CN105388146B - Kit that is a kind of while detecting sodium in urine, creatinine and microalbumin - Google Patents

Abstract

The invention provides a kit capable of simultaneously detecting sodium, creatinine and microalbumin in urine. The kit of the invention comprises: a reaction base plate with a sodium reaction hole, a creatinine reaction hole and a microalbumin reaction hole; a sodium diluent, a creatinine diluent and a standard color comparison plate of the three substances. The present invention realizes semi-quantitative detection of sodium, creatinine and microalbumin in urine by adding reaction pads containing differently configured substrates and enzyme solutions to the above three reaction wells. The preparation method of the kit of the present invention is simple, the performance is stable, and it is easy to store. The color scale corresponding to different urine sodium, creatinine, and trace albumin contents is obvious, which is convenient for judging whether the salt content of the tested person exceeds the standard and whether the kidney is damaged. It is also suitable for hospitals. Laboratory and non-professional inspection personnel use.

Description

A kit for simultaneously detecting sodium, creatinine and microalbumin in urine

technical field

The invention relates to a method for measuring sodium, creatinine and microalbumin in urine by an enzymatic method, and a dry chemical kit for simultaneously detecting urine sodium, creatinine and microalbumin prepared by using the method, which belongs to the medical examination and measurement technology field.

Background technique

Excessive intake of sodium can cause left ventricular hypertrophy, proteinuria, arteriosclerosis, etc., and plays an important role in the progression of end-stage renal failure. While reducing sodium intake, it can not only lower blood pressure, but also reduce the excretion of urinary protein. Sodium plays a central role in maintaining normal water distribution and osmotic pressure. The kidney is the main organ that regulates sodium and water and maintains acid-base balance. Sodium is freely filtered out by the kidneys, and most of it is reabsorbed. Urinary sodium excretion is also closely related to dietary sodium salt and body water status. Abnormal results: decreased: seen in adrenal hyperfunction, Cushing's syndrome, primary aldosteronism, congestive heart failure, etc. In addition, vomiting, diarrhea, gastrointestinal surgical fistula, extensive burns, etc. can also reduce urinary sodium excretion. Elevated: seen in severe pyelonephritis, renal tubular injury, diabetes, acute renal tubular necrosis (oliguria), diabetes insipidus, adrenal insufficiency, etc.

At present, the determination of electrolyte sodium includes chemical method, flame photometry and ion selective electrode method, with the development of modern analysis technology. Chemical methods have gradually been replaced by sensitive and rapid instrumental methods. At present, most hospitals use flame photometry and ion selective electrode method, and the latter is more and more favored by laboratory personnel because of its fast response (suitable for emergency), sensitive and accurate results. In recent years, an electrolyte enzyme method has emerged, which can be tested with a fully automatic analyzer together with other routine testing items, which is accurate and convenient, and also reduces the cost of testing. Enzymatic sodium determination kits have been marketed at home and abroad, mainly liquid double reagents, but only a few manufacturers can produce them, and the liquid reagents are very unstable.

Creatinine (creatinine, Cre) is the product of muscle metabolism in the human body, and every 20g of muscle metabolism can produce 1mg of creatinine. Creatinine is excreted mainly by glomerular filtration. Blood creatinine comes from both exogenous and endogenous sources. Exogenous creatinine is the product of meat food metabolism in the body; endogenous creatinine is the product of muscle tissue metabolism in the body. When meat food intake is stable. Without major changes in the body's muscle metabolism, the production of creatinine will be relatively constant. Urinary creatinine mainly comes from the blood and is excreted with urine after being filtered by the glomerulus. The urine creatinine test can measure the amount of creatinine excreted by the blood through glomerular filtration. Normal range of urine creatinine value: 8.4-13.25mmol/24h urine or 40-130mg/dl urine. The daily excretion of urinary creatinine in normal people is quite stable, basically not affected by food protein content and urine output. When the creatinine content in the urine is significantly increased or decreased, it reflects that the renal function has been damaged to a certain extent. Increased urinary creatinine excretion: seen in hypothyroidism, certain wasting diseases, liver diseases, diabetes, acromegaly, gigantism, fever, and hunger. Decreased urinary creatinine excretion: seen in renal insufficiency, hyperthyroidism, anemia, paralysis, typhoid, tetanus, tuberculosis and other wasting diseases, muscle atrophy and muscular dystrophy. Therefore, checking the changes of urine creatinine content can play an important reference role in the changes and treatment of renal function.

There are two main types of routine biochemical detection methods for creatinine. One is the chemical method—the basic picric acid endpoint method and the basic picric acid two-point kinetic method. Because the endpoint method is affected by serum endogenous false positive and false negative interference There are too many types, some of these interferers react faster with picric acid than creatinine, called "fast interferers", such as acetic acid, acetoacetic acid; some react slower than creatinine, called "slow interferers", such as glucose, Certain protein residues, etc. In addition to false positive interferences, there are also false negative interferences, the most common being bilirubin. Bilirubin will lower the creatinine measurement results, and the interference will be obvious when the bilirubin concentration rises to 35mg/dl (600μmol/L). Nearly half of the clinical chemistry laboratories in the national tertiary first-class hospitals have applied the creatinine enzyme method to the routine detection of blood/urine creatinine. The application of enzymatic methods in the development of clinical chemistry methodology has made a qualitative leap in clinical chemistry assay technology. Its core is the high specificity of enzyme action, which greatly improves the accuracy, sensitivity, repeatability, and linear measurement range of the determination. The popularization of enzymatic methods makes chemical methods using strong acids and strong bases gradually eliminated. The enzymatic reaction conditions are mild. With the development of commercial kits, it is matched with semi-automatic or fully automatic biochemical analyzers to make the detection fast, simple and efficient. The principle of creatinine enzymatic determination developed in recent years is available in commercial kits, including single reagents and double reagents in different dosage forms and specifications. Enzymatic methods improve the specificity of detection, but not absolute specificity. Therefore, although they belong to the same enzyme method, there are different types and sizes of interference in the selection of tool enzymes due to different specific assay principles and differences in the source, purity, specific activity, and especially the specificity of tool enzyme action. The enzymatic analysis of creatinine has developed rapidly in recent years. Enzymatic creatinine determination kits have been marketed at home and abroad, mainly liquid double reagents, but only a few manufacturers can produce them, and the liquid reagents are very unstable.

Urinary microalbumin is an index reflecting glomerular filtration function, which indicates early kidney damage, and also reflects vascular endothelial dysfunction in the body. Microalbumin in urine can reflect the functional status of blood vessels. If it can be treated early Kidney function may still recover. However, without early treatment, these patients develop from microalbuminuria to macroalbuminuria, which indicates that the renal function of the patients deteriorates and is irreversible. Therefore, predicting early kidney damage and vascular function by detecting microalbuminuria, and performing early intervention therapy have important clinical significance for improving kidney damage caused by hypertension or diabetes.

Albumin detection methods include colloidal gold method, immunoturbidimetric method, dye affinity chemical method, etc. At present, related products are on the market at home and abroad. Colloidal gold method requires professional operation, and the production process is complicated. The cost is high and supporting equipment is required. The simplest method is the dye affinity method. Albumin in urine reacts with sulfonphthalein dyes to form a blue product, and the color depth is proportional to the concentration of albumin in urine. The dye is immobilized on the chromatographic membrane and read by instrument or naked eyes.

In conclusion, predicting water and sodium retention, glomerular excretion, and sodium intake by detecting urinary sodium excretion has a good predictive effect on nephrogenic hypertension, especially volume-dependent hypertension; Urinary microalbuminuria is used to predict whether the kidneys are damaged, and whether renal vascular lesions change the function of the kidneys to filter proteins (especially albumin); microalbuminuria is also an early indication of changes in the kidney and cardiovascular system. It plays an important role in preventing nephrogenic hypertension. At present, there is no dry chemical kit or similar method that can detect sodium, creatinine and microalbumin in urine at the same time. Due to the insufficient detection hardware of the detection unit, it is urgent to provide a fast, simple and semi-quantitative detection kit for semi-quantitative detection of sodium, creatinine and microalbumin in urine.

Contents of the invention

The purpose of the present invention is to provide a dry chemical kit for simultaneously detecting urine sodium, creatinine and microalbumin.

The present invention provides a kit for simultaneously detecting sodium, creatinine and microalbumin in urine, comprising: (1) a reaction substrate with sodium reaction wells, creatinine reaction wells and microalbumin reaction wells;

(2) Sodium diluent, creatinine diluent;

(3) Standard color guides with different concentrations of sodium ion standard color development, different concentrations of creatinine standard color development and different concentrations of microalbumin standard color development.

The sodium reaction wells are sequentially provided with substrate pads and enzyme pads from the bottom of the wells;

The substrate pad is prepared by dripping the substrate solution on the carrier; the substrate solution uses 100-1000mmol/L pH7.5-9.0 buffer as solvent and contains 0.001-0.01g/mL of β-galactose Glycosidase substrate, 20-80mmol/L cryptin, 0.005%-50% stabilizer and 0.005%-50% surfactant;

The enzyme pad is prepared by dripping the enzyme solution on the carrier; the enzyme solution uses 100-1000mmol/L pH 7.0-9.0 buffer as the solvent, contains 200-3000U/mL β-galactosidase and 0.01- 100g/L enzyme stabilizer.

Preferably, the enzyme solution uses 100-1000mmol/L buffer solution with pH 7.0-9.0 as solvent, contains 500-1500U/mL β-galactosidase and 10-20g/L stabilizer.

The above buffers can be selected from one or more of potassium phosphate buffer, Tris-HCl buffer or PIPES buffer.

Preferably, the buffer of the substrate solution is 800-1000mmol/L Tris-HCl buffer at pH 8.0-9.0; the buffer of the enzyme solution is 100-200mmol/L Tris-HCl buffer at pH 7.0-8.0.

The β-galactosidase substrates contained in the substrate pad are O-nitro-β-D-pyranoside (ONPG), 5-bromo-4-chloro-3-indole-β-D-pyranoside Galactoside (x-gal), chlorophenol red-β-D-galactopyranoside (CPRG), PNPG, IPTG.

Preferably, the β-galactosidase substrates are 5-bromo-4-chloro-3-indole-β-D-galactopyranoside (x-gal) and chlorophenol red-β-D-pyranoside Galactoside (CPRG).

The cryptane contained in the substrate pad is 4,7,13,16,21-pentoxa-1,10-diazabicyclo[8.8.5]tricosane, 18-Crown-8 or 15-Crown One or more of -5. Preferably, the cryptane is 4,7,13,16,21-pentoxa-1,10-diazabicyclo[8.8.5]tricosane (Kryptofix)

The stabilizers contained in the substrate pad are D-trehalose, sucrose, dextran T70, β-cyclodextrin, bovine serum albumin, mannitol, α-lactose, polyethylene glycol, gelatin, inositol, wood One or more of sugar or arabitol, the preferred stabilizers are sucrose and D-trehalose;

The surfactant contained in the substrate pad is one or more of Tween 80, Tween 20, Triton X-100, NP40 or sorbitol, preferably the surfactant is Tween 80.

The stabilizers contained in the enzyme pad are D-trehalose, sucrose, dextran T70, β-cyclodextrin, bovine serum albumin, mannitol, polyethylene glycol, gelatin, inositol, xylose, arabitol , one or more of α-lactose, maleic acid, tartaric acid or citric acid, preferably the stabilizer is α-lactose and dextran T70.

The creatinine reaction hole in the reaction substrate in the kit for simultaneously detecting sodium, creatinine and microalbumin in urine provided by the present invention has a lower pad and an upper pad sequentially from the bottom of the hole;

The underpad is prepared by dripping the underpad solution on the carrier; the underpad solution uses water as a solvent, contains 10-2000U/mL peroxidase, 10-1000U/mL creatinine amide hydrolase, 0.1%- 10% chromogen 1, 5-500mmol/L pH 6.0-8.0 buffer, 0.005%-50% stabilizer;

Preferably, the activity of peroxidase in the underlying solution is 800-1500U/mL, and the activity of creatinine amide hydrolase is 500-1000U/mL;

The color developer 1 is one or more of 4-aminoantipyridine, 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate (MBTH); preferably the color developer 1 0.5%-2% of 4-aminoantipyridine;

The buffer of 5-500mmol/L pH 6.0-8.0 is one or more of sodium phosphate buffer, potassium phosphate buffer, Tris-HCl buffer or PIPES buffer, preferably the buffer is 50-200mmol /L potassium phosphate buffer solution with pH 7.0-8.0;

The 0.005%-50% stabilizer is D-trehalose, sucrose, dextran T70, β-cyclodextrin, bovine serum albumin, mannitol, polyethylene glycol, gelatin, inositol, xylose, One or more of arabitol, α-lactose, maleic acid, tartaric acid or citric acid, preferably the stabilizer is 1%-20% sucrose and inositol.

The upper pad of the creatinine reaction hole in the reaction substrate in the kit for simultaneously detecting sodium, creatinine and microalbumin in urine of the present invention is prepared by dropping the upper pad solution on the carrier; the upper pad solution uses water as a solvent , containing 10-2000U/mL sarcosine oxidase, 10-2000U/mL creatine amidinohydrolase, 0.1%-10% chromogen 2, 5-500mmol/L pH6.0-8.0 buffer Liquid, 0.005%-50% stabilizer;

Preferably, the activity of sarcosine oxidase in the upper pad solution is 10-500U/mL, and the activity of creatine amidinohydrolase is 20-500U/mL.

The developer 2 is 2,4,6-tribromo-3-hydroxybenzoic acid (TBHB), 3,5-dichloro-2-hydroxybenzenesulfonic acid (DCHBS), N-ethyl-N-( 2-Hydroxy-3-sulfopropyl)-3-methylaniline (TOOS), N-ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS), N,N-bis( One or more of 4-sulfobutyl)-3-methylaniline disodium salts. Preferably, the developer 2 is one or more of 1%-10% TBHB and DCHBS.

The buffer of the 5-500mmol/L pH6.0-8.0 is one or more of sodium phosphate buffer, potassium phosphate buffer, Tris-HCl buffer or PIPES buffer, preferably the buffer is 50-200mmol/L Potassium phosphate buffer solution of L pH7.0-8.0;

The 0.005%-50% stabilizer is D-trehalose, sucrose, dextran T70, β-cyclodextrin, bovine serum albumin, mannitol, polyethylene glycol, gelatin, inositol, xylose, One or more of arabitol, α-lactose, maleic acid, tartaric acid or citric acid, preferably the stabilizer is 1%-20% sucrose and dextran T70.

In a kit for simultaneously detecting sodium, creatinine and microalbumin in urine provided by the present invention, there is a chromogenic pad in the microalbumin reaction hole in the reaction substrate; the chromogenic pad is added dropwise by a chromogenic dye solution Prepared on a carrier; the chromogenic dye solution uses water as a solvent, contains 0.02-20mg/mL dye, 0.1-2mol/L pH 1.5-3.0 buffer, and 0.1-2% dispersant;

The dye is selected from 5'5"-2-nitro-3',3"-diiodo-3,4,5,6-tetrabromophenolsulfonphthalein (DIDNTB), bromophenol blue, and tetrabromophenol blue One or more of DIDNTB; the preferred dye is 1-5 mg/mL of DIDNTB.

The buffer is one or more of sodium phosphate buffer, potassium phosphate buffer, citrate buffer, Tris-HCl buffer or PIPES buffer; the preferred buffer is 1.5-2mol/L pH 1.5-3.0 citrate buffer.

The dispersant is selected from one or more of polyvinyl alcohol, triethylhexyl phosphoric acid, sodium lauryl sulfate, methyl amyl alcohol, cellulose derivatives, polyacrylamide, and gull gum. Preferably, the dispersant is 0.5-1.5% polyvinyl alcohol.

The carrier used in the sodium reaction well, creatinine reaction well and microalbumin reaction well in the reaction substrate in a kit for simultaneously detecting sodium, creatinine and microalbumin in urine of the present invention is filter paper, glass fiber or layer Analysis paper.

The size of the sodium reaction well, the creatinine reaction well and the microalbumin reaction well of the kit of the present invention can be preferred

In a kit for simultaneously detecting sodium, creatinine and microalbumin in urine provided by the present invention, the sodium diluent is 0.1-1.5 mol/L sodium phosphate buffer and potassium phosphate buffer with a pH of 8.0-9.0 , one or more of citric acid buffer solution, Tris-HCl buffer or PIPES buffer; preferably the buffer is 0.5-1.2mol/L Tris-HCl buffer with pH 8.0-8.8.

The creatinine diluent is one or more of 0.1-1.2 mol/L sodium phosphate buffer solution, potassium phosphate buffer solution, citric acid buffer solution, Tris-HCl buffer solution or PIPES buffer solution with a pH of 6.5-8.0. Preferably, the buffer is a 0.5-1.2 mol/L potassium phosphate buffer with a pH of 7.0-8.0.

The invention provides the application of the above reagent kit in simultaneous detection of sodium content, creatinine content and microalbumin content in urine.

Those skilled in the art should understand that in this field, detecting sodium content in urine is detecting sodium ion content in urine. Considering the needs of kit preservation, the prepared reaction pad must have a relatively high stability. On the basis of a large number of experimental studies, the inventor has determined the composition of the above-mentioned enzyme solution, chromogenic solution and various substrate solutions and their The formula makes the kit have ideal stability and can be popularized in practical applications.

In the present invention, the sodium ion and creatinine indexes are used to measure and predict the change of sodium intake in 24 hours, and the creatinine and microalbumin indexes are used to measure kidney damage, which is an auxiliary diagnosis of risk factors for hypertension and cardiovascular patients. Based on these research results, the present invention establishes a simple and rapid dry chemical assay kit for simultaneous detection of urinary sodium, creatinine and microalbumin.

Adopting the above technical scheme, the advantage of the present invention is that: the present invention solves the instability problem of liquid reagents, and by adding activators, enzyme stabilizers and other substances, it can quickly improve the reaction rate under the premise of ensuring the stability of the reagents on the reagent pad. Sensitivity and accuracy; the kit is easy and fast to operate, with high accuracy, and is suitable for routine clinical testing, especially bedside testing of clinical patients, so as to guide rational clinical medication. It only needs to dilute the collected urine, add it to the corresponding reaction well, react for a period of time for colorimetry, measure the sodium value (creatinine and microalbumin value) range of the tested urine, and evaluate the sodium intake of the tester. How much, no need for large-scale equipment, easy to operate, can be used in hospital emergency rooms, primary medical and health care units and patients' families. The kit of the invention avoids the one-sidedness and interference factors caused by observing a single index alone; and relatively, the collection of a single urine has the advantages of simplicity and speed.

Detailed ways

The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention fall within the scope of the present invention.

Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

Embodiment 1 simultaneously detects the preparation of the dry chemical kit of urine sodium, creatinine and trace albumin

The kit for simultaneous detection of urinary sodium, creatinine and microalbumin in this embodiment includes: a reaction substrate with sodium reaction wells, creatinine reaction wells and microalbumin reaction wells; sodium diluent and creatinine diluent; sodium ions with different concentrations Standard color guides for standard color development, standard color development of different concentrations of creatinine and standard color development of different concentrations of microalbumin.

The preparation method of the above-mentioned simultaneous detection of urine sodium, creatinine and microalbumin test kit is as follows:

(1) The structure of the reaction substrate:

The reaction substrate of the present embodiment is selected from ABS engineering plastics and is provided with sodium reaction holes, creatinine reaction holes and microalbumin reaction holes; the size of the reaction holes is

(2) Preparation of the substrate pad in the sodium reaction well:

The sodium reaction wells are provided with substrate pads and enzyme pads sequentially from the bottom of the wells.

The substrate pad was dropwise added with 1 μL of substrate solution on On chromatography paper, it was prepared after drying in flowing air at 25°C for 4 hours under dark conditions, and sealed and stored at 4°C.

The substrate solution uses 1000mmol/L Tris-HCl buffer with pH 8.7 as solvent, contains 0.005g/mL β-galactosidase substrate CPRG, 50mmol/L cryptin [4,7,13,16,21 - Pentaoxa-1,10-diazabicyclo[8.8.5]tricosane (Kryptofix)], 10% D-trehalose and 0.05% Tween 80.

Enzyme pads were added dropwise with 1 μL of enzyme solution on On chromatography paper, it is prepared after drying in the flowing air at 25°C for 4 hours under the condition of avoiding light, and it is sealed and stored at 4°C.

The enzyme solution uses 100mmol/L Tris-HCl buffer solution with pH 7.5 as solvent, contains 800U/mL β-galactosidase and 10g/L enzyme stabilizer

(3) Preparation of lower pad and upper pad in the creatinine reaction well:

The creatinine reaction well has a lower pad and an upper pad in order from the bottom of the well.

Preparation of underpad: Add 1 μL of underpad solution dropwise on On the chromatographic paper, under the condition of avoiding light, dry at 25°C with flowing air for 4 hours, and then seal and store at 4°C.

The underlying solution uses water as a solvent, containing 1000U/mL peroxidase, 800U/mL creatinine amide hydrolase, 1% 4-aminoantipyridine, 100mmol/L potassium phosphate buffer at pH 7.3, 10% of sucrose.

Preparation of the upper pad: Add 1 μL of the upper pad solution dropwise on the On the chromatographic paper, under the condition of avoiding light, dry at 25°C with flowing air for 4 hours, and then seal and store at 4°C.

The upper pad solution uses water as the solvent, contains 300U/mL sarcosine oxidase, 200U/mL creatine amidinohydrolase, 1% TBHB, 100mmol/L potassium phosphate buffer at pH 7.3, 10% sucrose .

(4) Preparation of chromogenic pads in microalbumin reaction wells: 1 μL chromogenic dye solution was added dropwise to the chromogenic pads. On the chromatographic paper, under the condition of avoiding light, dry at 25°C with flowing air for 4 hours, and then seal and store at 4°C.

The chromogenic dye solution uses water as a solvent and contains 2 mg/mL of dye 5'5"-2-nitro-3',3"-diiodo-3,4,5,6-tetrabromophenolsulfonphthalein (DIDNTB), 1mol/L pH 2.5 citrate buffer, 1% polyvinyl alcohol.

(5) Fill the reaction pads prepared in steps (2) to (4) into the corresponding reaction wells, compact them with a punching machine, then paste aluminum foil strips, and put them into aluminum foil bags to obtain sodium reaction wells. , creatinine reaction wells and reaction substrates for microalbumin reaction wells.

(6) Preparation of sodium diluent: 1000 mL of 0.8 mol/L Tris-HCl buffer solution with pH 8.7, mix thoroughly, and store in dropper bottles.

Preparation of creatinine dilution: 1000mL of 1mol/L potassium phosphate buffer solution with pH 7.3, mix thoroughly, and store in dropper bottles.

(7) Preparation of standard color guide: prepare standard solution of sodium, creatinine and microalbumin, the specific preparation method is as follows:

1) Basic urine preparation Collect urine: collect 5 copies of high-sodium urine that have not been ingested within three days (8:00-9:00 in the morning urine is the best), use this product to measure the sodium concentration in urine according to the instructions (should be < 20mmol/L), creatinine concentration (should be <0.15g/L), albumin concentration (should be <30mg/L), mix thoroughly, filter through a 0.45μm membrane, use as basic urine, and store at 2°C to 8°C. At the same time, basic urine can be used as a "negative sample".

2) Preparation of standard solution

(a) Preparation of sodium chloride standard solution Weigh 0.585 g of sodium chloride and add it to 2 ml of purified water for dissolution to prepare a 5000 mmol/L sodium chloride standard solution.

(b) Preparation of creatinine standard solution Weigh 0.1g of creatinine and add it into 2ml of purified water to dissolve to prepare 50g/L creatinine standard solution.

(c) Preparation of albumin standard solution Weigh 10 mg of human serum albumin and add it to 2 ml of purified water to dissolve to prepare a 5000 mg/L human serum albumin standard solution.

3) Preparation of color standard specimens

(a) Preparation of sodium standard swatches: Measure 4, 20, and 40 μl of basic urine into three centrifuge tubes in sequence, add 996, 980, and 960 μl of 5000 mmol/L sodium chloride standard solution in sequence, and mix well , sealed and stored at 2°C to 8°C, and used within 24 hours, the final concentration is shown in Table 1 below.

(b) Preparation of samples of creatinine standard swatches: Measure 3, 10, 20, and 40 μl of basic urine in turn and put them into four centrifuge tubes, add 997, 990, 980, and 960 μl of 50 g/L creatinine standard solution in sequence, mix Mix well, seal and store at 2°C to 8°C, and use within 24 hours. The final concentration is shown in Table 1 below.

(c) Preparation of samples of human albumin standard color plate: Measure 6, 16, 32, and 60 μl of basic urine into four centrifuge tubes, and add 994, 984, 968, and 940 μl of 5000 mg/L human serum in sequence Albumin standard solution, mixed well, sealed and stored at 2°C to 8°C, used within 24 hours, the final concentration is shown in Table 1 below.

Table 1 Concentration table of each color standard sample

project Color scale 1 Color code 2 Color code 3 Color scale 4 sodium 20mmol/L 100mmol/L 200mmol/L —— creatinine 0.15g/L 0.5g/L 1g/L 2g/L microalbumin 30mg/L 80mg/L 160mg/L 300mg/L

Add the above-mentioned standard solutions of different color standards to the reaction wells for color development, visually inspect by 3-5 people, and compare with the standard color guide, select the standard color, print it, and verify it. After repeated several times, Finalize the standard color guide.

Embodiment 2 Application of the dry chemical kit capable of simultaneously detecting urine sodium, creatinine and microalbumin of the present invention

The kit of the present invention is a dry chemical kit for semi-quantitative detection of urine sodium, creatinine and microalbumin, which can detect clinically the urine samples of hypertensive patients, and can also detect the urine samples of healthy people. It can be used as an auxiliary indicator for the diagnosis, differential diagnosis and curative effect evaluation of hypertensive patients, and can also be used as an auxiliary tool for healthy people to control their salt intake in their daily diet.

The specific method of use of the kit prepared in Example 1 of the present invention is as follows:

1) When in use, first take the kit out of the storage temperature, equilibrate to room temperature and start using;

2) Tear off the aluminum foil packaging and take out the reaction substrate;

3) Urine treatment: Take 25 μl of urine and add sodium and creatinine diluent respectively: mix thoroughly; the volume ratio of diluent to urine is 1:20-35 (1:30 is used in this embodiment).

4) Pipette 25 μl of the above-mentioned diluted urine into the sodium reaction well and the creatinine reaction well respectively; directly measure microalbumin.

5) React in a dry bath (set temperature at 48°C) for 7 minutes, compare the reaction result with the color guide, and obtain the concentration of the analyte semi-quantitatively. Those skilled in the art should understand that, according to the existing technology in this field, after using other heat preservation containers such as incubators, ovens, water bath containers, etc., to react at a temperature of 35-50 ° C, the comparative judgment of the results can also be realized .

The test kit of the present invention is used to detect clinically 90 volunteers (30 corresponding to different salt groups) whose daily salt content is less than 6 grams, 6-12 grams and >12 grams, and the accuracy of the detection results is up to 100%.

The performance of the kit of the invention is stable, and the shelf life is 18 months.

The kit of the present invention is used to detect clinical urine sodium, creatinine and microalbumin, and compared with commercialized urine sodium, creatinine and microalbumin quantitative kits, the accuracy of the detection results of the kit of the present invention is up to above 95.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (9)

1. kit that is a kind of while detecting sodium in urine, creatinine and microalbumin, which is characterized in that including：

(1) the reaction substrate with sodium reacting hole, creatinine reacting hole and microalbumin reacting hole；

(2) sodium dilution, creatinine dilution；

(3) there is various concentration sodium ion standard coloration, various concentration creatinine standard coloration and various concentration microalbumin mark The standard colorimetric plate of quasi- colour developing；

Wherein step (1) the sodium reacting hole is placed with substrate pad and enzyme pad successively from the bottom of hole；

The substrate pad is added dropwise by substrate solution and is prepared on carrier；Substrate solution is with 100-1000mmol/L pH 7.5- 9.0 buffer solution be solvent, the cave ether of beta galactosidase substrate, 20-80mmol/L containing 0.001-0.01g/mL, The stabilizer of 0.005%-50% and the surfactant of 0.005%-50%；

The enzyme pad is added dropwise by enzyme solutions and is prepared on carrier；Enzyme solutions are with 100-1000mmol/L pH 7.0-9.0's Buffer solution is solvent, the beta galactosidase containing 200-3000U/mL and 0.01-100g/L enzyme stabilizers.

2. kit according to claim 1, which is characterized in that buffer solution is selected from kaliumphosphate buffer, Tris-HCl is buffered The one or more of liquid or PIPES buffer solutions.

3. kit according to claim 2, which is characterized in that the buffer solution of substrate solution is 800-1000mmol/L pH 8.0-9.0 Tris-HCl buffer solutions；The Tris-HCl that the buffer solution of enzyme solutions is 100-200mmol/L pH 7.0-8.0 is slow Fliud flushing.

4. kit according to claim 1, which is characterized in that the beta galactosidase substrate is O- nitro-β-D- pyrroles Mutter the chloro- 3- indoles-β-D- galactopyranosides of the bromo- 4- of glucosides, 5-, chlorophenol red-β-D- galactopyranosides, PNPG, IPTG.

5. kit according to claim 1, which is characterized in that the cave ether is 4,7,13,16,21- five oxa-s -1,10- One or more of diazabicyclo [8.8.5] tricosane, 18-Crown-8 either 15-Crown-5.

6. kit according to claim 1, which is characterized in that the creatinine reacting hole is placed with underlay successively from the bottom of hole With upper pad；

The underlay is added dropwise by underlay solution and is prepared on carrier；Underlay solution contains 10-2000U/mL using water as solvent Peroxidase, 10-1000U/mL creatinine amidohydrolase hydrolases, the color developing agent 1 of 0.1%-10%, 5-500mmol/L pH The buffer solution of 6.0-8.0, the stabilizer of 0.005%-50%；

The color developing agent 1 be 4- amino antipyrine, one kind in 3- methyl-2-benzothiazolinone hydrazone hydrochloride hydrates or It is a variety of；

The upper pad is added dropwise by upper pad solution to be prepared on carrier；Upper pad solution contains 10-2000U/mL using water as solvent Sarcosine oxidase, 10-2000U/mL creatine amidino groups hydrolase, the color developing agent 2 of 0.1%-10%, 5-500mmol/L pH The buffer solution of 6.0-8.0, the stabilizer of 0.005%-50%；

The color developing agent 2 is tri- bromo- 3- hydroxybenzoic acids of 2,4,6-, bis- chloro- 2- hydroxy benzene sulfonic acids of 3,5-, N- ethyls-N- (2- hydroxyls Base -3- sulfopropyls) -3- methylanilines, N- ethyls-N- (3- sulfopropyls) -3- methylanilines, N, bis- (4- the sulphur butyls) -3- first of N- It is one or more in base aniline disodium salt.

7. kit according to claim 1, which is characterized in that the microalbumin reacting hole has colour developing to pad；It is described aobvious Color pad is added dropwise by developing dye solution and is prepared on carrier；The developing dye solution contains 0.02- using water as solvent The dispersant of the dyestuff of 20mg/mL, the buffer solution of 0.1-2mol/L pH 1.5-3.0,0.1-2%；

The dyestuff is selected from 5 ' 5 " -2- nitros -3 ', and 3 "-two iodo- 3, in 4,5,6- tetrabromophenol sulfonphthaleins, bromophenol blue, Tetrabromophenol Blue It is one or more；

The buffer solution be sodium phosphate buffer, kaliumphosphate buffer, citric acid solution, Tris-HCl buffer solutions or The one or more of PIPES buffer solutions；

The dispersant is selected from polyvinyl alcohol, triethyl group hexyl phosphoric acid, lauryl sodium sulfate, methyl anyl alcohol, cellulose and derives One or more of object, polyacrylamide, guar gum.

8. according to any kits of claim 1-7, which is characterized in that the carrier be filter paper, glass fibre or Chromatographic paper.

9. claim the 1-7 any kit sodium content, creatinine content and microalbumin content in detecting urine In application.