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 PBS buffer solution has the pH value of 8.0 to 8.2 and the concentration of 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.